Isolation,purification and partial characterization of chloragocytes from the earthworm species Lumbricus terrestris

Chloragocytes were isolated from the earthworm species Lumbricus terrestris. After mechanical dissociation and sedimentation through Percoll, a highly purified fraction of viable chloragocytes was obtained. The isolated chloragocytes accumulated the vital dye neutral red and reduced the tetrazolium dye MTT, thereby indicating cellular integrity. Time of flight flow cytometric analyses revealed a main population of large and highly granulated cells in the 30-33 m size range. Hydrolase measurements showed that -D-N-acetyl-glucosaminidase and acid phosphatase exhibited the highest activities (146.6 and 24.9 mU/mg of protein, respectively), possibly indicating a major role for these 2 hydrolases in the physiological function of chloragocytes. In contrast, other acid hydrolases such as -D-galactosidase and -D-glucuronidase had specific activities of respectively 26 and 182 times lower than the glucosaminidase. The specific activity of the membrane-bound alkaline phosphatase was comparable to that of its acid counterpart (18.9 vs. 24.9 mU/mg of protein, respectively) and this level of activity may show an important trans-membrane activity in chloragocytes. The cytoplasmic and mitochondrial enzyme isocitrate dehydrogenase had a level of activity comparable to that of the exclusively cytoplasmic enzyme lactate dehydrogenase (6.6 vs. 8.1 mIU/mg of protein, respectively). When L. terrestris chloragocyte homogenates were separated on Percoll, results showed that hydrolases and dehydrogenases were mainly associated with the lighter materials that remained above the Percoll layer. Nonetheless, the detection of significant proportions (15-25%) of the total recovered activity of acid phosphatase and-galactosidase in the enriched chloragosome fraction supports the notion that some chloragosomes may be 'lysosome-like' organelles.     

Microsatellite markers for the earthworm Lumbricus rubellus

We describe eight microsatellite markers for the earthworm Lumbricus rubellus, a commonly used ‘biological sentinel’ species in ecotoxicology studies. Thirty‐four individuals from a single population were tested for polymorphism. Markers possessed between two and 15 alleles per locus, and expected heterozygosity ranged between 0.06 and 0.92 (observed heterozygosity 0.03–0.92). Unfortunately, no consistent cross‐species amplification was observed in the related congeners Lumbricus castaneus and Lumbricus terrestris.     

Effects of transgenic Bt corn litter on the earthworm Lumbricus terrestris

A 200-day study was carried out to investigate the impact of transgenic Bacillus thuringiensis (Bt) corn on immature and adult Lumbricus terrestris in the field and in the laboratory. Another objective of this study was to develop test methods that could be used for standard testing of the impact of transgenic plants on different earthworm species in the field and in the laboratory. For this purpose two different experiments were involved, a laboratory experiment with adult L. terrestris and a field experiment with immature L. terrestris. No lethal effects of transgenic Bt corn on immature and adult earthworms were observed. Immature L. terrestris in the field had a very similar growth pattern when fed either (Bt+) or (Bt-) corn litter. No significant differences in relative weights of (Bt+) and (Bt-) corn-fed adult L. terrestris were observed during the first 160 days of the laboratory trial, but after 200 days adult L. terrestris had a significant weight loss of 18% of their initial weight when fed (Bt+) corn litter compared to a weight gain of 4% of the initial weight of (Bt-) corn-fed earthworms. Further studies are necessary to see whether or not this difference in relative weight was due to the Bt toxin or other factors discussed in the study. Degradation of Cry1Ab toxin in corn residues was significantly slower in the field than at 10 degrees C in the laboratory. Enzyme-linked immunosorbent assay results indicated that earthworms in both experiments were exposed to the Bt toxin throughout the whole experimental time.     

Fine structural analysis of the basal epidermal receptor cells in the earthworm (Lumbricus terrestris L.)

