A reassessment of the systematics and a proposal for the phylogeny of some cosmopolitan Lineus species (Nemertea)

Morphological, behavioural and biochemical characters of 9 cosmopolitan, benthic species of the nemertean genus Lineus are used both to clarify their taxonomy and to discuss their phylogenetic relationships. Data analysis shows that all fissiparous species of Lineus collected in the world seas up to date can be placed in a single species, L. sanguineus, with three recognisable subspecies, L. s. sanguineus, L. s. nigricans and L. s. pseudolacteus. These taxa were originally described as separate species mainly according to their geographic origin under the names L. sanguineus (shores of European seas), L. socialis (Atlantic North American shores), L. vegetus (Pacific North American shores), L. pseudolacteus (shores of the English Channel) and L. nigricans (Mediterranean coasts of Italy and France). Examination of the character value matrix suggests a phylogenetic tree developing from two ancestral branches. The first branch includes the two related species L. ruber and L. viridis, the second branch the three species L. longissimus, L. lacteus and L. sanguineus. This approach to the taxonomic and phylogenetic relationships of Lineus nemertean species is in agreement with the intra- and interspecies histocompatibility data in nemerteans: (i) grafts transplanted from donors to recipients of the same species succeed and grafts transplanted from donors to recipients of different species fail; (ii) grafts are rejected more rapidly when the donor and recipient species diverged earlier in evolution and, a contrario, grafts are accepted better when the donor and recipient species have been more recently isolated.     

Lineus as a model for studying developmental processes in animals reconstructed from adult pieces

The difficulties that prevent reconstruction of animals by piecing together body fragments from several adults are overcome by using nemerteans of the genus Lineus. For 25 years I have managed to make viable composite worms by grafting body parts cut out of Lineus specimens either from the same clonal strain (syngeneically reconstructed worms) or from the same species (allogeneically reconstructed worms) or else from different species (xenogeneically reconstructed worms). Body reconstruction has usually been carried out orthotopically, i.e., the components of composite animals have been selected so as to be anatomically complementary. However, reconstruction has been made heterotopically when it was essential to obtain morphogenetic events in the adults. Here, I shall review some of the developmental processes that took place in such reconstructed animals. First, I shall report immune responses in composite worms derived from various combinations of body pieces grafted together. Second, I shall study sex differentiation during gonad development in growing or regenerating chimeric worms made by the grafting of male and female components. I shall refer also to gonadogenesis in the asexual progeny of bipartite chimeras derived from lateral body halves of both sexes fused together (clones of bilaterally allophenic worms). Third, I shall analyze regulative processes (regeneration, transgeneration) during localized morphogenesis occurring in heterotopically reconstructed worms. The data show how reconstructed Lineus may be exploited to increase our knowledge of developmental mechanisms, especially in the misunderstood field of organismal pattern homeostasis.     

Molecular Phylogeny of Some European Heteronemertean (Nemertea) Species and the Monophyletic Status ofRiseriellus, Lineus,andMicrura

The 16S rRNA mitochondrial gene was used to reconstruct the relationships among 10 heteronemertean species (subclass Heteronemertea, phylum Nemertea);Lineus ruberandL. viridisare represented by more than one specimen to assess intraspecific variation in these enigmatic species, and the analysis includes in total 14 terminal taxa incorporating one palaeonemertean species (Tubulanus annulatus) for outgroup rooting. The aligned sequences were subjected to maximum parsimony, maximum-likelihood, and neighbor-joining analyses to estimate the phylogenetic relationship of the species. The results were concordant from all analyses and indicate that neitherLineusnorMicruraare monophyletic taxa, and that there is no support from a phylogenetic point of view to establish the monotypic genusRiseriellus.     

