Plant composition changes in a small-scale community have a large effect on the performance of an economically important grassland pest.

BACKGROUND: The grasshopper Oedaleus asiaticus Bey-Bienko (Acrididae: Oedipodinae) is a dominant and economically important pest that is widely distributed across the Mongolian plateau. This herbivore pest causes major damage to the grassland of the Inner Mongolian steppe in China. The population dynamics of herbivore pests is affected by grassland management practices (e.g., mowing and heavy livestock grazing) that alter plant community structures and stoichiometric characteristics. For example, O. asiaticus outbreak is closely associated with plant preference changes caused by nitrogen loss from heavy livestock grazing. However, the manner by which small-scale variation in vegetation affects grasshopper performance and promotes outbreak is poorly characterized. To address this question, we investigated the relationship between small-scale (1 m2) vegetation variability and measures of O. asiaticus performance associated with plant stoichiometric characteristics. RESULTS: We found that food preferences of O. asiaticus varied significantly, but maintained a specific dietary structure for different plant compositions. Notably, small-scale changes in plant community composition significantly affected grasshopper food preference and body size. Partial least-square modeling indicated that plant proportion and biomass affected grasshopper body size and density. We found that this effect differed between sexes. Specifically, female body mass positively correlated with the proportion of Stipa krylovii grass, whereas male mass positively correlated with the proportion of Artemisia frigida grass. Further analyses indicated that grasshopper performance is closely associated with plant stoichiometric traits that might be responsible for the pest's plague. CONCLUSIONS: This study provides valuable information for managing grasshoppers using rational grassland management practices.

Involvement of nonbinding site proteinases in the development of resistance of Helicoverpa armigera (Lepidoptera: Noctuidae) to Cry1Ac.

Development of resistance to transgenic crops expressing the Cry toxin from Bacterium thuringiensis (Bt) has been the major concern for the long-term success of Bt crops. Alterations in nonbinding site proteinases and Bt toxin receptors are the two types of mechanisms responsible for Bt resistance in resistant insects. However, little is known about the relative contributions of the two types of mechanisms in the early and late phases of the development of Bt resistance. To address the relative contributions of four nonbinding site proteinases including esterase, total protease, chymotrypsin, and glutathione S-transferase in the early and late phases of the development of Cry1Ac resistance, we analyzed the relationships between nonbinding site proteinases and resistance of three groups of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) strains with different resistance levels because of different geographic origins and selection pressures. Positive correlation (esterase, glutathione-S-tranferases [GST], and chymotrypsin) and negative correlation (total midgut protease) were observed within the low to moderate group II resistant strains. Such correlations were less obvious within the low to moderate group III resistant strains because of only threefold differences in LC50 values. Relative to the unselected susceptible 96S strain, the two highly resistant group I resistant strains BtI and BtR have the same amounts of esterase, GST, and chymotrypsin and disproportionally decreased the amount of total midgut protease. Overall, the low to moderate resistant strains had the lowest amount of the nonbinding site proteinases. The results obtained suggest that alternations in the nonbinding site proteinases probably can only confer low to moderate levels of resistance and thus are enriched in the early phase of the development of Cry1Ac resistance.

Changes of sex pheromone communication systems associated with tebufenozide and abamectin resistance in diamondback moth, Plutella xylostella (L.).

Many insect pests have evolved resistance to insecticides. Along with this evolution, the sex pheromone communication system of insects also may change, and subsequently reproductive isolation may occur between resistant and susceptible populations. In this study of the diamondback moth, we found that resistant females (especially Abamectin resistant females) produced less sex pheromone and displayed a lower level of calling behavior. Resistant males showed higher EAG responsiveness to the sex pheromone mixture of females, and responded to a broader range of ratios between the two major components compared to the responses of susceptible moths. In addition, wind tunnel experiments indicated that changes associated with insecticide resistance in the Abamectin resistant strain (Aba-R) significantly reduced female attractiveness to susceptible males. Furthermore, mating choice experiments confirmed that non-random mating occurred between the two different strains. Aba-R females with an abnormal pheromone production and blend ratio exhibited significantly lower mating percentages with males from either their own strain or other strains, which corroborates the results obtained by the wind tunnel experiments. The implications of this non-random mating for insect speciation and insecticide resistance management are discussed.

