Effects of a Neonicotinoid on Indigenous Earthworm Perionyx excavatus Biochemical and Histopathological Alterations.

Acetamiprid is a broad-spectrum insecticide, belonging to the neonicotinoid compounds group, which has been extensively applied throughout the globe. Recently, indiscriminate use of these compounds was reported to cause fatal impacts on non-targeted soil organisms. Hence, the present study aimed to examine the impact of acetamiprid on Indian indigenous earthworm, Perionyx excavatus. Acute toxicity revealed an LC50 concentration of 0.25 µg/cm2 for filter paper test/72 h and 400 µg/kg for artificial soil test/14 days. Oxidative stress (ROS) and various biomarkers including superoxide dismutase, catalase, glutathione S-transferase, malondialdehyde content and DNA damage were measured. The results of the biomarker responses confirmed the acetamiprid exposure can cause toxicity to P. excavatus. In addition, cell density (20 × 102 cell mL/mg) and cell viability (40%) were significantly (p < 0.05) reduced. Further, the ecotoxicological assessment made through this study can be utilized as good evidence to toxicity of neonicotinoids to non-targeted indigenous organisms.

Environment-friendly management of textile mill wastewater sludge using epigeic earthworms: Bioaccumulation of heavy metals and metallothionein production.

In the present study, Eudrilus eugeniae and Perionyx excavatus were used for vermistabilization of textile mill sludge in different combinations with cowdung for 60 days. A higher percentage of metal removal was observed in earthworm treated mixtures for cadmium (54.5%) followed by copper (36.0%), chromium (37.0%) and zinc (35.9%). Vermistabilized textile mill sludge + cowdung (1:1) showed a maximum percentage increase in total NPK, a significant (P < 0.05) increase in bacteria, fungi, and actinomycetes with a better earthworm survival rate. A higher amount of metallothionein protein was produced by E. eugeniae than P. excavatus. Further, 100% textile mill sludge showed a number of histological abnormalities like degeneration of cells, cellular debris, and uneven cellular compartmentation while textile mill sludge with cowdung showed normal earthworm histology. Results suggest that textile mill sludge + cowdung (1:1) combination is suitable for vermistabilization of textile mill sludge.

Isolation, identification, and characterization of gut microflora of Perionyx excavatus collected from Midnapore, West Bengal.

Agriculture is an important part of the economy of the undivided Midnapore district. Agricultural land is its asset and most importantly its means of sustenance as well as survival. Earthworms are invertebrates that play a key role in recycling organic matters in soils. Since the intestines of earthworms harbor wide ranges of microorganisms, enzymes, hormones etc., these half digested materials decompose rapidly and are transformed into a stabilized material called vermicompost which is very useful for increasing the soil fertility. One has to look for these characters before recommending any species for vermiculture. In the present study, Perionyx excavatus specimens were collected from the undivided Midnapore district and from the Earthworms gut, bacteria, fungus, actinobacteria, and yeast were isolated and identified using various morphological and biochemical tests. All the bacterial isolates were identified using morphological study, staining techniques, and different biochemical tests such as catalase test, KOH test, H2 SO4 test, Starch hydrolysis test, oxidase test, and sucrose hydrolysis test. All the fungal, actinobacteria, and yeast isolates were subjected to staining and morphological characterization (color and texture of fungal colony). Bacterial isolates of genus Bacillus sp., Staphylococcus sp., Enterococci, Micrococcus sp., Enterobacter sp., and Citrobacter sp. were identified. Among the fungal isolates Aspergilus sp., and P. boydii were identified. Streptomyces sp., Nocardia sp. among the actinobacteria and Candida sp. among yeast were also found to be present in earthworm gut and these might play an important role along with the earthworm to increase the quality and fertility of soil.

Taxonomic and functional diversity of the culturable microbiomes of epigeic earthworms and their prospects in agriculture.