Regeneration of Rapid Escape Reflex Pathways in Earthworms

SYNOPSIS. The medial and lateral giant nerve fibers in the earthworm,Eisenia foetida, regenerate cell-specific connections and recoverthrough-conduction capabilities in as little as 1–2 daysafter ventral nerve cord (VNC) transection Similar cell-specificreconnections between giant fibers occur approximately 4–10days after grafting together two posterior pieces of worms ortransplanting lengths of VNC from donor to recipient worms fromwhich a comparable length of VNC has been removed In the lattercase, touch-sensory and giant motor neurons within the transplantedVNC also regenerate, leading to restoration of escape reflexfunction in segments receiving the transplant Results from heterotopicallytransplanted VNC indicate that both central and peripheral regenerationis cell-specific, but specificity is sufficiently broad to includesegmentally homologous target cells from body regions otherthan those of the transplant origin E. foetida and related speciesmay be useful for studying the extent to which differentiatednervous systems, composed of serially homologous neuronal networks,can be remodelled by experimental manipulations such as graftsand transplants.     

Synthesis and Characterization of Nicotiana-Solanum Graft Chimeras

Intergeneric Nicotiana tabacum L./Solanum laciniatum Ait. graftchimeras were produced from decapitated grafts made betweenthese two graft compatible species. Graft unions were treatedwith or without either the auxin, p-chlorophenoxyacetic acid(p-CPA) or the cytokinin, benzylaminopurine (BAP). While BAPwas inhibitory to shoot formation, p-CPA increased the numberof adventitious shoots and raised the frequencies of shoot-forminggrafts obtained. Approximately 9% (14/151) of the shoots producedat p-CPA-treated graft unions were intergeneric chimeras. Theauxin application significantly increased levels of chimeralshoot recovery numbers thus indicating a direct auxin effecton chimeral shoot production. The types and numbers of chimerasproduced were independent of the scion/stock graft combinationemployed. All chimeras appeared to arise initially as mericlinalor sectorial chimeras, with a proportion of the former ones(5/14) stabilizing into periclinal chimeras. The morphologicalcharacteristics of these latter chimeras were compared withthose of the two parental species. The LII layer of speciesdetermined the characteristics of the vegetative and floralcomponents. However, the LI layer modified the qualitative traitsof both components. The LIII layer on the other hand alteredthe quantitative traits of the vegetative organs and the flower.This layer also determined the growth type, breeding behaviourand inflorescence type of synthesized chimeras. These organizationalfunctions of the LIII layer have not been reported previously. Chimeras, intergeneric chimera synthesis, grafting, graft chimeras, Nicotiana tabacum, Solanum aviculare, p-chlorophenoxyacetic acid, peroxidase isoenzymes     

Experimentally Synthesized Plant Chimeras 3. Qualitative and Quantitative Characteristics of the Flowers of Interspecific Nicotiana Chimeras

The flowers from a series of interspecific periclinal chimerasbetween Nicotiana glauca Grahm. and N. tabacum Su/su L. werequantitatively and qualitatively compared with the flowers oftheir component species and the sexual hybrid. Results indicatethat the epidermal component of the chimeral flowers has thegreatest influence on flower morphology and that each histogenicarrangement results in a unique flower morphology. When comparedto interspecific hybrids a greater diversity of flower shapes,sizes and colours exists with periclinal chimeras, demonstratingthat the experimental synthesis of chimeras between morphologicallydistinct components can be an important source of new phenotypes. Nicotiana glauca Grahm., Nicotiana tabacum L., tobacco, chimeras, graft chimeras, floral morphology, flowers     

Experimentally Synthesized Plant Chimeras 2. A Comparison of In vitro and In vivo Techniques for the Production of Interspecific Nicotiana Chimeras