Identification of two Lynx proteins in Nilaparvata lugens and the modulation on insect nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect brain and are targets for neonicotinoid insecticides. Some proteins, other than nAChRs themselves, might play important roles in insect nAChRs function in vivo and in vitro, such as the chaperone, regulator and modulator. Here we report the identification of two nAChR modulators (Nl-lynx1 and Nl-lynx2) in the brown planthopper, Nilaparvata lugens. Analysis of amino acid sequences of Nl-lynx1 and Nl-lynx2 reveals that they are two members of the Ly-6/neurotoxin superfamily, with a cysteine-rich consensus signature motif. Nl-lynx1 and Nl-lynx2 only increased agonist-evoked macroscopic currents of hybrid receptors Nlalpha1/beta2 expressed in Xenopus oocytes, but not change the agonist sensitivity and desensitization properties. For example, Nl-lynx1 increased I(max) of acetylcholine and imidacloprid to 3.56-fold and 1.72-fold of that of Nlalpha1/beta2 alone, and these folds for Nl-lynx2 were 3.25 and 1.51. When the previously identified Nlalpha1(Y151S) mutation was included (Nlalpha1(Y151S)/beta2), the effects of Nl-lynx1 and Nl-lynx2 on imidacloprid responses, but not acetylcholine response, were different from that in Nlalpha1/beta2. The increased folds in imidacloprid responses by Nl-lynx1 and Nl-lynx2 were much higher in Nlalpha1(Y151S)/beta2 (3.25-fold and 2.86-fold) than in Nlalpha1/beta2 (1.72-fold and 1.51-fold), which indicated Nl-lynx1 and Nl-lynx2 might also serve as an influencing factor in target-site insensitivity in N. lugens. These findings indicate that nAChRs chaperone, regulator and modulator may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance.

Correction: Different Effects of Metarhizium anisopliae Strains IMI330189 and IBC200614 on Enzymes Activities and Hemocytes of Locusta migratoria L.

[This corrects the article DOI: 10.1371/journal.pone.0155257.].

Response of peanut Arachis hypogaea roots to the presence of beneficial and pathogenic fungi by transcriptome analysis.

Entomopathogenic fungus Metarhizium anisopliae obtain survival benefit meanwhile promote the nutrient absorption of root as an endophyte. However, little is known concerning molecular mechanisms in the process. We performed the transcriptome sequencing of A. hypogaea roots inoculated M. anisopliae and pathogenic Fusarium axysporum, respectively. There were 81323 unigenes from 132023 transcripts. Total 203 differentially expressed genes (DEGs) respond to the two fungi, including specific 76 and 34 DEGs distributed respectively in M. anisopliae and F. axysporum treatment. KEGG pathway enrichment for DEGs showed the two top2 were signal transductions of plant-pathogen interaction and plant hormone. By qRT-PCR, the mRNA level of 26 genes involved in plant-fungus interaction confirmed the reliability of the RNA-Seq data. The expression pattern of the key DEGs on jasmonic acid (JA) or salicylic acid (SA) signaling pathway presented regulating consistency with JA or SA concentration detected by HPLC-MS. Those significantly stronger down-regulated DEGs by M. anisopliae thanby F. axysporum linking to hypersensitive response and negative regulation of defense, and those specific up-regulated genes in M. anisopliae treatment may predict that the less immunity is conducive to symbiosis F. axysporum may trigger JA-mediated defense regulated by ERF branch of JA signaling pathway, whereas M. anisopliae does not.

Biology, physiology and gene expression of grasshopper Oedaleus asiaticus exposed to diet stress from plant secondary compounds.

