Transintegumentary absorption of acidic amino acids in the oligochaete annelid Enchytraeus albidus

• 1.1. Transintegumentary absorption of acidic amino acids in Enchytraeus albidus amounts to only a few per cent of neutral amino acid uptake.
• 2.2. Influx of acidic amino acids is composed of a mediated route with high affinity and an apparent diffusional entry.
• 3.3. Glutamic acid influx is susceptible to metabolic inhibition, reduced salinity and Na⁺-levels and the presence of additional acidic amino acids.
• 4.4. Inhibition experiments suggest the presence of a distinct integumentary uptake system for acidic amino acids.

     

Structural adaptation of enzymes to low temperatures

A systematic comparative analysis of 21 psychrophilic enzymes belonging to different structural families from prokaryotic and eukaryotic organisms is reported. The sequences of these enzymes were multiply aligned to 427 homologous proteins from mesophiles and thermophiles. The net flux of amino acid exchanges from meso/thermophilic to psychrophilic enzymes was measured. To assign the observed preferred exchanges to different structural environments, such as secondary structure, solvent accessibility and subunit interfaces, homology modeling was utilized to predict the secondary structure and accessibility of amino acid residues for the psychrophilic enzymes for which no experimental three-dimensional structure is available. Our results show a clear tendency for the charged residues Arg and Glu to be replaced at exposed sites on alpha-helices by Lys and Ala, respectively, in the direction from 'hot' to 'cold' enzymes. Val is replaced by Ala at buried regions in alpha-helices. Compositional analysis of psychrophilic enzymes shows a significant increase in Ala and Asn and a decrease in Arg at exposed sites. Buried sites in beta-strands tend to be depleted of VAL: Possible implications of the observed structural variations for protein stability and engineering are discussed.     

Ultrastructure of the nephrostome in Enchytraeus albidus (Annelida, Clitellata)

 The nephrostome of Enchytraeus albidus exhibits a longitudinal slit-like opening on the dorsal side of a bulbous organ which is mainly composed of three cells, one flame cell situated centrally and endowed with a ciliary flame, and two cells lying superficially, called the mantle cell and the accessory mantle cell. The mantle cell occupies the ventral side of the organ extending on both sides up to the opening to constitute its immediate border on the frontal and lateral sides. Here it forms a kind of crest which is heavily subdivided into many protrusions and infoldings endowed with long cilia which exclusively spread into the coelomic cavity. The accessory mantle cell borders the narrow posterior slit of the opening, forming the roof of the initial canal which is devoid of cilia. From its anterior region a projection arises extending above the opening. The flame cell forms a groove from which the ciliary flame arises which extends into the canal. At its posterior region it replaces the accessory mantle cell displacing it onto the dorsal surface of the organ. It is argued that the mantle cell and the accessory mantle cell have presumbly originated from coelothelial cells. Thus the metanephridium may be a composite organ developing from a nephridioblastic and a coeloblastic source. A discussion of results in other annelid species indicates that metanephridia in the Annelida may have evolved more than once. Accepted: 13 October 1997     

Oogenesis in the annelid Enchytraeus albidus with special reference to the origin and cytochemistry of yolk

In the annelid Enchytraeus albidus the ovary is composed of packets containing eight synchronously developing oocytes. Each oocyte in the packet is connected, via a bridge, to a common cytoplasmic mass. Developmental synchrony of oocytes within individual packets is probably related to the ooplasmic continuity. The young previtellogenic oocyte contains many polysomes, a few cisternae of smooth and rough endoplasmic reticulum, small Golgi complexes, and mitochondria. Many of the mitochondria are dumbbell-shaped and may thus represent division stages. Vitellogenesis is marked by the appearance of peripherally located lipid yolk and small, densely staining granules scattered throughout the ooplasm. There is an increase of smooth endoplasmic reticulum, mitochondria, and enlarged Golgi elements. Small multivesicular-like bodies, the early stages of developing yolk, are derived from the Golgi complex. The mature yolk sphere is bipartite and consists of (a) a variable number of dense spheres, the core bodies, which are produced in the ooplasm by the Golgi complex and which become embedded in (b) a dense matrix. The electron opaque tracer, horseradish peroxidase is incorporated into the oocyte and deposited in the matrix suggesting that this component of the yolk sphere is obtained by micropinocytosis. Enzyme digestions and various cytochemical techniques suggest that the core bodies are rich in carbohydrate, probably as glyco- or mucoproteins, and that the matrix is rich in lipid.     

