Reaction of microorganisms to the digestive fluid of earthworms

The reaction of soil bacteria and fungi to the digestive fluid of the earthworm Aporrectodea caliginosa was studied. The fluid was obtained by centrifugation of the native enzymes of the digestive tract. The inhibition of growth of certain bacteria, spores, and fungal hyphae under the effect of extracts from the anterior and middle sections of the digestive tract of A. caliginosa was discovered for the first time. In bacteria, microcolony formation was inhibited as early as 20–30 s after the application of the gut extracts, which may indicate the nonenzymatic nature of the effect. The digestive fluid exhibited the same microbicidal activity whether the earthworms were feeding on soil or sterile sand. This indicates that the microbicidal agents are formed within the earthworm’s body, rather than by soil microorganisms. The effect of the digestive fluid from the anterior and middle divisions is selective in relation to different microorganisms. Of 42 strains of soil bacteria, seven were susceptible to the microbicidal action of the fluid (Alcaligenes faecalis 345-1, Microbacterium sp. 423-1, Arthrobacter sp. 430-1, Bacillus megaterium 401-1, B. megaterium 413-1, Kluyvera ascorbata 301-1, Pseudomonas reactans 387-2). The remaining bacteria did not die in the digestive fluid. Of 13 micromycetes, the digestive fluid inhibited spore germination in Aspergillus terreus and Paecilomyces lilacinus and the growth of hyphae in Trichoderma harzianum and Penicillium decumbens. The digestive fluid stimulated spore germination in Alternaria alternata and the growth of hyphae in Penicillium chrysogenum. The reaction of the remaining micromycetes was neutral. The gut fluid from the posterior division of the abdominal tract did not possess microbicidal activity. No relation was found between the reaction of microorganisms to the effects of the digestive fluid and the taxonomic position of the microorganisms. The effects revealed are similar to those shown earlier for millipedes and wood lice in the following parameters: quick action of the digestive fluid on microorganisms, and the selectivity of the action on microorganisms revealed at the strain level. The selective effect of the digestive gut fluid of the earthworms on soil microorganisms is important for animal feeding, maintaining the homeostasis of the gut microbial community, and the formation of microbial communities in soils.     

In silico characterization and transcriptomic analysis of nif family genes from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.     

Changes in the composition and physiological and biochemical properties of fungi during passage through the digestive tract of earthworms

It was established that the biomass of fungal mycelium decreased by 30–50% during passage through the intestine of the Aporrectodea caliginosa and Lumbricus terrestris earthworms, while its content in empty intestines was 40–60% less than in the soil. It was found that the amount of mycelium increases again in three-day-old coprolites due to the rapid growth of the species. It was demonstrated that the physiological activity of fungi (estimated according to the time of the appearance of colonies on the medium and probability of propagation) is lower in the intestine content, digestive tract, and fresh excrement of the worms than in the soil. It was noted that the activity and diversity of organic substrates (utilized by fungi), as well as proteolytic activity is lower in fungal isolates from the intestine than from the soil. It was registered that the death of a part of the fungi occurs in the worm intestine, while the physiological state changes in the animals withstanding the effect of the digestive medium.     

Culturable microorganisms from the earthworm digestive tract

The cultured aerobic copiotrophic bacteria and fungi from food-free digestive tracts of Aporrectodea caliginosa, Lumbricus terrestris, and Eisenia fetida earthworms, soil (compost), and fresh earthworm excrements were investigated. The microorganisms were isolated on nutrient media and identified by sequencing the fragments of bacterial 16S rRNA and fungal 28S rRNA (D1/D2 domain) gene sequences with subsequent phylogenetic analysis. Bacteria isolated from the digestive tracts of earthworms belonged to the families Aeromonadaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Moraxellaceae, Pseudomonadaceae, and Sphingobacteriaceae (Bacteroidetes), as well as Actinobacteria. For five strains, namely Ochrobactrum sp. 341-2 (α-Proteobacteria), Massilia sp. 557-1 (β-Proteobacteria), Sphingobacterium sp. 611-2 (Bacteroidetes), Leifsonia sp. 555-1, and a bacterium from the family Microbacteriaceae, isolate 521-1 (Actinobacteria), the similarity to known 16S rRNA sequences was 93–97%; they therefore, probably belong to new species and genera. Bacterial groups isolated from the digestive tracts of earthworms were significantly different from those isolated from soil and excrements. Some bacterial taxa occurred in different sections of A. caliginosa intestine and in intestines of different earthworm species; however, the overall composition of bacterial communities in these objects is different. Existence of bacterial groupings symbiotically associated with intestines is proposed. Among the fungi, Bjerkandera adusta and Syspastospora parasitica were isolated from the cleaned digestive tracts as light-colored, sterile mycelium, as well as Geotrichum candidum, Acremonium murorum (A. murorum var. felina), Alternaria alternata, Aspergillus candidus, A. versicolor, Cladosporium cladosporioides, Rhizomucor racemosus, Mucor hiemalis, Fusarium (F. oxysporum, Fusarium sp.), and Penicillium spp. These fungi survive for a long time in the earthworm’s digestive environment. Investigation of the functional characteristics and role in the host organism is required to confirm the symbiotic status of the microorganisms associated with the earthworm digestive tract.     

