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This study using hexadecyl trimethyl ammonium bromide (HDTMA) modified zeolite as a component of bioretention substrate, to investigate the effect of HDTMA modification on the basic physical and hydraulic properties of substrate layer. Two different levels of HDTMA modified zeolite (ZHD10 and ZHD50) were mixed with a mixture consists of peat soil, river sand and compost (fixed volumetric proportion at 5:4:1) with varying volumetric percentage (25%, 50%, and 75%) to form substrate media. The modification only changes the physical properties of zeolite and media with zeolite slightly, while significant changes in surface hydrophobicity and hydraulic properties were observed. A distinct decline of saturated hydraulic conductivity (

The robust innate immune system of the earthworm provides a potential source of natural antimicrobial peptides (AMPs). However, the cost and high rediscovery rate of direct separation and purification limits their discovery. Genome sequencing of numerous earthworm species facilitates the discovery of new antimicrobial peptides. Through predicting potential antimicrobial peptides in the open reading frames of the Eisenia andrei genome and sequence optimization, a novel antimicrobial peptide, named EWAMP-R (RIWWSGGWRRWRW), was identified. EWAMP-R demonstrated good activity against various bacteria, including drug-resistant strains. The antibacterial mechanisms of EWAMP-R were explored through molecular simulation and wet-laboratory experiments. These experiments demonstrated that the bacterial membrane may be one of the targets of EWAMP-R but that there may be different interactions with Gram-negative and Gram-positive bacterial membranes. EWAMP-R can disrupt bacterial membrane integrity; however, at low concentrations, it appears that EWAMP-R may get through the membrane of Escherichia coli instead of damaging it directly, implying the existence of a secondary response. Gene expression studies identified that in E. coli, only the apoptosis-like cell death (ALD) pathway was activated, while in Staphylococcus aureus, the MazEF pathway was also upregulated, limiting the influence of the ALD pathway. The different antimicrobial actions against Gram-positive and -negative bacteria can provide important information on the structure-activity relationship of AMPs and facilitate AMP design with higher specificity. This study identified a new source of antibacterial agents that has the potential to address the increasingly serious issue of antibiotic resistance.

Yuan, Zhu, Dong, Yan, Jiang, Jibao, Qiu, Jiangping (2019): Three new species of earthworms belonging to the genera Amynthas and Metaphire (Oligochaeta: Megascolecidae) from Yunnanı China. Journal of Natural History 53 (31): 1961-1974, DOI: 10.1080/00222933.2019.1680760

Arsenic (As) is a widely distributed contaminant in soil and is toxic to plants, animals and humans. Many studies have explored the effects of As on various physiological and biochemical processes in soil fauna. However, there is no report on the effect of sodium arsenite on the behavior of soil fauna such as earthworms. In this study, two earthworm species, Metaphire californica and Eisenia fetida, were exposed to sodium arsenite in farm soils at concentrations of 5, 10, 20, 40 and 80 mg As kg−1 soil for 7 days to investigate the effect on burrowing behavior. Earthworm burrowing behavior responded significantly to both arsenite concentration and exposure time. Both species displayed an initially similar burrowing rate in farm soil with no added As. With added As, M. californica displayed an ‘escape response’ of increased burrowing activity while E. fetida displayed an ‘exposure avoidance’ response of decreased burrowing activity and increased burrow reuse. Data are consistent with an early, concentration dependent sensory response, followed by addition of a physiological response component as As bioaccumulates in earthworm tissues with time. Any bioassay using earthworms to detect As contamination would need to be designed with awareness of the complex behavioral response.

Steroid estrogens (SEs) are mainly derived from livestock manure, and composting is the common method of bioresource utilization. In this study, an open-air composting experiment with dairy manure was conducted to observe the degradation and loss of five SEs under the influence of different precipitation intensities and additional mixed fermentation strains. SE determination results for dairy manure after 30 days of composting indicated that the average removal rates of 17α-estradiol (E2α), estrone (E1), 17β-estradiol (E2β), 17α-ethinyl estradiol (EE2), estriol (E3), and estradiol equivalent (EEQ) were 76.67%, 71.07%, 73.88%, 92.02%, 98.77%, and 88.11%, respectively, partly due to SE runoff. The rates of SEs leaching from the open-air composting dairy manure ranged from 0.05% to 4.75% after 10 rounds of 5–60 mm/d simulated rainfall. The total leaching amount of SEs was positively correlated with rainfall, but the leaching concentrations of SEs were just the opposite. As a result of its role as a degradation intermediate of other SEs, E3 was the most prone to run off. By strengthening the action of microorganisms, the total leaching amount of EEQ increased by 5%, E3 increased by five times, and E2β also underwent a transition from a conjugated form to free. However, there were also fewer final SEs remaining in the composted product, as well as the environmental risks of conjugated SEs. These conclusions can provide beneficial suggestions and references for controlling the environmental risks of SEs in the process of composting livestock and poultry manure.

Tylosin fermentation residues (TFR) pose an ecotoxicological risk through antibiotic resistant bacteria (ARBs) and their corresponding genes (ARGs). This study evaluated the ecotoxicity of TFR to soil biological activity, and further explored the mechanisms of vermicomposting to reduce the toxicological risk. The results showed that tylosin (TYL) was moderately degradable with a half-life (t1/2) of 37.5 d, inducing 28-44% inhibition rate of nitrogen transformation in soil, and the EC50 of earthworm avoidance was 880 mg/kg. The 30-d vermicomposting reduced the pH and OM content, while increased the EC and TN content, accelerated compost maturation (C/N ratio up to 20), and enriched the microbial community. ARGs were reduced by earthworm through removal of TYL (>70% degradation, t1/2 of

Three new earthworm species are described from South China, namely Amynthas rusticanus sp. nov., Amynthas scaberulus sp. nov., and Amynthas yuanjiangensis sp. nov. They are widely distributed in South China, and be polymorphic in reproductive organs. Of three new species, A. rusticanus sp. nov. is a small species with three pairs of spermathecal pores in 5/6–7/8 or two pairs of spermathecal pores in 5/6–6/7; it has developed prostate glands in XVI–XX. A. scaberulus sp. nov. is a medium-sized species with two pairs of spermathecal pores in 7/8–8/9; the lower edge of each spermathecal pore forms a tiny papilla, and the prostate glands are either degenerated or developed. A. yuanjiangensis sp. nov. is a medium-sized species with four pairs of spermathecal pores in 5/6–8/9 and has degenerated or developed prostate glands. Barcode data for each species (partial sequence of mitochondrial cytochrome c oxidase subunit I) are provided as well.