Summary A fine structural investigation was performed on receptor cells lying at the base of the epidermis in the earthworm, Lumbricus terrestris. Two types of receptor cells with many similarities, but also with major differences, were discriminated.One receptor is of the microvillar receptor type, that appears to be identical with the photoreceptor cell described earlier by Röhlich et al. (1970). Proximal to the nucleus is a large vacuole (phaosome, Binnenkörper) with many daughter cavities containing a large number of microvilli and several cilia with the 9 × 2 + 0 microtubular pattern. The intracellular cavity has no connection with the surface membrane, in contrast to that in hirudineans (White and Walther, 1969) and pogonophores (Nørrevang, 1974).The other receptor is the ciliated receptor type, that is presently described for the first time. This receptor also has a comparatively large uniform cavity, few microvilli and about 20 cilia with the 9 × 2 + 2 microtubular pattern. The cilia leave the cell in the proximal part through a wide opening, make a turn of 180 °, and proceed toward the epidermal surface. Receptors of a similar type have been described by Golding and Whittle (1975) in the cerebral ganglion of four limicole oligochaete annelids; they presumed that these cells have an osmoreceptor function. The new epidermal receptor type described in the present investigation probably has a chemoreceptor function of hitherto unknown kind.This investigation was supported by the Royal Physiographic Society at Lund, Sweden. The author would like to express his thanks to Mrs. Lena Sandell for skilful technical assistance     

Glutaraldehyde cross‐linking increases the stability of Lumbricus terrestris erythrocruorin

Since donated red blood cells must be constantly refrigerated, they are not available in remote areas and battlefields. We have previously shown that the hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an effective and safe substitute for donated blood that is stable enough to be stored for long periods at the relatively high temperatures that may be encountered in remote areas. The goal of this study was to further increase the thermal stability of LtEc by covalently cross‐linking LtEc with glutaraldehyde (gLtEc). Our results show that the melting temperatures of the gLtEc samples steadily increase as the molar ratio of glutaraldehyde to heme increases (from T_m = 57°C for native LtEc up to T_m = 68°C at a ratio of 128:1). In addition, while native LtEc is susceptible to subunit dissociation at alkaline pH (8–10), cross‐linking with glutaraldehyde completely prevents dissociation of gLtEc at pH 10. Increasing the molar ratio of glutaraldehyde:heme also significantly increased the oxygen affinity of gLtEc, but this effect was decreased by cross‐linking gLtEc in the deoxygenated T state. Finally, while gLtEc samples cross‐linked at low G:H ratios (e.g., 2:1) exhibited slight increases in oxidation rate in Tris buffer, no significant difference in oxidation rate was observed between native LtEc and the gLtEc samples in Ringer's Solution, which contains antioxidants. Overall, cross‐linking LtEc with glutaraldehyde significantly increases its thermal and structural stability without any loss of function, making gLtEc an attractive blood substitute for deployment in remote areas and battlefields. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:521–528, 2018     

Development of microsatellite markers for mango (Mangifera indica L.)

Microsatellite markers for mango (Mangifera indica L.) were developed using a genomic library enriched for (GA)_n and (GT)_n dinucleotide repeats. A subset of 41 positive clones was sequenced and primers were designed. Twenty‐eight primer pairs produced polymorphic amplification products for a diversity sample including 15 mango cultivars and two accessions from the related species Mangifera laurina and Mangifera applanata. Nineteen simple sequence repeat (SSR) loci with clear scorable patterns were chosen to study diversity in the mango germplasm bank of Guadalupe (FWI). The number of alleles ranged from three to 13 with observed levels of heterozygosity ranging from 0.059 to 0.857.     

Microsatellite markers for the drought-resistant earthworm Hormogaster elisae

We developed and characterized 10 highly polymorphic microsatellite loci from a simple sequence repeat-enriched genomic DNA library of the earthworm Hormogaster elisae. Characterization of these loci using 26 individuals revealed eight to 25 alleles per locus and high levels of heterozygosity. These loci will be used for paternity analysis and population genetic studies.     

Patterns of serotonin-immunoreactive neurons in the central nervous system of the earthworm Lumbricus terrestris L.

Summary The distribution patterns of serotonin-immunoreactive somata in the cerebral and subpharyngeal ganglion, and in the head and tail ganglia of the nerve cord of Lumbricus terrestris are described from whole-mount preparations. A small number of serotonin-immunoreactive neurons occurs in the cerebral ganglion, in contrast to the large population of serotonin-immunoreactive neurons that exists in all parts of the ventral nerve cord. From the arrangement of serotonin-immunoreactive somata in the subpharyngeal ganglion, we suggest that this ganglion arises from the fusion of two primordial ganglia. In head and tail ganglia, the distribution of serotonin-immunoreactive somata resembles that in midbody segments. Segmental variations in the pattern and number of serotonin-immunoreactive somata in the different body regions are discussed on the background of known developmental mechanisms that result in metameric neuronal populations in annelids and arthropods.Abbreviations CG1, CG2 cerebral soma group 1, 2 - CNS central nervous system - GINs giant interneurons - 5-HT 5-hydroxytryptamine, serotonin - 5-HTi 5-HT-immunoreactive - N side nerve - SG19 subpharyngeal soma group 1–9 - SN segmental nerve     

A multiplex panel of dinucleotide microsatellite markers for the water vole,Arvicola terrestris

We isolated and characterized nine polymorphic dinucleotide microsatellite loci in the fossorial vole Arvicola terrestris Scherman (Shaw). A multiplex panel comprising all nine loci was developed and its application to a set of 31 individuals allowed clear and easy characterization of allele sizes. The number of alleles range from three to 14 per locus with the observed heterozygosity ranging from 0.42 to 0.90. These markers will be useful for analysis of questions concerning population genetic structure and reproductive behaviour.     