Life history of the oligochaete Enchytraeus coronatus (Annelida, Enchytraeidae) in agar culture

Abstract. Life-history attributes and the reproductive biology of Enchytraeus coronatus (Oligochaeta, Enchytraeidae) were studied in agar cultures, as a basis for using this species in toxicity tests and other experimental studies. The use of agar allows daily examination of the behavior, reproduction, and mortality of the worms. At 20°C, E. coronatus had a generation time of 21–22 days and an embryonic period of 7–10 days. Hatch rate was high (>80%) and worms became sexually mature 8–10 days after hatching. Cocoon production and number of eggs per cocoon were positively correlated. Mortality in the first stages of the life cycle (embryonic period and first week after hatching) was about 38%. Three different periods of cocoon production were distinguished: a period characterized by a regular increase in the number of cocoons produced (weeks 4–9), a period of production at a constant high rate of ∼5 cocoons per week (weeks 10–20), and a period of decline in production rate (weeks 21–32), down to a mean value of 1.6 cocoons per adult in the last week. This study showed that synchronized agar cultures of cohorts can provide adults for long-term experimental tests, with the age for optimum reproduction being 8–20 weeks.     

Sequence and structural parameters enhancing adaptation of proteins to low temperatures

A systematic analysis compared sequence and structural parameters distributions between 13 pairs of psychrophilic and mesophilic proteins for elucidating the cold adaptation parameters. The results of statistical test (t-test) revealed that helical content, tight turn content, disulfide bonds and hydrogen bonds do not show significant difference between psychrophilic and mesophilic proteins. However, it was demonstrated in this study that a larger proportion of open beta-turn in psychrophilic proteins is an effective parameter in specific activity at low temperature. In addition, substitution of amino acids of charged and aliphatic groups with amino acids of tiny and small groups in protein chains, tight turns and alpha-helices in the direction from mesophilic to psychrophilic proteins is one of the mechanisms of low temperature adaptation. Such sequence and structural parameter differences would help to develop a strategy for designing cold-adapted proteins.     

The possible mechanisms of the adaptation of microbial populations to low positive temperatures

Biologically low temperature was studied for its effect on the survival of populations and their biochemical and molecular-genetical state. The research was carried out on Yersinia pseudotuberculosis and Shigella sonnei models. It is established that the process of low-temperature adaptation of the populations may occur either at the level of the whole population with participation of inducible cold isoenzymes or at the level of a part of population, the temperature being a selection factor of the adapted cells.     

A comparative account of the life cycles of Lumbricillus lineatus Mull. and Enchytraeus albidus Henle in relation to temperature

Compared with Lumbricillus lineatus the cocoons of Enchytraeus albidus are smaller and contain fewer eggs whilst the egg production per worm is higher and sterility among the eggs lower. The fecundity of E. albidus is greater over a temperature range 1–25 and is not so adversely affected at the higher or lower temperatures as L. lineatus. Incubation in E. albidus is longer than in L. lineatus but the time required for maturation is much shorter, the periods depending upon the temperature. There is a similar relation to temperature as in the case of fecundity, with E. albidus again more tolerant to heat and cold. There is no relation between size and temperature but unlike L. lineatus , growth occurs after maturation in E. albidus and its cocoons (after the first) and their egg content are larger. There is no apparent relation between the setae length and temperature. The temperature relations favour a more rapid increase of E. albidus in the bacteria beds than of L. lineatus but they are well suited to the distribution of the species under natural conditions, E. albidus favouring a terrestrial habitat whereas L. lineatus exists in moister places. Further, they show specific physiological differences distinguishing the two species of worms.     

Freeze tolerance and accumulation of cryoprotectants in the enchytraeid Enchytraeus albidus (Oligochaeta) from Greenland and Europe

The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at −15 °C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at −2 °C, approximately 50 μg glucose mg⁻¹ tissue dry weight (DW). At −14 °C the glucose concentrations increased to between 110 and 170 μg mg⁻¹ DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300 μg mg⁻¹ DW, but only 230 μg mg⁻¹ DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at −2 °C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant.     

Embryonic development of the oligochaete Enchytraeus coronatus: an SEM and histological study of embryogenesis from one-cell stage to hatching

We describe the embryonic development of the soil-living oligochaete Enchytraeus coronatus (Enchytraeidae, Oligochaeta, Annelida). Enchytraeus coronatus is a direct developer. It follows the typical spiral cleavage mode of development that is highly conserved among annelids and a large number of other lophotrochozoan taxa that are collectively named "Spiralia." Scanning electron microscopy (SEM) was combined with light microscopic analysis of wholemounted and sectioned embryos, differentially processed through histological stainings, to reconstruct and document cellular movements and organogenesis from early cleavage stages until hatching. With the help of these data we have established a scheme of morphologically defined stages in order to facilitate future studies on the molecular and histological level that will allow a detailed cross-species comparison among annelids and other phyla.     