We studied the role of plant primary and secondary metabolites in mediating plant-insect interactions by conducting a no-choice single-plant species field experiment to compare the suitability, enzyme activities, and gene expression of Oedaleus asiaticus grasshoppers feeding on four host and non-host plants with different chemical traits. O. asiaticus growth showed a positive relationship to food nutrition content and a negative relationship to secondary compounds content. Grasshopper amylase, chymotrypsin, and lipase activities were positively related to food starch, crude protein, and lipid content, respectively. Activity of cytochrome P450s, glutathione-S-transferase, and carboxylesterase were positively related to levels of secondary plant compounds. Gene expression of UDP-glucuronosyltransferase 2C1, cytochrome P450 6K1 were also positively related to secondary compounds content in the diet. Grasshoppers feeding on Artemisia frigida, a species with low nutrient content and a high level of secondary compounds, had reduced growth and digestive enzyme activity. They also had higher detoxification enzyme activity and gene expression compared to grasshoppers feeding on the grasses Cleistogenes squarrosa, Leymus chinensis, or Stipa krylovii. These results illustrated Oedaleus asiaticus adaptive responses to diet stress resulting from toxic chemicals, and support the hypothesis that nutritious food benefits insect growth, but plant secondary compounds are detrimental for insect growth.

Gut Transcriptome Analysis Shows Different Food Utilization Efficiency by the Grasshopper Oedaleous asiaticus (Orthoptera: Acrididae).

Oedaleus asiaticus B. Bienko is a persistent pest occurring in north Asian grasslands. We found that O. asiaticus feeding on Stipa krylovii Roshev. had higher approximate digestibility (AD), efficiency of conversion of ingested food (ECI), and efficiency of conversion of digested food (ECD), compared with cohorts feeding on Leymus chinensis (Trin.) Tzvel, Artemisia frigida Willd., or Cleistogenes squarrosa (Trin.) Keng. Although this indicated high food utilization efficiency for S. krylovii, the physiological processes and molecular mechanisms underlying these biological observations are not well understood. Transcriptome analysis was used to examine how gene expression levels in O. asiaticus gut are altered by feeding on the four plant species. Nymphs (fifth-instar female) that fed on S. krylovii had the largest variation in gene expression profiles, with a total of 88 genes significantly upregulated compared with those feeding on the other three plants, mainly including nutrition digestive genes of protein, carbohydrate, and lipid digestion. GO and KEGG enrichment also showed that feeding S. krylovii could upregulate the nutrition digestion-related molecular function, biological process, and pathways. These changes in transcripts levels indicate that the physiological processes of activating nutrition digestive enzymes and metabolism pathways can well explain the high food utilization of S. krylovii by O. asiaticus.

Molecular Ecological Basis of Grasshopper (Oedaleus asiaticus) Phenotypic Plasticity under Environmental Selection.

While ecological adaptation in insects can be reflected by plasticity of phenotype, determining the causes and molecular mechanisms for phenotypic plasticity (PP) remains a crucial and still difficult question in ecology, especially where control of insect pests is involved. Oedaleus asiaticus is one of the most dominant pests in the Inner Mongolia steppe and represents an excellent system to study phenotypic plasticity. To better understand ecological factors affecting grasshopper phenotypic plasticity and its molecular control, we conducted a full transcriptional screening of O. asiaticus grasshoppers reared in four different grassland patches in Inner Mongolia. Grasshoppers showed different degrees of PP associated with unique gene expressions and different habitat plant community compositions. Grasshopper performance variables were susceptible to habitat environment conditions and closely associated with plant architectures. Intriguingly, eco-transcriptome analysis revealed five potential candidate genes playing important roles in grasshopper performance, with gene expression closely relating to PP and plant community factors. By linking the grasshopper performances to gene profiles and ecological factors using canonical regression, we first demonstrated the eco-transcriptomic architecture (ETA) of grasshopper phenotypic traits (ETAGPTs). ETAGPTs revealed plant food type, plant density, coverage, and height were the main ecological factors influencing PP, while insect cuticle protein (ICP), negative elongation factor A (NELFA), and lactase-phlorizin hydrolase (LCT) were the key genes associated with PP. Our study gives a clear picture of gene-environment interaction in the formation and maintenance of PP and enriches our understanding of the transcriptional events underlying molecular control of rapid phenotypic plasticity associated with environmental variability. The findings of this study may also provide new targets for pest control and highlight the significance of ecological management practice on grassland conservation.

Tebufenozide resistance selected in Plutella xylostella and its cross-resistance and fitness cost.