Arsenate (As-V) is a ubiquitous contaminant in soil as a result of excessive use of veterinary drugs and pesticides, causing enormous environmental risks. Multiple biomarkers have been used to assess the ecotoxicity of arsenic, however, the mechanisms of toxicity remain unclear. This paper describes the exposure of the earthworm (Eisenia fetida) to natural soil with different As-V concentrations for 28 days, then biomarkers from oxidative stress and burrowing behavior were quantified to evaluate As-V stress. Dynamic changes in reactive oxygen species (ROS), lipid peroxidation (MDA), adenosine triphosphate (ATP) content and antioxidant enzymes activity (Gpx, SOD, CAT) implied two stages of intensified stress responses and physiological adaptability. The transcriptional expression and regulation of antioxidant enzymes showed different responses. The mRNA expression of sod1 was up-regulated, while that of cat showed no significant change. The related regulators, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), showed dose-dependent activation, suggesting antioxidant defense induced by Nrf2 signaling. The burrowing behavior after 14-day exposure indicated that As-V inhibited burrowing activity, especially the burrow length and maximum burrow depth. These multiple biomarkers were integrated using a biomarker response index (BRI) model, which showed significant dose-effect relationship especially on day 28, and suggested that ATP was a sensitive and representative biomarker. This study provided evidence that burrowing activity, Nrf2 and HO-1 were useful biomarkers warranting inclusion into the BRI model. Arsenic toxicity was comprehensively understood through redox homeostasis regulation, biochemical and behavioral changes, and these results suggested new strategies for soil pollutants diagnosis.

Metaphire remanens sp. nov. is widely distributed throughout Hunan Province, China. We sequenced the mitochondrial DNA to investigate its population genetic structure and genetic diversity, including cytochrome c oxidase subunit I, cytochrome c oxidase subunit II, 12S ribosomal (r)RNA, 16S rRNA, and nicotinamide adenine dinucleotide dehydrogenase subunit 1, derived from 39 individuals from seven geographic locations in Hunan Province. The genetic diversity indices showed that populations of M. remanens have a strong genetic structure and obvious dispersal histories. M. remanens did not experience population expansion, except in Xiangtan City. This may be because of its evolution toward parthenogenesis. The divergence time estimates indicated that M. remanens originated at 19.2055 Ma and then generated two main lineages at 1.7334 Ma (Quaternary glaciation). These results indicate that glaciation, geographic isolation, and dispersal ability are significant factors that influence the differentiation and dispersal of M. remanens. In this study, we describe Metaphire remanens sp. nov. in morphology.

Background Electron beam (eBeam) irradiation and hermetic storage are safe and effective technologies to protect stored products. Although hypoxic environment improves performance of some irradiated insects, whether hypoxia affects irradiation of storage insects and impacts pest control efficacy remains to be investigated. Results Using cowpea bruchid (Callosobruchus maculatus) larvae, we showed that, relative to eBeam irradiation under normoxia, the adult emergence rate increased substantially if they were exposed to hypoxia prior to and during eBeam treatment. Conversely, exposure to hypoxia only after eBeam irradiation did not have this protective effect. eBeam irradiation caused an increase in reactive oxygen species (ROS) in normoxic larvae but not in hypoxic larvae. The activity of citrate synthase, a pace-making enzyme in the citric acid cycle, was suppressed under hypoxia but resumed normal function within hours of reoxygenation, suggesting that reduced mitochondrial activity, and thus less ROS production under hypoxia increased insect tolerance to irradiation. Furthermore, reoxygenation accelerated eBeam-induced glutathione-S-transferase activation and potentiated eBeam-enhanced catalase activities. Faster and stronger detoxification capacity in eBeam-irradiated, hypoxic larvae may have protected them from oxidative damage. Conclusion Hypoxic environment enhanced radiotolerance of bruchid larvae, presumably due to limited ROS production and elevated antioxidant enzymatic activities after reoxygenation. © 2018 Society of Chemical Industry.

Cytochrome P450 (CYP450) enzymes are a family of hemoproteins primarily responsible for detoxification functions. Earthworms have been used as a bioindicator of soil pollution in numerous studies, but no CYP450 gene has so far been cloned. RT-PCR and RACE-PCR were employed to construct and sequence the CYP450 gene DNA from the extracted mRNA in the earthworm Eisenia fetida. The cloned gene (EW1) has an open reading frame of 477 bp. The 3′-terminal region contained both the consensus and the signature sequences characteristic of CYP450. It was closely related to the CYP450 gene from the flatworm genus Opisthorchis felineus with 87% homology. The predicted structure of the putative protein was 97% homologous to human CYP450 family 27. This gene has been deposited in GenBank (accession no. KM881474 ). Earthworms (E. fetida) were then exposed to 1, 10, 100, and 500 mg kg−1 enrofloxacin in soils to explore the mRNA expression by real time qPCR. The effect of enrofloxacin on mRNA expression levels of EW1 exhibited a marked hormesis pattern across the enrofloxacin dose range tested. This is believed to be the first reported CYP450 gene in earthworms, with reference value for molecular studies on detoxification processes in earthworms.

