Small-angle X-ray studies of Lumbricus terrestris haemoglobin

Small-angle X-ray scattering of Lumbricus terrestris haemoglobin was measured in dilute solutions in 0.1 M Tris HCl buffer, pH 7.0. The following molecular parameters were determined: radius of gyration 11.2 nm, volume 7700 nm³, maximum diameter 29 nm, molecular weight 3.95 × 10⁶. The experimental scattering curve was compared with the scattering curves and distance distribution functions calculated for various models. The overall shape of the haemoglobin could be approximated by a hollow cylinder with the following dimensions: outer radius 13.5 nm, inner radius 5.4 nm, height 16.0 nm. The best fit was obtained with a model which consists of 12 large subunits arranged in two superimposed hexagonal rings with a number of smaller subunits between the large subunits and in the centre of the molecule.     

The self-association of the giant hemoglobin from the earthworm, Lumbricus terrestris

Background: The crystallographic structure of the gigantic hemoglobin (erythrocruorin) of the annelid worm, Lumbricus terrestris, provides a molar mass of 3.6 MDa for the hexagonal bilayer structure. Prior to this determination, some light-scattering and ultracentrifugal measurements indicated higher masses: 4.1–4.4 MDa. Values of 3.6 MDa were attributed to dissociation or subunit loss. However, early electron microscopy of the giant hemoglobin from a related annelid, Eumenia crassa by Öster Levin, showed that the hexagonal bilayer molecules were present mostly as oligomers; few were monomeric. Methods: Measurements by light-scattering of solutions of Lumbricus hemoglobin resolved by size-exclusion chromatography have been used to determine the weight-average molar mass of self-associating proteins. The X-ray structure has been re-examined. Results: Our measurements show that both 3.6 MDa monomers and self-association products are present as a mixture. Analysis of the X-ray structure indicates several different kinds of monomer–monomer interactions. Conclusions: We propose that the measured masses of Lumbricus hemoglobin as high as 4.4 MDa, result from oligomerization. These masses would result from the presence of an array of oligomers of various sizes together with monomers of 3.6 MDa. Furthermore, several different kinds of monomer–monomer interactions are clearly evident in the X-ray structure as well as in solution. General significance: The results demonstrate that self-association of monomers of the hemoglobin of Lumbricus terrestris explains the high molar masses of 4.1–4.4 MDa previously observed.     

Tissue-specific inhibition and recovery of esterase activities in Lumbricus terrestris experimentally exposed to chlorpyrifos

Exposure and effect assessment of organophosphate (OP) pesticides generally involves the use of cholinesterase (ChE) inhibition. In earthworm, this enzyme activity is often measured in homogenates from the whole organism. Here we examine the tissue-specific response of ChE and carboxylesterase (CE) activities in Lumbricus terrestris experimentally exposed to chlorpyrifos-spiked field soils. Esterases were measured in different gut segments and in the seminal vesicles of earthworms following acute exposure (2 d) to the OP and during 35 d of a recovery period. We found that inhibition of both esterase activities was dependent on the tissue. Cholinesterase activity decreased in the pharynx, crop, foregut and seminal vesicles in a concentration-dependent way, whereas CE activity (4-nitrophenyl valerate) was strongly inhibited in these tissues. Gizzard CE activity was not inhibited by the OP, even an increase of enzyme activity was evident during the recovery period. These results suggest that both esterases should be determined jointly in selected tissues of earthworms. Moreover, the high levels of gut CE activity and its inhibition and recovery dynamic following OP exposure suggest that this esterase could play an important role as an enzymatic barrier against OP uptake from the ingested contaminated soil.     