In vitro and in vivo techniques were compared for synthesizingchimeras between Nicotiana glauca Grahm and N tabacum L Interspecificchimeral callus, produced from mixed callus cultures in vitro,was placed on media which favoured only N tabacum shoot formationNone of the 474 regenerated N tabacum shoots incorporated Nglauca cells into their meristems When chimeral callus was regeneratedunder hormonal conditions favouring simultaneous organogenesis,of 397 shoots, only non-chimeral shoots of both species aroseIn vivo, reciprocal splice grafts between species were decapitatedjust above the graft union and treated with or without auxin—lanolinpastes Auxin increased callus formation but inhibited adventitiousshoot formation Three of 209 adventitious shoots arising fromthe graft union were interspecific mericlinal chimeras whichwere later stabilized as periclinal chimeras All three chimerasformed when N glauca was the understock Two of the chimerasarose on untreated shoots which produced no visible callus,indicating that excessive callus formation may be unnecessaryfor multiple cell origin of adventitious shoots to occur Chimeras, tobacco, Nicotiana glauca, Nicotiana tabacum, tissue culture, graft chimeras, callus cultures     

The fate of the larval epidermis in the Desor-larva of <Emphasis Type="Italic">Lineus viridis</Emphasis> (Pilidiophora,Nemertea) displays a historically constrained functional shift from planktotrophy to lecithotrophy

Pilidiophora constitutes a clade of nemerteans characterized by a peculiar larval type, the pilidium. A characteristic of this larva is the transitory epidermis in which the juvenile develops from imaginal discs. The primary function of this larval envelope is assumed to be feeding and dispersal. When juvenile development is complete, the larval epidermis is ruptured and swallowed by the juvenile. According to recent cladistic and molecular analyses of the Nemertea, the intracapsular Desor-larva of the sibling species Lineus viridis and L. ruber is thought to have evolved from a pelagic pilidium. The general course of development has been demonstrated to be similar to that of the pilidium, in which the juvenile forms from imaginal discs under the larval epidermis. The two Lineus species, however, differ in their mode of larval feeding: L. ruber being ootrophic and L. viridis being lecithotrophic. In order to elucidate the transition from the planktotrophic pilidum to lecithotrophic development, I studied the early cleavage and metamorphosis from intracapsular Desor-larva to juvenile stages in L. viridis from the island of Sylt, using light microscopical, electron microscopical, and fluorescent staining methods. Due to the specific cleavage pattern with equally sized 1st quartet animal blastomeres and vegetal blastomeres in L. viridis, the larval epidermis later contains a considerable amount of the yolk reserve. During metamorphosis, the larval epidermis is ingested by the juvenile thus displaying behavior similar to that of the pilidium larva. In contrast to the pilidium, the function of the larval epidermis of the Desor-larva has shifted from feeding and dispersal to direct food supply. Thus, the development of L. viridis is a perfect example for strong historical constraints that prevent ancestral larval structures from being lost.     

Studies of the immunological capacity of germ-free mouse radiation chimeras. IV. Cell-mediated immunity

The cell-mediated immune (CMI) response of germ-free mouse radiation chimeras was compared with that of conventional mice. Spleen or thymus cells from chimeric or normal mice were injected intravenously into lethally irradiated, allogeneic hosts. Spleens of the irradiated hosts were assayed for effector cells using the ⁵¹Cr release assay. Spleen cells from syngeneic and allogeneic chimeras and normal mice were equally active in giving rise to effector cells. However, thymus cells from allogeneic chimeras were completely inactive within 9 months post-bone marrow transplant while thymus cells from syngeneic chimeras and normal mice still remained functional. Although allogeneic chimeras contain cells potentially reactive toward host antigens, cells cytotoxic to host antigens were not detectable. In addition, these studies indicate the helper cell and effector cell, both associated with T-derived lymphocytes, represent two different populations.     

Function of being colorful in web spiders: attracting prey or camouflaging oneself?