Immunocytochemical demonstration of vertebrate neuropeptides in the earthworm Lumbricus terrestris (Annelida,Oligochaeta)

Summary The localisation and distribution of 10 vertebrate-derived neuropeptides in the earthworm, Lumbricus terrestris, have been determined by an indirect immunofluorescence technique. The peptides are pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), neuropeptide Y (NPY), glucagon (C-terminal), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), gastrinreleasing peptide (GRP), calcitonin gene-related peptide (CGRP), neurotensin (NT), and met-enkephalin. For 6 of the peptides — PYY, NPY, PHI, glucagon, GRP and CGRP — this is the first demonstration of their presence in any annelid, and NT has not previously been described in an oligochaete. Cell bodies and nerve fibres immunoreactive to the 10 peptides occur throughout the CNS. In the PNS, epidermal sensory cells displayed immunoreactivities to PP and PYY, and PP-, PYY-, NPY-, PHI- and GRP-like immunoreactivities occurred in nerve fibres supplying the main body muscles. Nerve fibres immunoreactive to PP and PYY are also associated with the innervation of the gut (pharynx, oesophageal glands, and mid and posterior regions of the intestine). No endocrine cells immunoreactive for any of the antisera tested could be identified in the gut epithelium, suggesting that dual location of peptides in the brain and gut epithelium is a phenomenon that occurred at a later stage in evolution. No immunoreactive elements were detected in any of the organs and ducts of the reproductive and excretory systems.     

水稻微卫星标记的发展和应用

微卫星又称简单序列重复。它是由几个核苷酸（一般２～４个）为重复单位组成的串联重复序列。相同座位上的重复序列由于重复次数的不同而造成序列长度的多态性。微卫星标记是一种共显性标记,具有等位基因丰富、检测技术简单等优点。微卫星标记在基因组作图、品种鉴定、种质保存、分子标记辅助选择等方面有着广泛的应用。目前水稻中已发展了３００多个微卫星标记。     

Development of polymorphic microsatellite markers in sesame (Sesamum indicum L.)

Fifty microsatellite sequences (SSRs) were isolated from an enriched library of sesame (Sesamum indicum L.) using a modified protocol. After screening, 10 polymorphic microsatellites were used to determine their usefulness in diversity analysis among 16 sesame accessions. The number of alleles ranged from three to six alleles per locus with an average of 4.6 alleles. The fragment size varied from 150 bp to 307 bp. Expected heterozygosites (H_E) and polymorphism information contents (PICs) ranged from 0.437 to 0.858 and 0.34 to 0.80, respectively, which indicates the highly informative nature of the microsatellites reported here. These microsatellite markers will be very useful in diversity analysis among a large germplasm collection of sesame present in our Korean gene bank and also in the establishment of its core collection.     

Formation of filopodia in earthworm (Lumbricus terrestris) coelomocytes in response to osmotic stress

Coelomocytes, the immunodefense cells of the earthworm Lumbricus terrestris, are exposed to changing osmotic pressures as the worm's coelomic fluid responds to fluctuating wet-dry conditions of the surrounding soil. Using light and fluorescence microscopy combined with actin and tubulin disrupting drugs, we determined the effects of changing osmotic pressure on coelomocyte morphology. The coelomocytes from L. terrestris respond to an increase in environmental osmotic pressure from isotonic conditions (170 mOsm) to hypertonic conditions (715 mOsm) by changing from a round/petalloid morphology to a filopodial morphology. Cytoskeletal fluorescent staining studies indicate that for filopodia to form, the actin cortical ring, present in most coelomocytes in isotonic conditions, must be disrupted. Breakdown of the actin ring by exposure to a hypertonic environment or actin disrupting drugs allows the formation of actin or tubulin-based filopodia. The filopodia, or podial-like extensions formed by earthworm coelomocytes, may enable the cells to better explore their environment.     