赤子爱胜蚓和毛利远盲蚓对添加造纸污泥土壤的化学和生物学特征的影响

将赤子爱胜蚓(Eisenia foetida)和毛利远盲蚓(Amynthas morrisi)接种于混合15%造纸污泥的旱地土和水稻土中,研究不同蚯蚓对不同混合污泥土壤的化学和生物学性状的作用。主成分分析结果显示蚯蚓对混合污泥土壤化学和生物学性质影响与土壤类型和蚯蚓种间差异密切相关(P0.05)。方差分析结果显示:(1)在混合污泥旱地土处理中,与对照相比,E.foetida处理的混合污泥土壤pH和Eh分别显著降低了0.22和13个单位(P0.05),有机碳和微生物量碳含量,以及过氧化氢酶,N-乙酰-氨基葡萄糖苷酶、脲酶和酸性磷酸酶活性分别降低了22.8%、43.8%、12.4%、48.4%、44.0%和40.7%(P0.05),而荧光素二乙酸酯酶活性增加了57.4%(P0.05);A.morrisi处理的混合污泥土壤碱解氮含量、过氧化氢酶、N-乙酰-氨基葡萄糖苷酶和脲酶活性分别明显下降了16.5%,12.4%、33.9%和45.5%(P0.05)。另外,两种蚯蚓相比较,E.foetida活动后混合污泥旱地土壤pH和Eh值、有机碳含量和碳氮比分别比A.morrisi活动后土壤降低0.17和10个单位、24.9%和2.26个单位,而代谢熵和荧光素二乙酸酯酶活性显著增高(P0.05)。(2)在混合污泥水稻土处理中,与对照相比,E.foetida处理的混合土壤有机碳含量和碳氮比显著提高20.9%和1.02个单位(P0.05),全钾含量和呼吸速率明显降低6.11%和33.8%(P0.05);而与对照相比,A.morrisi处理的混合土壤的速效钾含量和过氧化氢酶活性显著提高13.2%和10.8%(P0.05),但β-葡萄糖苷酶和荧光素二乙酸酯酶活性分别下降46.7%和34.4%(P0.05)。两种蚯蚓相比较,E.foetida处理后混合污泥水稻土的有机碳含量比A.morrisi处理显著增加了15.7%,碳氮比增加0.84个单位,速效钾、呼吸速率和过氧化氢酶活性减少11.4%、36.5%和5.51%(P0.05)。综上所述,蚯蚓能显著影响混合造纸污泥土壤的化学和生物学特征。蚯蚓在高有机碳和低粘粒含量旱地土壤中活动,能够加速有机碳的分解和释放,降低与土壤有机质分解和养分转化相关的酶活性和微生物量,但增加微生物总体活性;而蚯蚓在低有机碳和高粘粒含量水稻土壤中活动,则能够有助于土壤有机碳储存,对养分和微生物活性的影响相对较小。与A.morrisi相比较,E.foetida对混合污泥旱地土有机碳的分解和释放、微生物活性的提高,以及混合污泥水稻土有机碳的储存等方面的能力均较强,而对混合污泥水稻土钾素转化能力相对较弱。由于造纸污泥具有高有机碳和低养分含量特征,因此污泥农用仍需考虑按一定比例配施高养分含量有机物料。同时,进一步根据土壤类型和选择适宜的蚯蚓品种进行较长期的小区和大田试验,将能够为污泥农用提供更多科学参考。     

Correlation between the activity of digestive enzymes and nonself recognition in the gut of Eisenia andrei earthworms

Earthworms Eisenia andrei, similarly to other invertebrates, rely on innate defense mechanisms based on the capability to recognize and respond to nonself. Here, we show a correlation between the expression of CCF, a crucial pattern-recognition receptor, and lysozyme, with enzyme activities in the gut of E. andrei earthworms following a microbial challenge. These data suggest that enzyme activities important for the release and recognition of molecular patterns by pattern-recognition molecules, as well as enzymes involved in effector pathways, are modulated during the microbial challenge. In particular, protease, laminarinase, and glucosaminidase activities were increased in parallel to up-regulated CCF and lysozyme expression.     