Eisenia foetida and Perionyx excavatus are potent vermicomposting earthworms having immense importance in organic matter recycling under tropical conditions, particularly in India. Comparative assessment of the cultivable gut microbiome of these two epigeic earthworms after growth on lignocellulosic biomass, revealed populations of 3.2-8.3 × 10(9) CFU. Diversity analyses using 16S rDNA sequences revealed that the major dominating classes were Firmicutes (50-60%), followed by Actinobacteria (26.7-33%), and Alphaproteobacteria (5.6-6.7%). Despite exhibiting similar diversity indices and species richness, Betaproteobacteria (6.7%) and Gammaproteobacteria (11.1%) were solely present in E. foetida and P. excavatus, respectively. A set of 33 distinct morphotypes, including 18 from E. foetida and 15 from P. excavatus were selected. Carbohydrate utilization profiles generated using Hi-Carbo™ kits revealed that the isolates from the gut of P. excavatus - Arthrobacter pascens IARI-L13 and Bacillus subtilis IARIC were able to utilize 54 and 51.4% of the carbohydrates tested. Sorbose was not utilized, while unusual carbohydrates - adonitol and methyl-d-mannoside were utilized only by members from the gut of P. excavatus, while melizitose was utilized by those uniquely by E. foetida microbiome. Functional characterization revealed that ß-glucosidase activity was most prevalent in the culturable microbial community. Alkaline and acid phosphatase activity was more widespread in the E. foetida gut microbiome. All the culturable gut bacterial isolates produced ammonia, but IAA was detected only in five cultures. The unique functional attributes of the two culturable microbiomes, grown on a similar diet, reveals the significance of proper selection of earthworm substrate combinations for effective vermicomposting.

Profiling microRNAs of earthworm, Perionyx excavatus and deciphering the expression of distinct novel miRNAs regulating epimorphosis regeneration.

Earthworm, P. excavatus, is an ideal model organism for studying regeneration. Due to its prodigious regeneration capability, the amputated head part of the earthworm can regenerate completely within 22 days. MicroRNAs (miRNAs) regulate specific genes and are involved in essential biological processes, including regeneration. In this study, we conducted a comprehensive analysis of miRNA profiling of the earthworm, P. excavatus, during the process of anterior regeneration. Our investigation involved in the identification of 55 miRNAs from 30 distinct miRNA families that exhibit significant relevance to wound healing and regeneration. Notably, we have identified 50 novel miRNAs and predicted their pre-miRNA secondary structures using MIREAP. Both Known and Novel miRNAs are validated using qPCR. In addition, we employed the miRanda algorithm to predict the interactions between these miRNAs and their target mRNA transcripts. Based on the miRanda target prediction results, we identified the target genes such as Wnt, Myc, MAPK, SoxB, IHH, Hox, and Notch. These findings indicate that the potential targets of these miRNAs might play crucial roles in various functions related to wound healing, tissue restoration, and regeneration. Furthermore, the acquisition of these findings provides a unique perspective on understanding the molecular mechanisms driving epimorphosis regeneration in connection with miRNAs for the development of miRNA-based therapeutics.

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture.

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

Comparative Study on Bacterial Population Dynamics of Foregut, Midgut, and Hindgut Content of Perionyx excavatus (Perrier) Isolated from Eco-friendly, Non-hazardous Vermicompost.

The ideal condition of earthworm gut promotes growth and multiplication of beneficial soil microorganisms eliminating pathogens and converts organic wastes into nutrients rich compost. The present study has been carried out to determine the population dynamics of earthworm gut bacteria and to find out relative abundance of different functional bacterial groups in the foregut, midgut, and hindgut of earthworm Perionyx excavatus. To assess bacterial diversity, a viable plate count method was adopted. In the different gut region of earthworm, aerobic heterotrophic, amylolytic, Bacillus, Gram-negative, proteolytic, fat hydrolyzing, nitrate-reducing, nitrifying, asymbiotic nitrogen-fixing, Azotobacter, and phosphate solubilizing bacterial populations ranged from 22.2 to 241.6 × 106, 8.0 to 171.60 × 106, 1.83 to 2.79 × 106, 10.68 to 23.04 × 104, 3.70 to 5.52 × 104, 59.60 to 208.40 × 104, 1.86 to 7.34 × 104, 10.94 to 19.78 × 104, 0.80 to 3.42 × 104, 7.83 to 13.70 × 104, 1.31 to 2.67 × 104 cfu/ml gut suspension, respectively. The results of the one-way ANOVA revealed that the bacterial load of most of the bacterial groups was significantly higher (p < 0.05) in the hindgut region, followed by midgut and foregut. Only the density of the proteolytic group was significantly higher (p < 0.05) in the midgut region followed by foregut and hindgut. Starch hydrolyzing bacteria constitute the largest group of bacteria in the gut content. From principal component analysis, two components were extracted with the eigenvalues of 8.485 and 1.132. Agglomerative hierarchical cluster analysis revealed that the bacterial populations were clustered into four different groups. Quantitative variation among bacterial groups in earthworm's gut seems to determine the soil health and composting efficiency; from this point of view, the present study will provide a better understanding about different functional bacterial groups of earthworm's guts and might be helpful in sustainable agriculture and waste management.