Immunoreactivity to molluskan neuropeptides in the central and stomatogastric nervous systems of the earthworm, Lumbricus terrestris L.

Polyclonal antisera against two related command neuropeptides (CNP2 and CNP4) described in neurons of the terrestrial snail Helix were used in a study of the nervous system of the earthworm Lumbricus. The CNP-like peptides belong to the same neuropeptide subfamily and bear a C-terminal signature sequence Tyr-Pro-Arg-X. The distribution patterns of immunoreactive (IR) neurons were studied in the central nervous system (CNS), skin, and stomatogastric nervous system of the earthworm. IR neurons were found in all CNS ganglia, the patterns being similar for both antibodies used. Several clusters of IR cells were observed in the cerebral and subesophageal ganglia. In the ventral cord ganglia, the number of IR cells decreased in the rostro-caudal direction, and the IR cells sent their fibers mostly into the median fiber bundle. Segmental nerves contained no IR fibers. After injury of the worm body, the number of IR neurons in the CNS significantly increased. In the skin, IR sensory neurons were present in sensory buds. The stomatogastric ganglia only contained IR fibers. Numerous scattered IR neurons were found in the inner subepithelial layer of the esophagus and formed the enteric plexus in which the cell bodies displayed a segmentally repeated pattern. Possible involvement of CNP-like-IR neurons in central integratory processes, sensory processes, and the regulation of feeding is discussed.This work was supported by INTAS (grant 01-2117), CRDF (grant RB1-2321-MO-02), and the Russian Foundation for Basic Research (grants 05-04-48724 and 03-04-48179).     

Isolation and characterization of sequence‐tagged microsatellite sites markers in chickpea (Cicer arietinum L.)

In this study we report the isolation of microsatellite sequences and their conversion to sequence‐tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.     

Isolation and characterization of polymorphic microsatellite markers from European pear (Pyrus communis L.)

This study reports the development and characterization of 19 microsatellite primer pairs developed from genomic DNA of European pear (Pyrus communis) and their transferability to other Pyrus and Malus material. The primers were designed from two different genomic libraries enriched for di‐ and trinucleotide repeats. When tested in six P. communis cultivars and 15 other Pyrus species, 13 primers revealed single‐locus polymorphism and six showed more complex patterns that suggest multiple loci. Two to 18 alleles were detected per locus and two primer pairs were sufficient to discriminate all accessions. Transferability of nine primer pairs to Malus was demonstrated through amplification of discrete products in two accessions.     

Characterization of microsatellite markers in sycamore (Acer pseudoplatanus L.)

Sycamore (Acer pseudoplatanus L.) is a tetraploid European hardwood tree species. The reproduction system of the insect‐pollinated trees and patterns of genetic variation are largely unknown. We isolated and characterized eight polymorphic microsatellite markers for Acer pseudoplatanus L. The high degree of polymorphism observed at these markers makes them useful to observe genetic variation patterns at various spatial scales and to analyse gene flow and the mating system. Primers developed for the amplification of microsatellites in A. pseudoplatanus were tested for 21 different species of genus Acer. Amplification products of the expected size were obtained in most cases.     

Trinucleotide repeat microsatellite markers for black poplar (Populus nigra L.)

Using an enrichment procedure, we have cloned microsatellite repeats from black poplar (Populus nigra L.) and developed primers for microsatellite marker analysis. Ten primer pairs, mostly for trinucleotide repeats, produced polymorphic fragments in P. nigra. Some of them also showed amplification in other poplar species. (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, P. lasiocarpa). The best six loci were tested on 23 P. nigra genotypes collected across Europe. The microsatellites produced up to 12 alleles per locus in this set, with observed heterozygosity between 0.32 and 0.91.     

Development of EST‐derived microsatellite markers for Arabidopsis lyrata subspecies petraea (L.)

A set of expressed sequence tag–simple sequence repeat (EST‐SSR) loci has been developed for Arabidopsis lyrata ssp. petraea. From 768 root cDNA clones, 126 microsatellites, including di‐, tri‐, tetra‐ and pentanucleotide repeat motifs were identified and primers were designed to 24 EST‐SSRs. Eleven loci were subsequently screened on 150 individuals sampled from five natural populations, which revealed three to nine alleles per locus (mean 5.36) and expected heterozygosity (H_E) estimates ranging from 0.046 to 0.698. Significant deviations from random mating were observed at 10 EST‐SSR loci, likely due to inbreeding (global F_IS = 0.151) and population structure (global F_ST = 0.246).