Arsenic (As) is widely distributed in soil and is toxic to plants, animals and humans. In this study, earthworms (Eisenia fetida) were exposed to five concentrations of sodium arsenite (5, 10, 20, 40, and 80 mg As kg−1) in farm soils for 28 d. With increasing soil As(III) concentrations, As bioaccumulation in earthworms increased (maximum bioaccumulation factor 3.77), and levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were elevated. The expression of nuclear factor erythroid 2- related factor 2 (Nrf2) and heme oxygenase -1 (HO-1) were upregulated by As in a dose-dependent pattern, and reached 5.93 and 2.94 times the control values for Nrf2 and HO-1 respectively, at 28 d in the 80 mg As kg−1 soil treatment. Similarly, DNA damage, as measured in earthworm sperm using the comet assay, increased with increasing As(III) concentrations, with ‘Olive tail moment’ values in the comet assay ranging from c. 0.5 in Control to c. 3.5 at 80 mg As kg−1 soil. In contrast, activity of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), decreased. These results indicate that As(III) caused oxidative stress that resulted in damage to lipids and DNA. Nrf2 and HO-1 protein expression was demonstrated in earthworms for the first time to our knowledge, and found to be a sensitive biomarker of arsenic contamination. The presence of earthworms was also found to change the distribution of As in soil, in particular, reducing the proportion in the residual fraction and increasing the proportion in As bound to Fe-oxides. This may result in increased bioavailability of bound arsenic. Soil organic matter, NH4+-N, NO3−-N and available K were indirectly changed by the As(III) through its toxicity to earthworms. This study helps to inform future assessments and biomonitoring of soil arsenic contamination.

Four new species of earthworm species collected from various parts of Hainan Island were found. The species belong to Amynthas and Metaphire and are characterized by having two pairs of spermathecae in 7/8–8/9. They are described here as Amynthas flexuosus sp. nov., A. lucidus sp. nov., A. haikouensis sp. nov. in the aeruhinosusgroup of species of the genus Amynthas, and Metaphire fortuita sp. nov. in the insulana-group of species of the genus Metaphire. Their anatomical and morphological characteristics are compared to similar species from Hainan Island, China mainland and South-East Asia.

The earthworm Amynthas morrisi (Beddard, 1892) is a cosmopolitan species. It is mainly distributed in the mainland of South China, and its genetic structures had never been concerned. The earthworm specimens were collected systematically in this study, hence 88 A. morrisi new records from 30 locations were discovered. The haplotype network analysis and phylogenetic analysis shows that there are 11 haplotypes and four lineages in A. morrisi.The earthworm Amynthas morrisi (Beddard, 1892) is a cosmopolitan species. It is mainly distributed in the mainland of South China, and its genetic structures had never been concerned. The earthworm specimens were collected systematically in this study, hence 88 A. morrisi new records from 30 locations were discovered. The haplotype network analysis and phylogenetic analysis shows that there are 11 haplotypes and four lineages in A. morrisi.

The earthworm Amynthas corticis (Kinberg, 1867) is a cosmopolitan earthworm species and is located in several areas in China, but its genetic diversity is still unknown. The genetic diversity, phylogenetic structure, and divergence time were analyzed in this study. The result shows that there are 20 haplotypes and four lineages in A. corticis, the divergence time of A. corticis and A. morrisi is on Miocene (7.76Ma), and the divergence event under the species level happened after Pliocene (2.65Ma).

Lincomycin, monensin, and roxarsone are commonly used veterinary drugs. This study investigated their behaviours in different soils and their toxic effects on environmental organisms. Sorption and mobility analyses were performed to detect the migration capacity of drugs in soils. Toxic effects were evaluated by inhibition or acute toxicity tests on six organism species: algae, plants, daphnia, fish, earthworms and quails. The log Kd values (Freundlich model) of drugs were: lincomycin in laterite soil was 1.82, monensin in laterite soil was 2.76, and roxarsone in black soil was 1.29. The Rf value of lincomycin, roxarsone, monensin were 0.4995, 0.4493 and 0.8348 in laterite soil, and 0.5258, 0.5835 and 0.8033 in black soil, respectively. The EC50 for Scenedesmus obliquus, Arabidopsis thaliana, Daphnia magna and LC50/LD50 for Eisenia fetida, Danio rerio, and Coturnix coturnix were: 13.15 mg/L,32.18 mg/kg dry soil,292.6 mg/L,452.7 mg/L,5.74 g/kg dry soil and 103.9 mg/kg (roxarsone), 1.085 mg/L, 25 mg/kg dry soil, 21.1 mg/L, 4.76 mg/L, 0.346 g/kg dry soil and 672.8 mg/kg (monensin), 0.813 mg/L, 35.40 mg/kg dry soil, &gt, 400 mg/L, &gt, 2800 mg/L, &gt, 15 g/kg dry soil, &gt, 2000 mg/kg (lincomycin). These results showed that the environmental effects of veterinary drug residues should not be neglected, due to their mobility in environmental media and potential toxic effects on environmental organisms.

Population growth and social changes have recently contributed to an exaggerated increase in kitchen wastes in China. Vermicomposting has recently been recognized as an effective and eco-friendly method of organic waste treatment through the combination of earthworms and microbes. However, the influence of salt in kitchen wastes on vermicomposting have been unknown. The goal of this study was to analyze the influence of different salinities on earthworms (Eisenia fetida) and the products during the vermicomposting of kitchen wastes. In our research, kitchen wastes were divided into four different salinities: 0% (A), 0.1% (B), 0.2% (C) and 0.3% (D). The chemical characters of substrates and earthworm growth were measured on the 14th day and the 28th day of composting. Our results show that the high salinity (measured &gt, 0.2%) prevented earthworms from properly growing and had negative effects on quality of products in composting. T2 (measured salinity = 0.2%) had the highest average body weight, nitrate nitrogen, and available phosphorus. Thus, the salinity of kitchen wastes should be pretreated to less than 0.2% before vermicomposting.