Characterization of Perionyx excavatus Development and Its Head Regeneration.

Regeneration is a biological process restoring lost or amputated body parts. The capability of regeneration varies among organisms and the regeneration of the central nervous system (CNS) is limited to specific animals, including the earthworm Perionyx excavatus. Thus, it is crucial to establish P. excavatus as a model system to investigate mechanisms of CNS regeneration. Here, we set up a culture system to sustain the life cycle of P. excavatus and characterize the development of P. excavatus, from embryo to juvenile, based on its morphology, myogenesis and neurogenesis. During development, embryos have EdU-positive proliferating cells throughout the whole body, whereas juveniles maintain proliferating cells exclusively in the head and tail regions, not in the trunk region. Interestingly, juveniles amputated at the trunk, which lacks proliferating cells, are able to regenerate the entire head. In this process, a group of cells, which are fully differentiated, reactivates cell proliferation. Our data suggest that P. excavatus is a model system to study CNS regeneration, which is dependent on the dedifferentiation of cells.

Growth and reproduction performances of earthworm (Perionyx excavatus) fed with different organic waste materials.

OBJECTIVE: The purpose of the experiment was to examine the effect of different food sources on the growth and reproduction performances of an epigeic earthworm Perionyx excavatus. MATERIALS AND METHODS: The experiment was carried out in 18 cylindrical plastic containers for 10 weeks. The study was designed with six treatments, each having three replicates. In control treatment (T1), cow dung was used as the only food source for the earthworm. In another five treatments, water hyacinth (T2), chopped banana plant trunk (T3), vegetable scrap (T4), paddy straw (T5), and sugarcane bagasse (T6) were used as food sources with cow dung as bedding material. RESULTS: The maximum weight gain of earthworm P. excavatus was 3,294.7 ± 4.5 mg for the food staff of vegetable scrap (T4). Earthworm P. excavatus fed with chopped banana plant trunk (T3) showed a very similar weight gain of 3,243.7 ± 3.8 mg. On the contrary, the minimum weight gain was 1,799.7 ± 3.5 mg for the food staff of paddy straw (T5). The maximum cocoon number of 137.33 ± 6.46 mg was observed in T3, whereas a minimum number of 36.67 ± 4.16 mg in T1. The highest number of hatchlings (12.33 ± 0.88 mg) was recorded in treatment T3, whereas the least number of hatchling (5.00 ± 0.58 mg) was observed in T1. CONCLUSION: It was concluded that, among the six different food sources, chopped banana plant trunk was preferable food source to the earthworm for growth and reproduction.

Nutrient recovery and vermicompost production from livestock solid wastes with epigeic earthworms.

The aim of this work was to study nutrient recovery and vermicompost production from livestock solid wastes of indigenous and exotic cow breeds with epigeic earthworms. Fourteen days pre-decomposed dung of Vechur native (CD1) and exotic Jersey (CD2) breeds were vermicomposted with Perionyx excavatus and Eudrilus eugeniae for 45 days (Cycle I) and 90 days (Cycle II) including respective controls without earthworms. Vermicomposts from CD1 and CD2 substrates during Cycle I and II showed increase in NPK, Ca and micro-nutrients (P < 0.05); whereas, pH, total organic carbon, C/N and C/P ratios disclosed decrease (P < 0.05) over initial levels for both the earthworms. E. eugeniae was found efficient in vermicomposting cattle solid wastes in 45 days and CD1 yielded nutrient rich vermicompost. The study concludes that Cycle I is suitable for nutrient recovery and vermicompost production, in addition to mass multiplication of earthworms in Cycle II.

Seaweeds as bioresources for vermicompost production using the earthworm, Perionyx excavatus (Perrier).

Fifteen days pre-decomposed seaweeds, Halimeda gracilis, Gracilaria corticata, Sargassum wightii and Sargassum swartzii spiked with cowdung (1:1) were vermicomposted using Perionyx excavatus for 60 days. The pH in the vermicompost showed insignificant reduction while electrical conductivity showed significant enhancement (P < 0.05). The reduction of organic carbon in vermicomposts ranged from -37.78 to -50.97% over worm-unworked composts. Total NPK contents showed significant increment (26.72-78.17%) in vermicompost over worm-unworked composts. The difference in percentage increase/decrease between physicochemical parameters was statistically significant (P < 0.001) and the same pattern was found between substrates. The total microbial population in vermicomposts was significantly higher than that of initial and composts of all seaweed + cowdung combinations (P < 0.001). The growth and reproduction of Perionyx excavatus in seaweed + cowdung combinations showed equivalent or higher rates when compared with cowdung signifying that Perionyx excavatus is well suited to convert seaweed and cowdung combinations into nutrient rich vermicompost.