Different dispersal histories of lineages of the earthworm <Emphasis Type="Italic">Aporrectodea caliginosa</Emphasis> (Lumbricidae,Annelida) in the Palearctic

Earthworms are among the most abundant and ecologically important invasive species, and are therefore a good object for studying genetic processes in invasive populations. Aporrectodea caliginosa is one of the most widespread invasive earthworms in the temperate zone. It is believed to have dispersed from Europe to all continents except Antarctica. It is known that A. caliginosa consists of three genetic lineages, and genetic diversity is high both among and between them. We attempted to use that high genetic diversity to study A. caliginosa dispersal in the Palearctic based on a sample of 40 localities ranging from eastern Europe to the Russian Far East, and to compare our data to other studies on this species in western Europe and North America. Two genetic lineages were found in the studied sample. Only negligible decrease in genetic diversity was observed for the lineage 2 of A. caliginosa from West Europe to the Far East, suggesting multiple human-mediated introductions. In contrast, lineage 3 is abundant in West Europe and Belarus, but is absent from the East European Plain, the Urals, and the Far East. However, it is present in West Siberia, where it has greatly reduced genetic diversity, indicating long-distance dispersal accompanied by a bottleneck event. Thus, although these two lineages of A. caliginosa are morphologically indistinguishable, they have dramatic differences in their distributions and dispersal histories.     

Biological fixation of nitrogen and growth of bacteria of the genus <Emphasis Type="Italic">Azotobacter</Emphasis> in liquid media in the presence of perfluorocarbons

The addition of perfluorocarbons (perfluorodecalin, carbogal, and perfluoromethyldecalin) to nitrogen-free liquid media during the submerged cultivation of bacteria of the genus Azotobacter was followed by (1) increases in biomass accumulation and nitrogenase activity and (2) fixation of molecular nitrogen. Addition of perfluorodecalin (5 vol %) to the culture medium of A. chroococcum ACB 121 contributed to increases in biomass accumulation, cell concentration (of more than by five times), nitrogenase activity (of 3.4 times), and total nitrogen content in the medium (of 4.5 times).     

Site of Nitrogenase Activity in the Blue-Green Alga <Emphasis Type="Italic">Anabaena</Emphasis> sp. L-31

MANY blue-green algae fix nitrogen, assimilate carbon dioxide and evolve oxygen and as algal nitrogenase is inhibited^1–3 by high oxygen pressure, enhanced nitrogen fixation accompanying photosynthesis is surprising. Heterocysts do not contain⁴ or have comparatively less amounts^4–7 of photosystem II (PS II) pigments, which are responsible for the evolution of oxygen. This tends to favour the suggestion of Fay et al.⁸ that these cells are the sites of nitrogenase activity. Until now, however, attempts at obtaining unequivocal evidence for heterocysts as principal loci for nitrogenase activity have yielded conflicting results. Stewart et al.⁷ first demonstrated nitrogenase activity in heterocysts incubated aerobically, a finding confirmed by Wolk and Wojciuch⁹ and Van Gorkom and Donze¹⁰. By contrast, Smith and Evans^3,11 and Kurz and La Rue¹² reported results favouring vegetative cells as the major site of nitrogenase activity. Other evidence^2,13 showed high nitrogenase activity in cell-free preparations of Anabaena cylindrica and the non-heterocystous alga Plectonema boryanum strain 594.     