Bright body colorations of orb-weaving spiders have been hypothesizedto be attractive to insects and thus function to increase foragingsuccess. However, the color signals of these spiders are alsoconsidered to be similar to those of the vegetation background,and thus the colorations function to camouflage the spiders.In this study, we evaluated these 2 hypotheses by field experimentsand by quantifying the spiders' visibility to insects. We firstcompared the insect interception rates of orbs constructed bythe orchid spider, Leucauge magnifica, with and without thespider. Orbs with spiders intercepted significantly more insectsthan orbs without. Such a result supported the prey attractionbut not the camouflaging hypothesis. We then tested whetherbright body colorations were responsible for L. magnifica'sattractiveness to insects by manipulating the spiders' colorsignals with paint. Alteration of color signals significantlyreduced L. magnifica's insect interception and consumption rates,indicating that these spiders' bright body parts were attractiveto insects. Congruent with the finding of field manipulationswere the color contrasts of various body parts of these spiders.When viewed against the vegetation background, the green bodyparts were lower, but the bright parts were significantly higherthan the discrimination threshold. Results of this study thusprovide direct evidence that bright body colorations of orbweavers function as visual lures to attract insects.     

Shoot Formation from Explant Cultures of Fourteen Potato Cultivars and Studies of the Cytology and Morphology of Regenerated Plants

Plants were regenerated in quantity from cultured excised stem,rachis or leaf pieces of 13 potato (Solarium tuberosum L.) cultivarsusing modifications of a two-step procedure. Pronounced differenceswere observed in the response of different cultivars and explantsources to medium formulations containing different auxins orcytokinins. A few plants were recovered from tuber pieces ofseven cultivars by a different two-step procedure. Morphologicaldifferences from control plants were observed in some of theprimary regenerants. Anthocyanin pigmentation of tubers wasscored and stable changes to red-splashed white or white tuberswere found in some plants regenerated from cv. Desiree. Regenerantsfrom the particoloured cultivars Cara and King Edward oftenlost this trait but it returned to varying extents in subsequentgenerations. Most of the regenerated plants (87 per cent) containedthe euploid number of chromosomes (2n = 4x = 48). The methodsare suitable for production of plants for assessment of somaclonalvariation in a range of genotypes. Potato breeding, somaclonal variation, chromosome variation, skin colour, explant culture, plant regeneration, solanum tuberosum L. potato     

Microscopic anatomy of the integument in the ribbon worm Micrura bella (Stimpson, 1857) (Pilidiophora,Nemertea)

The integument of ribbon worms in the order Heteronemertea is distinct from the integuments in the other taxa of nemerteans due to the presence of a special subepidermal glandular layer, the cutis. Among heteronemerteans, the ultrastructure of the cutis has been studied only in the Lineus ruber species complex. In the current study, ultrastructural (transmission electron microscopy) and histochemical studies of the epidermis and the cutis of Micrura bella from the basal Lineage A of the family Lineidae were performed. The epidermis consisted of ciliated and serous gland cells and is separated from the cutis by a layer of the subepidermal extracellular matrix; the basal lamina was not detected. The cutis comprised musculature, two types of mucous and four types of granular gland cells, and pigment cells with four types of granules. In the cutis of juvenile worms, type II granular gland cells and type II mucous cells were not observed. The integument of the caudal cirrus consisted of ciliated and serous gland cells and two intraepidermal lateral nerve cords; the cutis was absent. The compositions of the integument glands of M. bella and the L. ruber species complex are similar, except for the presence of type IV granular gland cells with narrow rod-shaped and lamellated granules exhibiting an alternating dark and light transverse layers and type II mucous cells found only in M. bella.     

Endocrine control of reproduction in two rhynchocoelan worms

This brief review of the reproductive endocrinology in two distant species of Rhynchocoela examines selected results of earlier studies on the heteronemertean, Lineus ruber, and reports current and unpublished research on the hoplonemertean, Amphiporus lactifloreus.

Experimental evidence is presented for existence in the brain of a gonad-inhibiting hormone (GIH) that controls reproduction in male and female worms of both species. In adults deprived of GIH during the quiescent phase of the reproductive cycle, sexual characteristics grow rapidly and precociously; GIH replacement therapy prevents the anticipated sexual development. In L. ruber, the removal of GIH stimulates macromolecular syntheses in sexual target cells of both sexes: RNA synthesis is promoted in young oocytes; DNA synthesis is increased in spermatogonia, and induced in primary spermatocytes; and protein synthesis is significantly increased in gametocytes and sex-specific skin glands. GIH might be a regulating hormone which would inhibit the secretion of sex-specific stimulating substances produced by cells in proximity to the gonads.     