Vermicomposting is the process of composting using worms and is applied in waste management to produce high-quality organic fertilizer. Garden waste (GW) is often mixed with other raw materials for vermicomposting. In the present study, the feasibility of vermicomposting using only GW was investigated in comparison with cow dung (CD). The total nitrogen (TN), total phosphorus (TP), and total potassium (TK) contents and the electrical conductivity increased, while total organic carbon (TOC) and the C/N ratio decreased in both substrates after vermicomposting. The nutrient content (TN, TP, and TK) of the GW vermicompost was promoted less than that in CD. Scanning electron microscopy images and specific surface area analysis showed that the vermicompost was strongly disaggregated and became more compacted and fragmented compared with the raw substrates. No mortality of earthworms was observed in GW; however, the earthworms had a higher mean body weight and reproduction rate in CD than that in GW. There were higher bacterial community richness and diversity in the vermicompost than that in the raw materials, and the dominant phylum species were Proteobacteria, Actinobacteria, and Bacteroidetes. Redundancy analysis demonstrated that TN, C/N ratio, and TOC play an important role in bacterial community dynamics. These data indicate that vermicomposting is a robust process that is suitable for the management of GW.

Green roof and bioretention have been frequently used as stormwater management practices to address urban hydrology issues, and researches regarding these technologies are increasing lately. However, a knowledge gap remains in characterizing the hydraulic properties of these practices. The hydrological process of green roof and bioretention is mainly determined by the substrate media of the practices. Based on current studies, this paper discussed factors affecting the hydrological response of substrate material for green roofs and bioretention. Most researches provide qualitative information on water movement within substrate, but few have further investigated the theoretical explanations on the hydrological process of substrate and give quantitative data. This study proposed that following factors affecting water flow dynamics of substrate media: type of materials and composition of the materials; depth of the substrate media; pore structure/network of the substrate materials. Many investigations focus on the effect of a single factor and generalizing the other, but the hydrological process of the substrate material is mostly under the effect of multiple factors simultaneously. And under different study conditions and different designs of green roof or bioretention, the importance of these factors to the hydrological performance of substrate media is different. In addition, interrelationships exist among the factors, changes in one factor could influence other factors. Current research on the factors influencing the hydrological process of the substrate material needs to be related. Future study needs to consider the effect of multiple factors and their interrelations, to create integrated approaches for a better understanding of the hydrological process of the substrate material, and for modeling the hydrological performance of the green roof and bioretention more accurately and designing the practices more efficient.

Alkaline hydrogen peroxide (AHP) pretreatment was applied to improve methane production from sweet sorghum bagasse under mesophilic conditions. After the pretreatment (solid-to-liquid ratio of 1 : 10, 2% NaOH at 121 °C for 60 min and then immersing with 5% (w/v) H2O2 for 24 h at room temperature), pretreated bagasse, the pretreatment effluent and the pretreatment mixture, together with the untreated bagasse, were used as substrates for methane production under the same volatile solid (VS) loading and digestion conditions. The AHP pretreatment resulted in lignin removal and different degrees of methane production. The methane production of bagasse was improved from 262.1 ± 7.3 mL CH4 per g VS to 330.4 ± 1.3 mL CH4 per g VS. The pretreatment mixture and pretreatment effluent could be successfully digested and both of the methane productions were higher than the untreated bagasse, although there were degrees of inhibition. In view of the bagasse recovery during the pretreatment, total methane production from separate digestion of pretreated bagasse and pretreatment effluent was 18.6% more than untreated bagasse based on the same mass of dry sweet sorghum bagasse. The kinetic analysis implied that the Cone model showed a better fit to the experimental data of untreated bagasse, pretreated bagasse and pretreatment effluent, and the Fitzhugh model was better to predict the methane production of the pretreatment mixture. AHP pretreatment could increase the methane production rate of sweet sorghum bagasse. These results will provide a reference to the methane production from AHP pretreated sweet sorghum bagasse in industry.

BACKGROUND Insect tolerance to low oxygen (hypoxia) and high carbon dioxide (hypercapnia) is critical for insect control. On the basis of bioassay, metabolism profiles were built to investigate adaptive mechanisms in bean weevil under hypoxia (2% O2), hypoxia/hypercapnia (2% O2 + 18% CO2) and normoxia (control, 20% O2 + 80% N2) using gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). RESULTS The growth and development of bean weevils were significantly suppressed by the two hypoxia conditions; hypercapnia enhanced the mortality, but after 24 days of exposure, the surviving insects emerged as adults earlier than those under hypoxia only. Metabolism profiles also showed striking differences in metabolites among the treatment and control groups, both quantitatively and qualitatively. Pairwise comparisons of the three groups showed that 61 metabolites changed significantly, 40 in the hypoxia group and 37 in the hypoxia/hypercapnia group relative to the control group, while only 16 were shared equally by the hypoxia and hypoxia/hypercapnia groups. Increased metabolites were mainly carbohydrates, amino acids and organic acids, while free fatty acids were decreased. Furthermore, the changes were strengthened by the addition of hypercapnia, but excluding free fatty acids. CONCLUSION The findings show that bean weevil has high tolerance to hypoxia or even hypoxia/hypercapnia at biologically achievable levels and provide more direct evidence for stored product insect mechanism regulation under hypoxia stress, especially free fatty acid regulation by hypercapnia but not by hypoxia. © 2016 Society of Chemical Industry

Four new species were collected from Mt. Emei, Sichuan Province, China. They are Amynthas leshanensis sp. nov., Amynthas gilvus sp. nov., Amynthas retortus sp. nov. and Amynthas orbicularis sp. nov. The first three species could be assigned to the Amynthas sieboldi group and the last species could be assigned to the Amynthas aeruginosus group according to the definition of Sims and Easton. A. leshanensis sp. nov. has the male pore porophore surrounded by a large irregular-shaped pad which extends from 17/18–½ XIX, and the intestinal caeca are complex. A. gilvus sp. nov. has each male pore on a slightly raised elliptical glandular flat-top pad, with a pointed extension lateral to each porophore. Amynthas retortus sp. nov. has a pair of small, round post-setal genital papillae medial to male porophores on XVII, spermathecal seminal chambers are twisted into zigzags and accessory glands are present on spermathecae and prostate gland areas. A. orbicularis sp. nov. has the spermathecal pores near dorso...