Features of the genetic structure of the earthworms <Emphasis Type="Italic">Aporrectodea</Emphasis> (Superspecies) <Emphasis Type="Italic">Caliginosa</Emphasis> (Oligochaeta: Lumbricidae) complex in Ukraine

An investigation of the genetic structure of the Aporrectodea caliginosa-A. trapezoids diploidpolyploid complex of earthworms found in the Ukraine is performed by means of biochemical genetic marking with respect to six loci (Aat, Es-1,-2,-3, and-4, and Mdh) and karyotyping. All 646 individuals from 21 samples are analyzed at the biochemical gene level and karyotype samples are obtained from 70 specimens. As a result, diploid amphimictic A. caliginosa individuals (2n = 36), which form panmictic populations, and triploid (2n = 54) A. trapezoides individuals, represented by 19 hypothetical clones discovered in the course of an analysis of 157 specimens, are clearly differentiated. A clear trend towards dominance of the triploid forms in the steppe zone of Ukraine is discovered. Here they represent roughly 70% as against 12% of all A. (superspecies) caliginosa individuals in the northern forest regions. Based on the stated nature of the heterozygosity of the loci studied and the behavior of the chromosomes in meiosis, it may be claimed that the cloned forms are allotriploids, formed as a result of hybridization of a series of related forms, which, judging from the allelic pools, does not include amphimictic species that now inhabit the territory of Ukraine. That is, either the apomictic clones of A. trapezoides lack a local origin and their appearance among the fauna of Ukraine is a consequence of settlement in arid steppe regions that are unfavorable for earthworms or, on the other hand, amphimictic A. caliginosa is an invasive species.     

A comparative study of gut microbiota profiles of earthworms fed in three different substrates

The gut microbiome of earthworms has a complex interdependence with the host. When the soil minerals pass through earthworm’s gut, they may affect the gut microbiota. To gain insight into the response of gut microbiota to the passed minerals, we fed earthworm (Eisenia fetida) on nutrient-poor soil and ore powder, and used high throughput sequencing to characterize the earthworm intestinal microbial community to find evidence for a core bacterial community of the E. fetida. The results showed that earthworms’ gut maintained a core microbiome that appeared in all samples. These core microbiota may play a significant role in a species’ environmental interactions. The composition of intestinal microbiomes varied with substrates. The earthworm guts from two nutrient-poor substrates had similar microbial communities and they were different from nutrient-rich substrate. Proteobacteria and Bacteroidetes were more abundant in the gut of earthworms kept on a nutrient-poor substrate such as ore powder or mineral soil than in the gut of earthworms kept in organic-rich compost soil; some of these microorganisms may help earthworms survive in nutrient-poor substrates.     

The effects of gibberellins and mepiquat chloride on nitrogenase activity in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA₃) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA₃ or PIX. GA₃ treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA₃-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA₃ and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA₃ treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA₃, respectively, in B. japonicum.     

Changes in Vegetation and Earthworm Populations under Free Grazing European Bison (<Emphasis Type="Italic">Bison bonasus</Emphasis>) in Broad-Leaved Forests of the Kaluzhskie Zaseki State Nature Reserve

This paper studies the impact of a free-ranging bison population on the vegetation and soil fauna in spatially adjacent biotopes (meadow-transition zone-surrounding forest) in the Kaluzhskie Zaseki State Nature Reserve. It is noted that bison maintains meadows in the absence of haymaking. Analysis of phytosociological relevés shows that the forest vegetation has been significantly transformed by bison over 10–15 years, as evident from (1) differences in the ecological-coenotic structure of the vegetation in the different biotopes, (2) a larger ecological-coenotic diversity of communities in the transition zones in comparison with surrounding forests, and (3) an increase in the participation of nitrophilous species in the vegetation. A fairly high earthworm species diversity (a total of seven species in the biotopes) is found; Aporrectodea caliginosa dominates in the earthworm density and biomass. The total earthworm density and biomass became lower in the transition zone than in neighboring forests and meadows, and the differences in earthworm density between the transition zone and the meadows were statistically significant.     