Biological and physiological responses of Perionyx excavatus to abamectin.

Biological and behavioral responses of the tropical earthworm Perionyx excavatus towards different concentrations of abamectin were evaluated. Abamectin significantly reduced the biomass and reproduction (cocoon production) of P. excavatus as well as inducing histopathological alterations in the cuticle. Biomass loss was recorded in P. excavatus exposed to abamectin at a concentration as low as 0.1 mg active ingredient (a.i.) kg-1, while atrophy, another physiological response, was observed at an abamectin concentration of 0.21 µg cm-2 in a filter paper test. Cocoon production was significantly reduced in the presence of abamectin, and no cocoons were produced at doses of 20 mg a.i. kg-1 or higher, while abamectin at 50 mg a.i. kg-1 induced extreme pathology, characterized by the loss of the integrity of the whole body wall and intestine of P. excavatus. Histopathological alterations can be used as a biomarker to evaluate the toxicological impact of exposure to abamectin.

In vitro cytotoxic and apoptotic induction effect of earthworm coelomic fluid of Eudrilus eugeniae, Eisenia foetida, and Perionyx excavatus on human oral squamous cell carcinoma-9 cell line.

The current protocol of cancer management includes surgery, radiotherapy and chemotherapy. However, these modalities have significant adverse effects and affect the quality of life. Further intensification of treatment is hindered as maximal toxicity levels are reached impeding improvement. Hence researchers are in the quest for adjunctive naturally available therapies that can alter tumor proliferation without causing significant adverse reactions. The present study aims to explore the cytotoxic potential of earthworm coelomic fluid (ECF) of Eudrilus eugeniae (EE), Eisenia foetida (EF), and Perionyx excavatus (PE) on oral cancer cell line SCC-9. The effect of ECF on cell cycle analysis and mechanism of cell death have also been investigated. All experiments reported in this paper were performed as 3 replicates per experiment. The results indicated that ECF of EE, EF and PE have potent variable cytotoxic effect on SCC-9 cells demonstrated through LDH, clonogenic and comet assay. An effective cell cycle arrest was observed at the G2M phase of cell cycle with apoptotic induction that was observed through an Annexin V - FITC/PI assay. ECF of EE was found to be superior in its cytotoxic action closely followed by ECF of PE. The present findings provide evidence for the first time that ECF of EE, EF and PE have potent cytotoxic effect on oral cancer cells in vitro. They significantly induce G2M cell cycle arrest and promote apoptosis in SCC-9 cell line. Gene expression studies have been planned to ascertain the pathways of cell death.

Differential expression of primary pair-rule genes during bidirectional regeneration in Perionyx excavatus.

The earthworm Perionyx excavatus is a species highly capable of bidirectional regeneration. Pair-rule genes are thought to have an ancestral function in arthropod segmentation. However, orthologs in annelids (i.e. Capitella teleta) do not exhibit segmental expression in the ectoderm or mesoderm. Their role in regeneration is currently unclear. Here, we report the expression profile of primary pair-rule genes (Pex-EvxA, Pex-EvxB, Pex-RuntA, Pex-RuntB, Pex-Hes1A, Pex-Hes1B, Pex-Hes4A, and Pex-Hey) found in P. excavatus using semi-quantitative reverse transcription-polymerase chain reaction. Our results indicated these genes showed variable expression during bidirectional regeneration. Six of these genes might play diverse and potentially critical roles in head and/or tail regeneration.

Metabolism of tri-n-butyl phosphate in earthworm Perionyx excavatus.

Tri-n-butyl phosphate (TBP) is widely used in various industrial processes and has been detected in all environmental matrices. So far, little work has been done regarding the metabolism of TBP on terrestrial invertebrates. We investigated the metabolism of TBP in the earthworm, Perionyx excavatus, after acute exposure to TBP for one and two days in filter paper contact test, as well as after chronic exposure for 28 days in soil experiment. Biotransformation products were identified by using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, and by exploiting the information dependent acquisition in tandem mass spectrometry. TBP exhibited low accumulation in earthworm-soil ecosystem at 10 mg/kg and 50 mg/kg. The presence of earthworms significantly enhanced TBP degradation at 50 mg/kg in soil. Dibutyl phosphate and hydroxylated TBP were the major phase I metabolites. Three novel phase II metabolites were identified: ethanol dibutyl phosphate and its sulfate conjugate, and the phosphate conjugate of hydroxylated TBP. Hydroxylation and further phosphorylation dominated metabolism in chronic exposure. An extensive metabolic pathway of TBP in earthworm was proposed. This is the first report of TBP metabolism in terrestrial invertebrates and highlights the necessity to identify metabolites of contaminants when evaluating their bioaccumulation and toxicity.