陆地革菌的化学成分研究

从陆地革菌(Thelephora terrestris)子实体中分离得到9个已知化合物,经波谱学分析鉴定为:(22E,24R)-麦角甾-7,22-二烯-3β -醇 (1),(22E, 24R)-麦角甾-7, 22-二烯-3β ,5α,6β -三醇 (2),(22E,24R)-麦角甾-4,6,8(14),22-四烯-3-酮 (3),24-亚甲基羊毛甾-8-烯-3β -醇 (4),熊果酸 (5),木栓酮 (6),cerebroside B (7),(2S,3S,4R,2'R)-2-(2'-羟基二十二碳酰氨基)-十八碳烷-1,3,4-三醇 (8),(2S,3S,4R,2'R)-2-(2'-羟基二十三碳酰氨基)-十八碳烷-1,3,4-三醇 (9)。     

Density of the New Zealand flatworm, a predator of earthworms, in allotments in Edinburgh between 1993 and 1995

The density of the New Zealand flatworm, Artioposthia triangulata, found between 1993 and 1995 on the surface of the soil under various types of debris scattered in four allotments, was 1–2 m^?2. The type of debris did not markedly affect the density of the flatworm and the distribution of the flatworm within the allotment studied was significantly clustered. Fluctuation in numbers of specimens under different sets of compost‐filled plastic sacks varied in a similar manner to one another. The flatworm was most abundant in the upper 100 mm of soil and its egg capsules were most numerous between 100 and 200 mm below the surface. The flatworm was as abundant under the soil surface shelters as it was beneath shelters buried below the soil surface.     

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.     

Rhabditis terricola: An opportunistic nematode parasite of earthworm cocoons

During a study on the breeding rate of the earthworms Lumbricus rubellus and Eisenia foetida, nematodes identified as Rhabditis terricola were found consistently in large numbers in earthworm cocoons that failed to hatch after an adequate incubation period. This nematode species, which was previously known as a saprophyte, was found to invade earthworm cocoons and reproduce within, causing extensive productivity losses in earthworm cultures. In this study, R. terricola was effectively eradicated from earthworm cultures by rinsing the earthworms in tap water and transferring them repeatedly to sterile bedding.     

Interactions of earthworms with Atrazine-degrading bacteria in an agricultural soil

In the last 10 years, accelerated mineralization of Atrazine (2-chloro-ethylamino-6-isopropylamino-s-triazine) has been evidenced in agricultural soils repeatedly treated with this herbicide. Here, we report on the interaction between earthworms, considered as soil engineers, and the Atrazine-degrading community. The impact of earthworm macrofauna on Atrazine mineralization was assessed in representative soil microsites of earthworm activities (gut contents, casts, burrow linings). Soil with or without earthworms, namely the anecic species Lumbricus terrestris and the endogenic species Aporrectodea caliginosa, was either inoculated or not inoculated with Pseudomonas sp. ADP, an Atrazine-degrading strain, and was either treated or not treated with Atrazine. The structure of the bacterial community, the Atrazine-degrading activity and the abundance of atzA, B and C sequences in soil microsites were investigated. Atrazine mineralization was found to be reduced in representative soil microsites of earthworm activities. Earthworms significantly affected the structure of soil bacterial communities. They also reduced the size of the inoculated population of Pseudomonas sp. ADP, thereby contributing to the diminution of the Atrazine-degrading genetic potential in representative soil microsites of earthworm activities. This study illustrates the regulation produced by the earthworms on functional bacterial communities involved in the fate of organic pollutants in soils.     

Antibacterial activity of the hemolytic system from the earthworm Eisenia fetida andrei

The coelomic fluid and the cocoon albumen of the earthworm Eisenia fetida andrei are demonstrated to possess an antibacterial activity. The antibacterial activity and the already known hemolytic activity are due, in fact, to the same lipoproteic molecules. The antibacterial activity (bacteriostatic effect) is only directed against the highly pathogenic soil bacteria. Only these pathogenic bacteria strains possess at least one common antigen with the sheep red blood cells.     