Local distribution of native and invasive earthworms and effects on a native salamander

North America is home to both native and invasive earthworms acting as ecosystem engineers as they build burrows that can serve as habitat for other species or otherwise alter soil structure, affecting nutrient cycling and other ecosystem processes. Here I determine where and what earthworm species commonly occur in my study area, and compare effects of native and invasive earthworms on the common woodland salamander, Plethodon cinereus, in field surveys and laboratory experiments. The native earthworm Eisenoides carolinensis was the most common earthworm, followed by two invasive species Dendrobaena octaedra and Octolasion tyrtaeum. The presence of O. tyrtaeum was associated with a narrower O-horizon (i.e., organic layer in the soil). Using structural equation modeling to explore direct and indirect pathways of these three most common earthworm species on salamanders, I found O. tyrtaeum occurrence was negatively correlated with nighttime salamander counts, a proxy for total salamander numbers, mediated by negative effects on O-horizon depth and microinvertebrate numbers. In the laboratory, O. tyrtaeum and D. octaedra consumed more leaf litter per gram of earthworm per day than the native E. carolinensis. However, salamanders consumed earthworms and used burrows of all native and invasive species of earthworms similarly. The potential for negative indirect effects of the invasive earthworm O. tyrtaeum on P. cinereus was demonstrated both in the field and laboratory, highlighting that seemingly small differences between native and invasive ecosystem engineers have the potential to significantly alter the effects of these closely related native and invasive organisms.     

Identification and characterization of a new agar-degrading strain with the novel properties of saccharides inhibition and nitrogen fixation

In this study, a new agar-degrading strain was isolated from soil with agar as a sole carbon source and energy. Based on its morphological, physiological, biochemical characterization and 16S rDNA sequence, the strain was identified as Streptomyces lavendulae UN-8. The extracellular agarase activity reached 0.03 U/ml after fermentation in shake flask (250 ml), which was close to other reported non-marine microorganisms. Furthermore, it is interesting that the growth of UN-8 would be inhibited by glucose (40 g/L) and maltose (40 g/L) with the inhibitory rate of 100% and 70%, respectively. Besides, UN-8 could be grown on the solid medium without any nitrogen sources, then the possible nitrogen fixation gene nifU was cloned from its genomic DNA. The deduced amino acid sequence of nifU has high similarity (98%) with nitrogen fixation protein NifU from Streptomyces sp. NRRL S-104 (KJY22454.1) and Streptomyces sp. NRRL F-4428 (KJK52526.1) based on NCBI blast. It is suggested that the nifU gene of UN-8 also encoded nitrogen fixation protein NifU. These results provided some new information for the further understanding of agar-degrading strain.     

Relationships between nitrogen fixation and photosynthesis as the main components of the productivity in alfalfa

Alfalfa (Medicago sativa L.) plants were inoculated with Sinorhizobium meliloti Tn-5 mutants featuring various nitrogen-fixing effectiveness and then grown in sand culture to study relations between CO₂ exchange, plant productivity, and nitrogen fixation. At the flowering stage, the relationship between nitrogen fixation and photosynthesis of whole alfalfa plants was described with the logarithmic curve. At the same stage of plant development, a close relationship was observed between nitrogen fixation rate and plant weight; this relationship showed a trend toward saturation at high rates of nitrogen fixation. The increase in nitrogenase activity of root nodules was accompanied by stimulation of root respiration; the relation of respiration to nitrogen-fixing activity was manifested stronger than its relation to the total root weight. It is concluded that highly effective strains of root nodule bacteria can realize their potential only in combination with complementary plant genotypes featuring active photosynthesis that provides a balanced supply of assimilates for both the symbiotic apparatus and growth processes in the macrosymbiont.     

Effects of non-native Asian earthworm invasion on temperate forest and prairie soils in the Midwestern US

Effects of invasive European earthworms in North America have been well documented, but less is known about ecological consequences of exotic Asian earthworm invasion, in particular Asian jumping worms (Amynthas) that are increasingly reported. Most earthworm invasion research has focused on forests; some Amynthas spp. are native to Asian grasslands and may thrive in prairies with unknown effects. We conducted an earthworm-addition mesocosm experiment with before–after control-impact (BACI) design and a complementary field study in southern Wisconsin, USA, in 2014 to investigate effects of a newly discovered invasion of two Asian jumping worms (Amynthas agrestis and Amynthas tokioensis) on forest and prairie litter and soil nutrient pools. In both studies, A. agrestis and A. tokioensis substantially reduced surface litter (84–95 % decline in foliage litter mass) and increased total carbon, total nitrogen, and available phosphorus in the upper 0–5 cm of soils over the 4-month period from July through October. Soil inorganic nitrogen (ammonium– and nitrate–N) concentration increased across soil depths of 0–25 cm, with greater effects on nitrate–N. Dissolved organic carbon concentration also increased, e.g., 71–108 % increase in the mesocosm experiment. Effects were observed in both forest and prairie soils, with stronger effects in forests. Effects were most pronounced late in the growing season when earthworm biomass likely peaked. Depletion of the litter layer and rapid mineralization of nutrients by non-native Asian jumping worms may make ecosystems more susceptible to nutrient losses, and effects may cascade to understory herbs and other soil biota.     