A susceptible strain of diamondback moth, Plutella xylostella (L.), was used to select for resistance to tebufenozide in the laboratory. After continuous selection with tebufenozide 17 times during 35 generations, a resistant strain was achieved with high resistance to tebufenozide (RR 93.8). Bioassay revealed that this strain showed high cross-resistance to abamectin (RR 35.7), methoxyfenozide (29.1) and JS118 (16.5), and a little to deltamethrin (3.9), but no obvious cross-resistance was found to cypermethrin (1.3), fipronil (1.3), trichlorfon (1.1), chlorfenapyr (1.0), phoxim (0.9) and acephate (0.8). The resistant and susceptible insects had similar development rates, but life table tests indicated that the resistant strain showed reproductive disadvantages, including decreased copulation rate, reproductivity and hatchability. When compared with the susceptible strain, the resistant insects had a relative fitness of only 0.3. This indicated that tebufenozide resistance selected under laboratory conditions had considerable fitness costs in this pest, and therefore rotational use of insecticides without cross-resistance is recommended to delay development of resistance.

Low Temperature Storage of Eggs Improve the Development and Reproduction of Locusta migratoria (Orthoptera: Acrididae).

Locusta migratoria L. is an insect with significant economic value. Improving the long-term storage of L. migratoria eggs will help promote the large-scale rearing of this insect. We assessed multiple fitness parameters and enzyme activities of locusts emerged from eggs exposed to 4 °C for 1-4 wk. Locusts emerged from eggs stored at 4 °C for 2 wk showed significantly improved development and reproduction compared with locusts emerged from eggs stored for other time periods. The preimaginal survival rate increased significantly after 2-wk storage while it decreased significantly after 4-wk storage compared with other storage times. The fecundity, hatching rate, and growth rate increased significantly after 2-wk storage, but decreased significantly after 1, 3, and 4 wk compared with the control. However, the preimaginal developmental duration decreased significantly after 2-wk storage but increased significantly after storage for 1, 3, and 4 wk compared with the control. The activities of esterase, glutathione-S-transferases, phenol oxidase, and chitinase were obviously fluctuated with changes in intrinsic rate of increase (rm). These results showed that eggs stored at 4 °C for 2 wk could improve the development and reproduction of locust emerged from eggs, and four enzymes activities in above could reflect the health of locust. Our results could be useful in developing large-scale rearing protocols for L. migratoria.

Different Effects of Metarhizium anisopliae Strains IMI330189 and IBC200614 on Enzymes Activities and Hemocytes of Locusta migratoria L.

BACKGROUND: Metarhizium is an important class of entomopathogenic fungi in the biocontrol of insects, but its virulence is affected by insect immunity. To clarify the mechanism in virulence of Metarhizium, we compared the immunological differences in Locusta migratoria L. when exposed to two strains of Metarhizium anisopliae (Ma). RESULTS: The virulence of Ma IMI330189 was significantly higher than that of Ma IBC200614 to locust, and IMI330189 overcame the hemocytes and began destroying the hemocytes of locust at 72 h after spray, while locust is immune to IBC200614. IMI330189 could overcome the humoral immunity of locust by inhibiting the activities of phenol oxidase (PO), esterases, multi-function oxidases (MFOs) and acetylcholinesterases in locust while increasing the activities of glutathione-S-transferases (GSTs), catalase and aryl-acylamidase (AA). However IBC200614 inhibit the activities of GSTs and AA in locust and increase the activities of MFOs, PO, superoxide dismutase, peroxidase and chitinase in locust. The changes of enzymes activities in period of infection showed that the time period between the 2nd and the 5th day after spray is critical in the pathogenic process. CONCLUSION: These results found the phenomenon that Ma initiatively broke host hemocytes, revealed the correlation between the virulence of Ma and the changes of enzymes activities in host induced by Ma, and clarified the critical period in the infection of Ma. So, these results should provide guidance for the construction of efficient biocontrol Ma strains.

Biochemical basis of synergism between pathogenic fungus Metarhizium anisopliae and insecticide chlorantraniliprole in Locusta migratoria (Meyen).