(1)H nuclear magnetic resonance-based metabolomics study of earthworm Perionyx excavatus in vermifiltration process.

In this study, (1)H nuclear magnetic resonance (NMR)-based metabolomics approach was used to characterize the metabolic response of the earthworm Perionyx excavatus in continuous vermifiltration for two months under hydraulic loading rates of 1m(3)m(-2)d(-1) (VF1) and 1.5m(3)m(-2)d(-1) (VF1.5). Both VF1 and VF1.5 showed higher removal of chemical oxygen demand and total nitrogen than the biofilter without earthworms. Principal component analysis of the NMR spectra of earthworm metabolites showed significant separations between those not subjected to wastewater filtration (control) and VF1 or VF1.5. Temporal variations of earthworm biomass, and the identified metabolites that are significantly different between control, VF1 and VF1.5 revealed that worms underwent increasing metabolic activity within 20days in VF1 and 14days in VF1.5, then decreasing metabolic activity. The use of NMR-based metabolomics in monitoring earthworm metabolism was demonstrated to be a novel approach in studying engineered vermifiltration systems.

Taxonomic and functional annotation of gut bacterial communities of Eisenia foetida and Perionyx excavatus.

Epigeic earthworms can significantly hasten the decomposition of organic matter, which is known to be mediated by gut associated microflora. However, there is scanty information on the abundance and diversity of the gut bacterial flora in different earthworm genera fed with a similar diet, particularly Eisenia foetida and Perionyx excavatus. In this context, 16S rDNA based clonal survey of gut metagenomic DNA was assessed after growth of these two earthworms on lignocellulosic biomass. A set of 67 clonal sequences belonging to E. foetida and 75 to P. excavatus were taxonomically annotated using MG-RAST and RDP pipeline servers. Highest number of sequences were annotated to Proteobacteria (38-44%), followed by unclassified bacteria (14-18%) and Firmicutes (9.3-11%). Comparative analyses revealed significantly higher abundance of Actinobacteria and Firmicutes in the gut of P. excavatus. The functional annotation for the 16S rDNA clonal libraries of both the metagenomes revealed a high abundance of xylan degraders (12.1-24.1%). However, chitin degraders (16.7%), ammonia oxidizers (24.1%) and nitrogen fixers (7.4%) were relatively higher in E. foetida, while in P. excavatus; sulphate reducers and sulphate oxidizers (12.1-29.6%) were more abundant. Lignin degradation was detected in 3.7% clones of E. foetida, while cellulose degraders represented 1.7%. The gut microbiomes showed relative abundance of dehalogenators (17.2-22.2%) and aromatic hydrocarbon degraders (1.7-5.6%), illustrating their role in bioremediation. This study highlights the significance of differences in the inherent microbiome of these two earthworms in shaping the metagenome for effective degradation of different types of biomass under tropical conditions.

Combined toxicities of methyl tert-butyl ether and its metabolite tert-butyl alcohol on earthworms via different exposure routes.

Methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) are among the major soil contaminants that threaten the health of soil ecosystems. Many MTBE-contaminated sites accumulate TBA, because TBA is the intermediate of MTBE biodegradation. To access the risk of MTBE and TBA in soil, we investigated the combined toxicities of MTBE and TBA using two earthworm species, Perionyx excavatus and Eisenia andrei, as well as the toxic effects via different exposure routes. The combined toxicity showed weak antagonistic effects (LC50mix values were slightly greater than 1.0), and sensitivity toward same pollutants differed in the two earthworm species. Moreover, the toxicity of MTBE and TBA was also affected by the exposure route; both filter paper and artificial soil tests showed that dermal-only exposure to MTBE had an even greater toxic effect than combined dermal and oral exposure. Thus, we suggest that diverse environmental factors including organic materials, the physicochemical properties of the contact media, and the exposure routes of the organism, should be taken into consideration when assessing the effects of pollutants on organisms in diverse environmental systems.