Production of gold nanoparticles by biogenesis using bacteria

Diazotrophic cyanobacteria Anabaena sp. PCC 7120, four Nostoc strains, and two Azotobacter species (A. vinelandii and A. chroococcum) were found to produce gold nanoparticles (GNP) under nitrogen fixation conditions. GNP biogenesis occurred at AuHCl₄ concentrations from 0.1 to 1 mM. In the cultures of unicellular cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 incapable of nitrogen fixation, no GNP were formed at the same concentrations of gold salts. The plasmon resonance band peak was located at 552 nm. This position is characteristic of spherical GNP 10 to 30 nm in size. Small amounts of GNP were also formed in the culture liquid supernatants of the tested nitrogen-fixing bacteria at AuHCl₄ concentrations from 0.25 to 0.5 mM.     

Effect of <Emphasis Type="Italic">Pseudomonas</Emphasis> Bacteria on Peroxidase Activity in Wheat Plants when Infected with <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis>

We investigated the effect of treating soft wheat seeds (Triticum aestivum L.) with two Pseudomonas bacteria strains, isolated from earthworm coprolites, showing a significant antifungal and growth-promoting action in preliminary screening on the activity of guaiacol-dependant peroxidase under phytopathogenic load in the presence of Bipolaris sorokiniana (Sacc.) Shoemaker as a mechanism for inducing plant resistance to the pathogen. We established a statistically significant decrease (P < 0.05) in root rot disease incidence and severity during bacterization, which is indicative both of antifungal activity of the used bacterial isolates and of their successful colonizing the rhizosphere of wheat plants. We noted a response of free and weakly bound peroxidase of wheat plants to infection with B. sorokiniana: the enzyme activity increased during pathogenesis. Bacterization also increased peroxidase activity in plant leaves and roots, the greatest differences from non-bacterized plants being observed in wheat roots in the presence of the pathogen. We detected a direct link between peroxidase activity in wheat roots and leaf tissues in the absence of the pathogen and the feedback between peroxidase activity and plant infestation by the root rot pathogen. In the presence of the phytopathogen, there is a lack of correlation between peroxidase activity in wheat roots and leaves, and there is a shift of activity towards its increase in roots, which plays an important role in the development of systemic resistance against the root rot pathogen that penetrates into plants through the roots and root collar.     

<Emphasis Type="Italic">Ochrobactrum intermedium</Emphasis> ANKI,a nitrogen-fixing bacterium able to decolorize azobenzene

Morphological and biochemical properties of the nitrogen-fixing strain Ochrobactrum intermedium ANKI, intensely growing on media with azo compounds, and its resistance to various common xenobiotics were investigated. The kinetics of azobenzene transformation by O. intermedium ANKI was studied. Under cometabolism conditions, up to 40 mg of azobenzene per liter of medium were decolorized within one week. It was shown that the strain possessed molybdenum-dependent nitrogenase activity, and its nitrogenase system was sensitive to oxygen and fixed nitrogen in the medium.     

Comparative bacterial survey in the gut of lepidopteran insects with different bionetwork

Insect gut is an untapped reservoir persist numerous unidentified gene responsible for the digestion of complex food material into a simpler nutrients, this activity of gut was enhanced by the microbial interaction. Insect gut-microbe interaction encodes beneficial activity to its host in several aspects, to illuminate the tactic of the interaction this study was framed and worked out in two insect species such as Spodoptera litura and Bombyx mori. The present study surveyed the comparative diversity of microbial population in the gut of a field pest Spodoptera litura and domesticated insect silkworm Bombyx mori at their active feeding larval stage. Both culture dependent and culture independent approaches exposed the dominance of Enterobacteriacae family and with majority of proteobacteria and firmicutes residing in the gut of both Spodoptera litura and Bombyx mori. Especially, the Enterococcus mundtii is a predominant affiliate in both processes of both insects.