We challenged Locusta migratoria (Meyen) grasshoppers with simultaneous doses of both the insecticide chlorantraniliprole and the fungal pathogen, Metarhizium anisopliae. Our results showed synergistic and antagonistic effects on host mortality and enzyme activities. To elucidate the biochemical mechanisms that underlie detoxification and pathogen-immune responses in insects, we monitored the activities of 10 enzymes. After administration of insecticide and fungus, activities of glutathione-S-transferase (GST), general esterases (ESTs) and phenol oxidase (PO) decreased in the insect during the initial time period, whereas those of aryl acylamidase (AA) and chitinase (CHI) increased during the initial period and that of acetylcholinesterase (AChE) increased during a later time period. Activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) decreased at a later time period post treatment. Interestingly, treatment with chlorantraniliprole and M. anisopliae relieved the convulsions that normally accompany M. anisopliae infection. We speculate that locust mortality increased as a result of synergism via a mechanism related to Ca(2+) disruption in the host. Our study illuminates the biochemical mechanisms involved in insect immunity to xenobiotics and pathogens as well as the mechanisms by which these factors disrupt host homeostasis and induce death. We expect this knowledge to lead to more effective pest control.

Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species.

Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management.

Molecular characterisation of the vitellogenin gene (AlVg) and its expression after Apolygus lucorum had fed on different hosts.

BACKGROUND: The polyphagous pest Apolygus lucorum is now the dominant pest of Bacillus thuringiensis (Bt) cotton in China. In this study, the transcriptional and translational profiles of AlVg influenced by different hosts were identified, and then the correlations between AlVg gene or AlVg protein expression and key population proliferation parameters of A. lucorum were further clarified. RESULTS: AlVg or AlVg expression can be significantly regulated by different host nutrients (P < 0.05). AlVg or AlVg expression was significantly higher in A. lucorum reared on Bt and conventional cotton than in A. lucorum reared on garland chrysanthemum and broad bean (P < 0.05), but there was no significant difference between AlVg or AlVg expression in A. lucorum reared on Bt and conventional cotton (P > 0.05). In addition, there were significant linear regression correlations between AlVg or AlVg expression and total mortality rate of nymphs, female lifespan, per female fecundity and egg hatching rates (P < 0.05). CONCLUSION: Our results confirm that AlVg or AlVg is the key parameter affecting female fertility of A. lucorum. AlVg and AlVg expression can be influenced by different host nutrients except for Bt toxin. © 2015 Society of Chemical Industry.

Tebufenozide resistance is associated with sex-linked inheritance in Plutella xylostella.

The diamondback moth (DBM), Plutella xylostella (L.), is a major pest of cruciferous crops. Tebufenozide, a novel nonsteroidal ecdysone agonist, exhibits good efficacy and has played an increasingly important role in the control of Lepidopteran pests in China. For its resistance management, the genetic basis of tebufenozide resistance was studied using a laboratory selected resistant strain of DBM (resistant ratio, RR = 268). A series of crosses with laboratory susceptible and resistant strains revealed that tebufenozide resistance in this pest was partially biased toward female heredity, with a large difference in RR for F1 (RR = 29) and rF1 progeny (RR = 147). The dominance calculated for these 2 cross progeny was -0.788 and 0.09, respectively. Further analysis showed that the susceptible male and female larvae were similar in their sensitivity to tebufenozide, but the resistant female larvae showed significantly higher resistance than the resistant male larvae. The heredity of tebufenozide resistance in DBM might be linked with the W sex chromosome, which suggested that DBM has the ability to develop high levels of resistance to tebufenozide. This is the first report of sex-linked inheritance of tebufenozide resistance in P. xylostella (L.).

Transcriptomic and proteomic analysis of pre-diapause and non-diapause eggs of migratory locust, Locusta migratoria L. (Orthoptera: Acridoidea).