Individual and combined effects of enrofloxacin (EF) and cadmium (Cd) on the earthworm Eisenia fetida at environmentally relevant concentrations were investigated. EF is a veterinary antibiotic; Cd is an impurity in phosphatic fertiliser. For both, residues may accumulate in farm soils. In laboratory tests, over 98% of spiked EF was adsorbed by farm soils, with a half-life >8 weeks. However, earthworms absorbed less than 20% of spiked EF. Earthworms in soil with EF concentration 10 mg kg(-1) soil experienced transient oxidative stress and exhibited reduced burrowing activity and respiration after an 8-week exposure; EF at 0.1 and 1.0 mg kg(-1) soil did not elicit toxicity symptoms. When both were added, Cd did not affect EF uptake, but each increment of spiked EF increased Cd bioaccumulation and associated oxidative stress of earthworms, and also caused decreased burrow length and CO2 production. However, metallothionein induction was not affected. The enhanced toxicity of Cd to earthworms in the presence of EF at low environmental concentrations may have implications for the health and reproductive success of earthworm populations and highlights the importance of understanding effects of antibiotic contamination of farm soils, and of awareness of environmental effects from interaction between multiple contaminants.

Despite the ecological function of earthworms being of undeniable importance, little is known about their population genetic diversity, dispersal, and gene flow patterns. Here, we applied 769,957 single-nucleotide polymorphisms (SNPs) gathered by restriction site-associated DNA sequencing (RAD-seq) to unearth the genetic variability of populations of the earthworm Amynthas_YN2017 sp. collected from Western Yunnan, China. The neighbor-joining (NJ) tree method revealed that 30 individuals strongly clustered into three lineages (G2, G3, and G5 lineages) that coincided with the geographic distribution proximity of the sampling locations. There was a low level of genetic differentiation among these lineages with an average genetic differentiation (FST) value ranging from 0.080 to 0.135. Clustering analysis demonstrated a clear population genetic structure with a low level of admixture. There was only one migration edge in the population from the G3 lineage to the G2 lineage. Therefore, we presume that a reduction in gene flow caused by geographic isolation influences the population genetic differentiation of Amynthas_YN2017 sp. Additionally, the presence of river (Nujiang River) as geographical barrier is likely to have limited the gene flow. Overall, the large number of SNPs gathered by RAD-seq reveals a significant population genetic structure over a restricted geographic scale, highlighting that the RAD-seq approach could be used to explore the population phylogenetic relationships and population genetic diversity of earthworms.

Three new species of earthworms belonging to the genus Amynthas are described from Hainan Island, China. They are named Amynthas wenchangensis sp. nov., Amynthas accessorius sp. nov., and Amynthas eumorphus sp. nov. A. wenchangensis sp. nov. keys to the Amynthas morrisi-group; it has several small genital papillae in the male pore region and a degenerated prostate. A. accessorius sp. nov. keys to the Amynthas hawayanus-group and has completely degenerated prostates and two large accessory glands. A. eumorphus sp. nov. keys to the Amynthas sieboldi-group.

Four new species belonging to genera Amyntha and Metaphire were discovered from an extensive area in southern China, covering the provinces Sichuan, Yunnan, Guizhou, Guangdong, Guangxi, Fujian, Hunan, Jiangxi and Anhui. The species are named Amynthas dispersus Sun & Qiu, sp. nov., Amynthas shanghangensis Sun & Qiu, sp. nov., Amynthas dentiformis Sun & Jiang, sp. nov. and Metaphire sanmingensis Sun & Jiang, sp. nov. The first two new species have four pairs of intersegmental spermathecal pores in 5/6–8/9. A. dispersus has elliptical and glandular male pore porophores raised on a low pad-like area surrounded by two to three shallow skin folds, the genital papillae are variable in the spermathecal pore and male pore regions, and prostate glands are vestigial or rudimentary. Amynthas shanghangensis has male porophores surrounded by three papillae, each prostate gland accompanied by an accessory gland, and the distal ½–⅓ of the spermathecal diverticulum dilated into a rod-shaped seminal chamber. Amynthas dentiformis has two pairs of spermathecal pores in 7/8–8/9, male pores always surrounded by two papillae, and small sacs on the dorsal and ventral margins of the intestinal caecae; the prostate gland occasionally has stalked accessory glands. M. sanmingensis is in the Metaphire houlleti-group, and has secondary male pores opening to copulatory pouches, rod-shaped spermathecal seminal chamber, as well as variable genital papillae in spermathecal and male pores regions. The GenBank accession numbers of DNA barcode data are attached under the description of each species.

Two new species of earthworms belonging to the genus Amynthas are described from Guangxi Province, China: Amynthas crassitubus sp. nov. and Amynthas stabilis sp. nov. from Shiwan Mountain Nature Reserve. The two species have three pairs of spermathecal pores in 6/7–8/9, and belong to the sieboldi-group. COI barcode sequences support their status as new species.