Freeze tolerance and accumulation of cryoprotectants in the enchytraeid Enchytraeus albidus (Oligochaeta) from Greenland and Europe

The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at −15 °C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at −2 °C, approximately 50 μg glucose mg⁻¹ tissue dry weight (DW). At −14 °C the glucose concentrations increased to between 110 and 170 μg mg⁻¹ DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300 μg mg⁻¹ DW, but only 230 μg mg⁻¹ DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at −2 °C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant.     

Cross adaptation of potato plants to low temperatures and potato cyst nematode infestation

Effect of short-term (2 h a day) and long-term (6 days) exposure to low temperature (5°C) on cold tolerance was investigated in two cultivars of potato (Solanum tuberosum L.): resistant (Sudarynya) and susceptible (Nevskii) to potato cyst nematode (Globodera rostochiensis Woll.). The extent of their infestation and changes in the expression of the genes of resistance to nematode (H1 and Gro1-4) were also analyzed. In both cultivars, exposure to low temperature enhanced cold resistance of potato plants. Enhancing cold resistance of cv. Sudarynya induced by a short-term exposure to chilling did not affect the extent of nematode infestation, whereas in susceptible cv. Nevskii, the extent of infestation decreased by almost three times. The level of expression of H1 gene in the leaves of the susceptible cultivar rose almost twofold both after short-term and long exposure to chilling, while in the resistant cultivar, gene expression increased only after a short-term effect of cold. The level of Gro1-4 gene expression increased after both temperature treatments only in the resistant cv. Sudarynya. Thus, the expression of genes for potato resistance to nematode infestation became more active in the susceptible cultivar as regards the gene H1 and in the resistant cultivar, regarding the gene Gro1-4. In the nematode-susceptible cv. Nevskii, the level of infestation decreased and cold resistance increased, apparently indicating cross adaptation to two factors of different nature.     

Protein psychrophilicity: role of residual structural properties in adaptation of proteins to low temperatures

In order to investigate the structural distribution responsible for protein psychrophilicity, a systematic comparative analysis of 13 pairs of psychrophilic and mesophilic proteins is reported. Three kinds of residue structural states such as exposed, intermediate and buried were considered for analyzing the structural patterns of single amino acids and amino acids in different groups. The statistical test revealed that higher frequency in exposed state of Ala, higher frequency in intermediate state of His, lower frequency in buried state of Lys, lower frequency in exposed state of Gln, higher frequency in exposed state and in intermediate state of Thr, higher frequency in exposed and intermediate state of tiny and small amino acids groups could be critical factors related with protein psychrophilicity. Such structure-based differences of residual properties would help to develop a strategy for designing psychrophilic proteins.     

Protein synthesis in the organs and tissues of frogs with local exposure to low temperatures]

Intensity of synthesis of total proteins and spectrum of synthesized proteins has been studied in various organs and tissues of sharp-faced frogs during local profound cooling of one of its limbs. It was shown that the effect of low temperatures on a certain part of a body of a poikilothermic animal has induced both a response of the whole organism and a much more acute response of the cooled tissues. The organism responded by a fall of the whole body temperature and a decrease in the activity of synthesis of total proteins of the viscera. Local cooling of a part of the frog body has induced a more significant decrease in the protein synthesis intensity in the cooled tissues alteration in the spectrum of synthesized proteins and appearance of specific proteins.     

Rapid responses to high temperature and desiccation but not to low temperature in the freeze tolerant sub-Antarctic caterpillar Pringleophaga marioni (Lepidoptera,Tineidae)

A broad definition of rapid cold hardening (RCH) is that it is the process whereby insects increase their survival of a sub-zero temperature after a brief (h) pre-exposure to a less severe low temperature. The effects of various pre-treatments on survival of two h at -7.9 degrees C were investigated in the freeze tolerant sub-Antarctic caterpillar Pringleophaga marioni (Lepidoptera: Tineidae), the first time RCH has been investigated in a freeze tolerant arthropod. All caterpillars froze when exposed to -7.9 degrees C, and none of the low temperature pre-treatments (-5, 0, 5 and 15 degrees C, as well as -5 degrees C and 0 degrees C with a delay before freezing) nor slow cooling (0.1 degrees C/min) elicited any improvement in survival of -7.9 degrees C as compared to controls. However, high temperature treatments (25, 30 and 35 degrees C), desiccation and acclimation for 5 days at 0 degrees C did result in significant increases in survival of the test temperature, possibly as a result of heat shock protein production. Haemolymph osmolality was elevated only by the 35 degrees C pre-treatment. It is suggested that the unpredictable environment of Marion Island means that P. marioni must always be physiologically prepared to survive cold snaps, and that this year-round cold hardiness therefore supersedes a rapid cold hardening response.