Low temperature induces diapause in locusts. However, the physiological processes and initiation mechanism of diapause are not well understood. To understand the molecular basis of diapause, 'omics' analyses were performed to examine the differences between diapause and non-diapause eggs at both transcriptional and translational levels. Results indicated that a total of 62,241 mRNAs and 212 proteins were differentially expressed. Among them, 116 transcripts had concurrent transcription and translation profiles. Up-regulated genes related to diapause included glutathiones-S-transferase et al., and down-regulated genes including juvenile hormone esterase-like protein et al. KEGG analysis mapped 7,243 and 99 differentially expressed genes and proteins, to 83 and 25 pathways, respectively. Correlation enriched pathways indicated that there were nine identical pathways related to diapause. Gene Ontology analysis placed these genes and proteins into three categories, and a higher proportion of genes related to metabolism was up-regulated than down-regulated. Furthermore, three up-regulated pathways were linked to cryoprotection. This study demonstrates the applicability of high-throughput omics tools to identify molecules linked to diapause in the locust. In addition, it reveals cellular metabolism in diapause eggs is more active than in non-diapause eggs, and up-regulated enzymes may play roles in cryoprotection and storing energy for diapause and post-diapause stages.

Quantitative analysis of fitness costs associated with the development of resistance to the Bt toxin Cry1Ac in Helicoverpa armigera.

Transgenic Bacillus thuringiensis (Bt) crops play an increasing role in pest control, and resistance management is a major issue in large-scale cultivation of Bt crops. The fitness cost of resistance in targeted pests is considered to be one of the main factors delaying resistance when using the refuge strategy. By comparing 10 resistant Helicoverpa armigera (Hubner) strains, showing various resistance levels to Bt toxin (Cry1Ac), to a susceptible strain, we showed an increasing fitness cost corresponding with increasing levels of resistance. The relationship between overall fitness cost C and the resistance ratio Rr could be described by C = 24.47/(1 + exp([1.57 - Log10Rr]/0.2)). This model predicted that the maximum overall fitness cost would be ~24% (± 5.22) in the strains with the highest resistance level. The overall fitness cost was closely linked to egg hatching rate, fecundity, emergence rate, larval survival rate, and developmental duration of adults. Among fitness components measured, fecundity was the most sensitive trait linked to the resistance selection. To integrate the results into simulation models would be valuable in evaluating how variation in fitness cost may influence the development of resistance in pest populations, thus helping to develop enhanced refuge strategies.

A constructed alkaline consortium and its dynamics in treating alkaline black liquor with very high pollution load.

BACKGROUND: Paper pulp wastewater resulting from alkaline extraction of wheat straw, known as black liquor, is very difficult to be treated and causes serious environmental problems due to its high pH value and chemical oxygen demand (COD) pollution load. Lignin, semicellulose and cellulose are the main contributors to the high COD values in black liquor. Very few microorganisms can survive in such harsh environments of the alkaline wheat straw black liquor. A naturally developed microbial community was found accidentally in a black liquor storing pool in a paper pulp mill of China. The community was effective in pH decreasing, color and COD removing from the high alkaline and high COD black liquor. FINDINGS: Thirty-eight strains of bacteria were isolated from the black liquor storing pool, and were grouped as eleven operational taxonomy units (OTUs) using random amplified polymorphic DNA-PCR profiles (RAPD). Eleven representative strains of each OTU, which were identified as genera of Halomonas and Bacillus, were used to construct a consortium to treat black liquor with a high pH value of 11.0 and very high COD pollution load of 142,600 mg l(-1). After treatment by the constructed consortium, about 35.4% of color and 39,000 mg l(-1) (27.3%) COD(cr) were removed and the pH decreased to 7.8. 16S rRNA gene polymerase chain reaction denaturant gradient gel electrophoresis (PCR-DGGE) and gas chromatography/mass spectrometry (GC/MS) analysis suggested a two-stage treatment mechanism to elucidate the interspecies collaboration: Halomonas isolates were important in the first stage to produce organic acids that contributed to the pH decline, while Bacillus isolates were involved in the degradation of lignin derivatives in the second stage under lower pH conditions. CONCLUSIONS/SIGNIFICANCE: Tolerance to the high alkaline environment and good controllability of the simple consortium suggested that the constructed consortium has good potential for black liquor treatment. Facilitating the treatment process by the constructed consortium would provide a promising opportunity to reduce the pollution, as well as to save forest resources and add value to a waste product.