Electron beam (eBeam) irradiation and hermetic storage are safe and effective technologies to protect stored products. Although hypoxic environment improves performance of some irradiated insects, whether hypoxia affects irradiation of storage insects and impacts pest control efficacy remains to be investigated.Using cowpea bruchid (Callosobruchus maculatus) larvae, we showed that, relative to eBeam irradiation under normoxia, the adult emergence rate increased substantially if they were exposed to hypoxia prior to and during eBeam treatment. Conversely, exposure to hypoxia only after eBeam irradiation did not have this protective effect. eBeam irradiation caused an increase in reactive oxygen species (ROS) in normoxic larvae but not in hypoxic larvae. The activity of citrate synthase, a pace-making enzyme in the citric acid cycle, was suppressed under hypoxia but resumed normal function within hours of reoxygenation, suggesting that reduced mitochondrial activity, and thus less ROS production under hypoxia increased insect tolerance to irradiation. Furthermore, reoxygenation accelerated eBeam-induced glutathione-S-transferase activation and potentiated eBeam-enhanced catalase activities. Faster and stronger detoxification capacity in eBeam-irradiated, hypoxic larvae may have protected them from oxidative damage.Hypoxic environment enhanced radiotolerance of bruchid larvae, presumably due to limited ROS production and elevated antioxidant enzymatic activities after reoxygenation. © 2018 Society of Chemical Industry.

Effects of severe and mild alkaline hydrogen peroxide (AHP) pretreatment on ethanol production from sweet sorghum bagasse via pre-simultaneous saccharification fermentation, and the chemical structure changes of the substrates were investigated. The results showed that the bagasse pretreated by severe AHP could produce more ethanol than that of mild AHP. The maximum ethanol concentration of the bagasses from mild and severe AHP pretreatment with 8% bagasse loading was 7.642±0.140 g/L and 19.330±0.085 g/L, respectively. Moreover, the FTIR and NMR analysis illustrated that the molecule and surface structures of the pretreated bagasse were significantly changed compared with the control. The potential biomass energy production of the effluent from the pretreatment was also briefly discussed for future utilization of waste solution.The heat energy potentials of waste solution with severe and mild AHP pretreatment were 367.2 kJ/L effluent and 327.6 kJ/L effluent , respectively.

Among oligochaetes, the Pheretima complex within the Megascolecidae is a major earthworm group. Recently, however, the systematics of the Pheretima complex based on morphology are challenged by molecular studies. Since little comparative analysis of earthworm complete mitochondrial genomes has been reported yet, we sequenced mitogenomes of four pheretimoid earthworm species to explore their phylogenetic relationships. The general earthworm genomic features are also found in four earthworms: all genes transcribed from the same strand, the same initiation codon ATG for each PCGs, and conserved structures of RNA genes. Interestingly we find an extra potential tRNA-leucine (CUN) in Amynthas longisiphonus. The earthworm mitochondrial ATP8 exhibits the highest evolutionary rate, while the gene CO1 evolves slowest. Phylogenetic analysis based on protein-coding genes (PCGs) strongly supports the monophyly of the Clitellata, Hirudinea, Oligochaeta, Megascolecidae and Pheretima complex. Our analysis, however, reveals non-monophyly within the genara Amynthas and Metaphire. Thus the generic divisions based on morphology in the Pheretima complex should be reconsidered.

Enrofloxacin (EF) is widely used in veterinary medicine while the bulk accumulation of EF residues and their metabolites have been raised more and more public concerns nowadays. The aim of this study was to explore the acute and sub-acute toxicity of EF on the earthworms (Eisenia fetida) in an artificial soil substrate. The 50% lethal concentration (LC50) of EF to earthworms at day 14 after exposure to different concentration of EF (0, 2.5, 5, 10, 15 and 25 g EF per kg soil dry weight) was calculated to examine the acute toxicity of EF. Sub-acute toxicity test was conducted through detecting the effects of EF on growth, reproduction, avoidance response and respiration on earthworms. The LC50 of EF at day 14 to earthworms was 11.01 g kg−1, which was far higher than the environmentally relevant concentration of EF residues. The inhibition of EF on the growth and reproduction of earthworms was shown in a dose-dependent manner and the high concentration of EF (above 1 g kg−1 on growth rate; above 0.05 g kg−1 on juvenile amounts) obviously disturbed the growth and reproduction of earthworms. Earthworms exposed to ≥0.25 g kg−1 of EF displayed avoidance response while the soil only spiked with ≥2 g kg−1 of EF showed decreased habitat function. The respiration of earthworms was inhibited when exposed to 2.0 g kg−1 of EF for more than 14 days or 1.0 g kg−1 of EF for more than 28 days. It is concluded that EF could cause acute and sub-acute toxicity to earthworms (E. fetida) at relatively high concentration. The long-term exposure of EF to earthworms and the test conducting on natural soil substrates specific to that region should be further studied.

The environmental and human health risks of veterinary drugs are becoming public health issues. Enrofloxacin (EF) is an extensively used animal-specific antibacterial agent that leaves drug residues in the environment. This study investigated the proteomic response of the earthworm Eisenia fetida to EF exposure. Earthworms were exposed to EF in soil at 1-500mg·kg-1, and samples were collected at intervals during a 28 day period. The extracted proteins were separated by two dimensional electrophoresis to detect differentially expressed proteins (DEPs) in EF-exposed earthworms. In total, 35 unique DEPs were found. These proteins were subjected to MALDI-TOF/TOF-MS analysis and identified through comparison of their mass spectra with those in protein databases. The DEPs were grouped on the basis of their function, into metabolism, stress-related, transport, transcription, and predicted/hypothetical protein categories. Knowledge of proteins that are induced or repressed by EF in earthworms could provide insight into mechanisms of sub-clinical physiological effects of xenobiotic residues in the environment, and may also help understand synergy between pollutants. As several DEPs in E. fetida showed similarity to human protein sequences, E. fetida has potential as an indicator species to assess the environmental and biological risks of drug residues.