The respiratory basis of locomotion in Drosophila

The respiratory system of insects has evolved to satisfy the oxygen supply during rest and energetically demanding processes such as locomotion. Flapping flight in particular is considered a key trait in insect evolution and requires an increase in metabolic activity of 10-15-fold the resting metabolism. Two major trade-offs are associated with the extensive development of the tracheal system and the function of spiracles in insects: the risk of desiccation because body water may leave the tracheal system when spiracles open for gas exchange and the risk of toxic tracheal oxygen levels at low metabolic activity. In resting animals there is an ongoing debate on the function and evolution of spiracle opening behavior, focusing mainly on discontinuous gas exchange patterns. During locomotion, large insects typically satisfy the increased respiratory requirements by various forms of ventilation, whereas in small insects such as Drosophila diffusive processes are thought to be sufficient. Recent data, however, have shown that during flight even small insects employ ventilatory mechanisms, potentially helping to balance respiratory currents inside the tracheal system. This review broadly summarizes our current knowledge on breathing strategies and spiracle function in the genus Drosophila, highlighting the gas exchange strategies in resting, running and flying animals.     

蚯蚓对湿地植物光合特性及净化污水能力的影响

以香蒲、芦苇和美人蕉为研究对象,并以土壤+沙子+有机质混合物为供试基质模拟人工湿地处理污水,采用向基质中加入蚯蚓与未加入蚯蚓2种处理。研究加入蚯蚓后,香蒲、芦苇和美人蕉光合速率、蒸腾速率、SPAD值和水分蒸发、蒸腾量的变化及其对净化污水能力的影响。结果表明:与未加入蚯蚓相比,加入蚯蚓后,香蒲、芦苇和美人蕉的净光合速率、蒸腾速率、SPAD值和水分蒸发、蒸腾量均增加,其中芦苇的净光合速率、蒸腾速率和水分蒸发、蒸腾量增加达到显著水平(P <0.05),而香蒲的水分蒸发、蒸腾量增加也达到显著水平(P <0.05);加入蚯蚓后,香蒲、芦苇和美人蕉对COD_Mn、NH₄⁺-N、NO₃^--N、TN和TP的去除率均增加,且香蒲和芦苇对COD_Mn的去除率显著增加 (P <0.05)。加入蚯蚓后,香蒲、芦苇和美人蕉的SPAD值均增加,说明蚯蚓能提高湿地植物对氮的吸收,增加植株中的氮含量,促进湿地植物的光合速率和蒸腾速率从而提高对污水的净化能力。     

The regeneration capacity of an earthworm, Eisenia fetida, in relation to the site of amputation along the body

It is well known that parts of earthworms can survive if they are cut off. Our aim was to link the regeneration capacity of an earthworm, Eisenia fetida (Oligochaeta, Annelida) with the site of the amputation, so we amputated earthworms at different body segment locations along the length of the body to examine the different survival rates and regeneration lengths of the anterior, posterior, and medial sections.
The greatest survival rates occurred for earthworms with the most body segments remaining after amputation. The anterior regeneration lengths were of two types. The lengths of regeneration of amputated from body segment 6/7 to further down the body posteriorly increased gradually (Type L_I). However, the regeneration lengths of earthworm which were amputated behind the 23rd segment, with less than a quarter of the total segments remaining, did not increase until the blastema and tail bud formation (Type L_II). These treatments were not completely regeneration. There were significant differences in both survival rates and lengths of regeneration lengths between immature earthworms and clitellate adult earthworms during the early stages of regeneration, but not at later stages of regeneration. The immature earthworms had a greater regeneration potential than clitellate adults amputated at the same segment. The survival rates of earthworms were correlated significantly with the number of body segments remaining after amputation, but not with the position of the amputation. The relationships between the survival rates and the numbers of remaining segments could be described by linear regressions. The anterior regeneration lengths were correlated with the position of the amputation, but not with the number of remaining segments; the posterior regeneration lengths, were not correlated with the number of segments remaining nor the amputation position. The anterior regeneration length was not related to the survival rates for all earthworm amputations after 30 days but was related in this way after 60 days.     