Silica and Ash in Tissues of Some Plants Growing in the Coastal Area of Mississippi, USA

Ash and silica contents and their depositional patterns in differenttissues of 27 plants growing in the Ocean Springs area of Mississippi(many grow elsewhere), were determined. Silica content of driedplant parts varied from no more than 0 per cent in Borrichiafrutescens (L.) D.C. stems to 18.76 per cent in Arundinariagigantea (Walt.) Muhl. leaves. Ash content varied from 0·73per cent in Cliftonia monophylla (Lam.) Britt. ex Sarg. stemsto 44·02 per cent in Batis maritima L. leaves. Plantssuch as Batis maritima L., Borrichia frutescens (L.) D.C., Salicorniabigelovii Torr. and Salicornia virginica L. which grew in salinemarshes had high ash contents due NaCl in their tissues. Morusrubra L. leaves had a high silica content for a dicotyledonousplant (3·12 per cent). Energy-dispersive X-ray analysisshows that the distribution of the element silicon is clearlyrelated to certain epidermal structures such as ridges, cellwalls, rows of irregular shaped structures lengthwise of theleaf, dumb-bell shaped ones and especially in trichomes. Therewas a high concentration of silica containing trichomes alongthe veins on the underside of Morus rubra L. leaves and thiswould protect them from insects. The outer parts of the inflorescencesof Ctenium aromaticum (Walt.) Wood, Elymus virginicus L., Juncuspolycephalus Michx. and phragmites communis Trin. were highlysilicified. This should give the seed some protection from insects.The sharp particles would be irritating to oesophageal tissuesand might be implicated in such a cancer.     

The influence of light and sea water temperature on the reproductive cycle of Lineus ruber (Heteronemertea)

The cerebral ganglia of Lineus ruber secretes a neurohormone named gonad-inhibiting hormone (GIH) controlling reproduction. As L. ruber undergoes repeated annual cycles of reproduction during its life, the way the neuroendocrine activity of the worm is seasonally regulated is an important ecophysiological question. In other words, what environmental ‘key factors’ influences the beginning of spawning season in L. ruber? In the present work we show that light is not a timer which controls the annual reproductive cycle progress of L. ruber. On the other hand our results suggest that thermoperiod is a good candidate for this function. Further work is still necessary to clarify the transduction mechanism of thermal stimuli in GIH discharge.     

Studies of the immunological capacity of germ-free mouse radiation chimeras. II. Thymus cell function in a humoral immune response to sheep erythrocytes

Experiments described here were undertaken to determine the reason for the depressed humoral immune response in germ-free mouse allogeneic radiation chimeras. Indirect immunofluorescence using the theta (θ) antigen as a marker demonstrated that about 10% of the nucleated cells in the spleen of both allogeneic and syngeneic chimeras bear the θ antigen. One type of in vivo cell transfer assay employed to determine the capacity for “helper” function of thymocytes revealed that allogeneic chimera thymocytes were only 7–18% as efficient in “helper” function as normal thymocytes. A second type of in vivo cell transfer assay demonstrated that the presence of intact normal thymic stroma had no effect on the “helper” inefficiency of thymocytes obtained from allogeneic radiation chimeras.     

Effects of the Over-Expression of the rolC Gene on Leaf Development in Transgenic Periclinal Chimeric Plants