Enrofloxacin (ENR) and roxarsone (ROX) have been widely used in animal breeding. In this study, the daily clinical dosage of ENR and daily additive amount of ROX were administrated to Bama pigs. After 5days, the activity and protein expression of three important enzymes in the cytochrome P450 family were measured in the porcine liver. CYP1A2 was induced by both ENR and ROX independently. CYP2E1 and CYP3A4 were inhibited by ENR, but not affected by ROX. The combined administration of ENR and ROX were antagonistic to CYP1A2 and CYP2E1, but not to CYP3A4. Drug-drug interactions should be considered during the administration of ENR, ROX and for their co-administration with other drugs to minimize adverse reactions.

This paper describes four new species of morrisi-group earthworms from Hainan and Guangdong Provinces, China: Amynthas instabilis sp. nov, Amynthas dilatatus sp. nov, Amynthas infuscuatus sp. nov. and Amynthas qiongzhongensis sp. nov. All four species have two pairs of spermathecal pores in 5/6–6/7; male pores in XVIII, 0.33 circumference ventrally apart, each on the top of a slightly raised porophore, surrounded by several tiny genital papillae, in a pulvinate pad with three to six circular folds. Characters of the spermathecae, prostate glands and other anatomical features easily distinguish the new species from earthworms previously reported from the morrisi-group.http://zoobank.org/urn:lsid:zoobank.org:pub:545E9152-C826-44A2-949E-402434D8493D.

The Hengduan Mountains (HMs), China, is a biodiversity hotspot that uplifted mainly between Late Miocene and Late Pliocene, the uplift movement might have a significant impact on the evolution of earthworms. Here, we investigated the species richness and the distribution of the earthworms in the HMs. A total of 170 species/subspecies belonging to four families and 12 genera were preliminary identified, species number in Megascolecidae account for more than 88%. Molecular phylogeny and divergence times analysis based on concatenated genes suggest that the divergence time of Moniligastridae (Outgroup) and Lumbricidae (Group 1) in the HMs was 215.84 million years ago (MYA). The divergence of first two clades in Megascolecidae (M405 and Group 2) can be date to around 44.42 MYA (95% HDP: 38.14–52.62 MYA). Dispersal of Megascolecidae from outside of the HMs to this region coincides with Indian-Eurasian collision between Eocene and Oligocene. The reconstruction of ancestral-area reveals the clades in Megascolecidae (Group 2–8) may have experienced different evolutionary histories following their divergence time during Cenozoic, which suggests that paleogeographical events might promote the dispersal and diversification of earthworm in the HMs. Unearthing the biogeographical pattern of earthworms provide insight into the evolution of soil invertebrate fauna in the temperate northern hemisphere and can be used for tracing the complex paleogeographical history of the HMs.

Three new species and one new subspecies of earthworms from Hainan Island, China, are described: Amynthas dinganensis sp. nov., Amynthas tenuis sp. nov., Metaphire wuzhimontis sp. nov. and Metaphire magna minuscula subsp. nov. Amynthas dinganensis and Amynthas tenuis have two pairs of spermathecal pores in 5/6–6/7, and Metaphire wuzhimontis and Metaphire magna minuscula have two pairs of spermathecal pores in 7/8–8/9. COI and 16S gene fragments of the new species taxa have been sequenced. We provide comparisons between these new species taxa and related species using morphological and molecular characters.

Cytochrome P450 (CYP450) is a hemoprotein superfamily, among which CYP1, CYP2 and CYP3 play a major role in the metabolism of vast array of xenobiotics and endobiotics. This paper reports on three CYP enzyme variants (CYP1A2, CYP2E1 and CYP3A4) in three species of earthworm (Eisenia fetida, Metaphire guillelmi and Amynthas carnosus). The relative expression levels and localization of the three associated proteins were investigated at three life-cycle points (juvenile, sub-adult and adult), through comparison of anterior and posterior body tissue and between specific organs (body wall, intestine and reproductive tissues) using western blot analysis. This study confirmed the presence of CYP3A4, CYP1A2 and CYP2E1 in all three species of earthworm tested. The levels of expression varied with earthworm species, age, and body location. These differences in occurrence of the three CYP enzymes appeared to reflect the ecological niche (the spatial and temporal location and functional relationship of each individual or population in populations or communities), and the likelihood of contact with soil contaminants of the respective species. These results may help to explain why earthworms are capable of adapting to very different and extensively polluted soil environments and provide important data for subsequent ecotoxicology and ecological adaptability studies.

Insect tolerance to low oxygen (hypoxia) and high carbon dioxide (hypercapnia) is critical for insect control. On the basis of bioassay, metabolism profiles were built to investigate adaptive mechanisms in bean weevil under hypoxia (2% OThe growth and development of bean weevils were significantly suppressed by the two hypoxia conditions; hypercapnia enhanced the mortality, but after 24 days of exposure, the surviving insects emerged as adults earlier than those under hypoxia only. Metabolism profiles also showed striking differences in metabolites among the treatment and control groups, both quantitatively and qualitatively. Pairwise comparisons of the three groups showed that 61 metabolites changed significantly, 40 in the hypoxia group and 37 in the hypoxia/hypercapnia group relative to the control group, while only 16 were shared equally by the hypoxia and hypoxia/hypercapnia groups. Increased metabolites were mainly carbohydrates, amino acids and organic acids, while free fatty acids were decreased. Furthermore, the changes were strengthened by the addition of hypercapnia, but excluding free fatty acids.The findings show that bean weevil has high tolerance to hypoxia or even hypoxia/hypercapnia at biologically achievable levels and provide more direct evidence for stored product insect mechanism regulation under hypoxia stress, especially free fatty acid regulation by hypercapnia but not by hypoxia. © 2016 Society of Chemical Industry.