Hybridization relics complicate barcode‐based identification of species in earthworms

Introgressive hybridization results in mito‐nuclear discordance which could obscure the delimitation of closely related taxa. Although such events are increasingly reported, they have been poorly studied in earthworms. Here, we propose a method for investigating the degree of introgressive hybridization between three taxa of the Allolobophora chlorotica aggregate within two field populations (N = 67 and N = 105) using a reference data set including published DNA barcoding and microsatellite data of all known A. chlorotica lineages (N = 85). For this, we used both molecular phylogenetic and population genetic approaches. The test of correspondence between mitochondrial cytochrome c oxidase I (COI) lineages and clusters of nuclear microsatellite genotypes allowed individuals to be sorted in three categories (matching, admixed and nonmatching) and additional markers (mitochondrial NADH dehydrogenase subunit 1, nuclear Histone 3 and Internal transcribed Spacer Region 2) were used for phylogenetic reconstructions in order to check assignments. Although 15 admixed individuals were observed, no early‐generation hybrids were detected within the two populations. Interestingly, 14 nonmatching individuals (i.e. with a mtDNA haplotype that did not correspond to their nuclear cluster) were detected, a pattern that would result after multiple generations of unidirectional hybridization of female from one taxon to male of the other taxon. Because earthworms are simultaneous hermaphrodites, these events of unidirectional hybridization suggest sterility of the male function in several crosses and highlight that some individuals can be misidentified if reliance is placed on COI barcodes alone. These findings could improve the use of these barcodes in earthworms for species delineation.     

Genetic structure of invasive earthworms Dendrobaena octaedra in the boreal forest of Alberta: insights into introduction mechanisms

Population genetic studies can help to determine whether invasive species are established via single vs. multiple introduction events and also to distinguish among various colonization scenarios. We used this approach to investigate the introduction of Dendrobaena octaedra , a non-native earthworm species, to the boreal forest of northern Alberta. The spread of non-native earthworms in forested systems is not well understood, although bait abandonment and vehicular transport are believed to be important. Mitochondrial DNA sequencing revealed that multiple introductions of this species have occurred in northern Alberta, although individual populations may have been established by either single or multiple invaders introduced on one or more occasions. There was no relationship between genetic distances and either geographical distances or distances along road networks, suggesting that human-mediated jump dispersal is more common than diffusive spread via road networks or via active dispersal. As well, genetic diversity was significantly greater at boat launches than roads, indicating that multiple introductions may be more likely to occur at those locations. Focusing management efforts on areas where multiple introductions are likely to occur may help to reduce invasive species' potential for adaptive evolution and subsequent rapid spread.     

Juvenile hormone control of oögenesis in bumblebee workers, Bombus terrestris

In queenright workers of Bombus terrestris oögenesis is inhibited by the queen, the activity of the corpora allata is suppressed, and the resulting JH titer in the haemolymph remains low. In contrast, in queenless workers the JH production is stimulated on the first day of adult emergence, the JH titer increases, and eggs are rapidly formed. After injection of JH I in newly emerged workers oögenesis can also be induced in the presence of a queen in the same way as in queenless workers. The induced oögenesis is JH dosage dependent, dosages of less than 8 μg stimulate the production of vitellogenins, whereas a complete oögenesis can be induced by high dosages of about 50 μg. From studies on the rate of JH excretion it can be concluded that such high dosages must be injected to obtain the required JH titer during the whole egg maturation of 5 days. It is evident, therefore, that the initiation and the maintenance of oögenesis depends on JH.The injected JH ester is completely degraded to JH acid and JH diol mainly in the hindgut. The excretion starts quickly after the injection, but only traces of unchanged JH are excreted.From these results it can be suggested that a queen inhibits egg maturation in workers by suppressing the JH production in the corpora allata and thus lowering the JH titer. This influence also enables a queen to block oögenesis once stimulated, for instance in queenless workers. Breakdown of JH and excretion are not under the control of the queen and therefore neither of them play any rôle in regulating egg maturation in the worker caste.