Using Agrobacterium tumefaciens harboring a vector that carriedthe rolC gene under the control of the 35S RNA promoter of cauliflowermosaic virus, we produced several transgenic plants. Two ofthem were periclinal chimeras with altered leaves that had wrinkleddark green margins and inner pale green regions. One chimericplant had shortened internodes, reduced apical dominance, smallflowers and exhibited male sterility, but the other had a normalphenotype. Analysis of proteins, RNA and DNA indicated thatthe inner pale green tissues consisted of transformed cellswhile the outer dark green tissues were composed of non-transformedcells. Histological analysis indicated that mesophyll cellswere distorted and larger intercellular spaces were presentin the transformed pale green regions. Furthermore, in youngleaves, transformed mesophyll cells were larger than non-transformedcells. However, the normal parts had larger numbers of cellsper unit area than the transformed parts. These observationssuggest that ths expression of 35S-rolC in leaves caused inhibitionof cell division in developing leaves and that the undulatingmargins, composed of non-transformed cells, were a consequenceof the requirement for accommodating more cells in less spacewithin the region of rolC-transformed cells. (Received January 29, 1993; Accepted May 17, 1993)     

Allogeneic bone marrow transplantation in conventional mice: I. Effect of antibiotic therapy on long term survival of allogeneic chimeras.

In the present communication the beneficial effect of long term antimicrobial treatment with poorly absorbable antiboitics on the survival of allogeneic bone marrow chimeras was investigated. The combination of C57Bl mice as bone marrow donors and CBA/CA mice as irradiated recipients (800 rad) was used because of their strong histoincompatibility on the H-2 loci. All allografted recipients received 10 X 10(6) bone marrow cells. The majority of the recipients, which were rendered gnotobiotic by an antimicrobial treatment, achieved stable long term chimerism. In contrast, the conventional chimeras died from secondary disease within 9 weeks after transplantation. As early as 14 days after allogeneic bone marrow grafting the gnotobiotic recipients tolerated the reassociation with a conventional microflora without a change in the rate of mortality. Bone marrow cells (8 X 10(6) i.v.) and spleen cells (2 X 10(6) i.v.) collected from allogeneic chimeras failed to induce graft-versus-host-reaction (GVH) in a second lethally irradiated host. The data indicate, that the high rate of mortality in murine allogeneic bone marrow chimeras results from delayed GVH-reaction and systemic infection. The marrow graft, once established seems to exert tolerance against the allogeneic host. The pathogenesis of the systemic infection has not yet been worked out. It is assumed that it originates from bacteremia, induced by radiation dependent lesions of the epithelial integrity and defected lymphatic tissue in the gut.     

CADMIUM, LEAD AND COPPER IN FRESHWATER MUSSELS FROM THE ASSINIBOINE RIVER, MANITOBA, CANADA

Cadmium, lead and copper levels were examined in adductor muscle,foot, gonad, mantle, gill, and heart of 9 species of freshwatermussels from the Assini-boine River, using anodic strippingvoltametry. Metal levels in most organs were lower than in bottomsediments. The metals generally showed significantly higherconcentrations in heart and hemolymph samples than in the othertissues examined. However in four species, gill tissues showedthe highest values forcopper, and Quadrula quadnda and Ligumiarecta accumulated the greatest amounts of copper overall. Cadmiumconcentrations were particularly high in heart and hemolymphof Lampsilis radiata sili-quoidea, L. vcntricosa, and Ligumiarecta. Metal concentrations in tissues of a given species showedvarious relationships to weight of soft parts, shell length,and shell weight, but inverse correlations predominated, indicatingthat metals in many organs were less concentrated in older individuals.In L. radiatasiliquoidea and L. vcntricosa, copper concentrationswere higher in males than in females. Lead levels were alsohigher in L. vcntricosamales. (Received 24 June 1994; accepted 8 September 1994)     

A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH

Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive abilities and diverse metabolic activities, and are therefore widely utilized in bioconversion, biosynthesis and bioremediation. However, due to the high GC-content of the genome (~70%), together with low transformation and recombination efficiency, the efficient genome editing of Rhodococcus remains challenging. In this study, we report for the first time the successful establishment of a CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, making it feasible to obtain enough colonies for screening of mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61, the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated triple-plasmid recombineering system was developed with high efficiency of gene deletion, insertion and mutation. Finally, this new genome editing method was successfully applied to engineer R. ruber for the bio-production of acrylamide. By deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained with reduced byproduct formation and enhanced catalytic stability. Compared with the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells could be recycled from 1 batch to 4 batches.