Regulating the air in low-oxygen environments protects hermetically stored grains from storage pests damage. However, pests that can tolerate hypoxic stress pose a huge challenge in terms of grain storage. We used various biological approaches to determine the fundamental mechanisms of Tribolium castaneum to cope with hypoxia. Our results indicated that limiting the available oxygen to T. castaneum increased glycolysis and inhibited the Krebs cycle, and that accumulated pyruvic acid was preferentially converted to lactic acid via anaerobic metabolism. Mitochondrial aerobic respiration was markedly suppressed for beetles under hypoxia, which also might have led to mitochondrial autophagy. The enzymatic activity of citrate synthase decreased in insects under hypoxia but recovered within 12 h, which suggested that the beetles recovered from the hypoxia. Moreover, hypoxia-reperfusion resulted in severe oxidative damage to insects, and antioxidant levels increased to defend against the high level of reactive oxygen species. In conclusion, our findings show that mitochondria were the main target in T. castaneum in response to low oxygen. The beetles under hypoxia inhibited mitochondrial respiration and increased antioxidant activity after reoxygenation. Our research advances the field of pest control and makes it possible to develop more efficient strategies for hermetic storage.

The performance of a pond–constructed wetland system in the treatment of municipal wastewater in Kiaochow city was studied; and comparison with oxidation ponds system was conducted. In the post-constructed wetland, the removal of COD, TN and TP is 24%, 58.5% and 24.8% respectively. The treated effluent from the constructed wetland can meet the Chinese National Agricultural and Irrigation Standard. The comparison between pond–constructed wetland system and oxidation pond system shows that total nitrogen removal in a constructed wetland is better than that in an oxidation pond and the TP removal is inferior. A possible reason is the low dissolved oxygen concentration in the wetland. Constructed wetlands can restrain the growth of algae effectively, and can produce obvious ecological and economical benefits.

We have determined the mitochondrial genome of the first Amynthas earthworm, Amynthas aspergillus (Perrier, 1872), which is a natural medical resource in Chinese traditional medicine. Its mitogenome is 15,115 bp in length containing 37 genes with the same contents and order as other sequenced earthworms. All genes are encoded by the same strand, all 13 PCGs use ATG as start codon. The content of A + T is 63.04% for A. aspergillus (33.41% A, 29.63% T, 14.56% G and 22.41% C). The complete mitochondrial genomes of A. aspergillus would be useful for the reconstruction of Oligochaeta polygenetic relationships.

We have determined the mitochondrial genome of the first Pheretimoid earthworm, Metaphire vulgaris (Chen, 1930). This mitogenome is 15,061 bp in length containing 37 genes typical of other annelid. All genes are encoded by the same strand, ATP8 is not adjacent to ATP6, all 13 PCGs use ATG as a start codon. These features are consistent with first determined earthworm Lumbricus terrestris, but unusual among animal mtDNAs.

Correction for ‘Methane production enhancement from products of alkaline hydrogen peroxide pretreated sweet sorghum bagasse’ by Weixing Cao et al., RSC Adv., 2017, 7, 5701–5707.

Roxarsone is a normal used feed additive in animal breeding. Its ecotoxicological effect has been noticed in recent years. However, very few studies have concerned its effect on the enzymatic activities in earthworms. In present study, we investigated the activities of catalase (CAT), acetylcholine esterase (T-CHE), as well as the content of MDA in earthworms exposed to roxarsone. 5 different concentration groups of roxarsone were designed to simulate the process that roxarsone was discharged into soil with animal manure. CAT, MDA and T-CHE in worms were respectively measured in different time after exposure. The results showed that CAT in worms was activated by roxarsone. This activation was dose-dependent to some extent. The content of MDA was almost unchanged during the treatment either in low or in high dose group. T-CHE could be inhibited by roxarsone. This inhibition was more obvious in the high dose level groups than the low ones. As a conclusion, CAT and T-CHE might be used as biomarker of roxarsone contamination in soils.

Oxytetracycline (OTC) is a normal used antibiotic in animal breeding and human clinic. There were large residues of this antibiotic discharged into the environment with the waste and manure. However, almost no study has concerned its effect on the enzymatic activities in earthworms. In present study, we investigated the activities of two important enzymes in cytochrome P450 2B subfamily: pentoxy resorufin -O dealkylase (PROD), benzoxyresorufin-O -dealkylase (BROD) in earthworm Eisenia andrei exposed to OTC. 4 different concentration groups of OTC were designed to simulate the process that OTC was discharged into soil with animal manure. BROD, PROD activities in worms were respectively measured in different exposure time. The results showed that the two enzymes in worms were inhibited by OTC in high contamination level, but not in low level. The inhibition showed time or dose dependent to some extent. The further study is necessary to indentify if BROD, PROD might be used as biomarker of OTC contamination.

Arsanilic acid is a normal used feed additive in animal breeding. Almost no studies have concerned its effect on the enzymatic activities in earthworms. In present study, we investigated the activities of three important enzymes in cytochrome P450 system: benzoxy- resorufin-O -dealkylase (BROD), Pentoxyresorufin -O- dealkylase (PROD), Methoxyresorufin-O -deethylase (MROD) in earthworm Eisenia andrei exposed to arsanilic acid. 5 different concentration groups of arsanilic acid were designed to simulate the process that arsanilic acid was discharged into soil with animal manure. BROD, PROD, MROD activities in worms were respectively measured in different time after exposure. The results showed that all the three enzymes in worms were inhibited by arsanilic acid. The inhibition extents were BROD>;PROD>; MROD. It also showed time or dose dependent is some treatments. As a conclusion, the three enzymes might be used as biomarker of arsanilic acid contamination in soils.