Surface topology and structural integrity of the Theromyzon tessulatum (Annelida: Hirudinea: Glossiphoniidae) cocoon

Cocoons secreted by the aquatic leech Theromyzon tessulatum comprise a tubular, membranous ovoid, sealed at each end by a glue‐like substance, called an operculum. Scanning electron microscopy showed surface features of the T. tessulatum cocoon that included a circuitous bulge, cups that conformed to the shape of embryos, relief folds that radiated from opercula, and asymmetric distributions of protuberances on the upper aspect of the cocoon surface. The structural integrity of the T. tessulatum cocoon was assessed after exposure to a variety of denaturing conditions (e.g., extreme heat, detergents, acids). Although both the fibrous cocoon membrane and opercula were strikingly resilient, the membrane/operculum boundary appeared to be the weakest structural component of the cocoon, consistent with its functional role as an escape hatch for juvenile leeches. The operculum itself was more sensitive to denaturation than the cocoon membrane, and thus was probably the source of a major protein component isolated from the T. tessulatum cocoon (i.e., Tcp; Theromyzon cocoon protein). J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Cell dynamics during cocoon secretion in the aquatic leech, Theromyzon tessulatum (Annelida: Clitellata: Glossiphoniidae)

One distinguishing feature of clitellate annelids is the presence of specialized segments comprising the clitellum, whose primary function is to secrete a cocoon. Using histological analyses, we have documented cell types (I-V) and cellular processes associated with cocoon secretion in the aquatic leech, Theromyzon tessulatum. Our data indicate that the bulk of the cocoon's biomass arises from precursor cells of a single type that hypertrophy and proliferate ∼1 week prior to egg laying, and then differentiate into either of two cell types (i.e., Type II or Type III) depending on their position within the clitellum. Type II cells are concentrated along the lateral edges and venter of the clitellum and secrete alcian blue-staining granules that form opercula (i.e., glue-like material that seals both cocoon ends), while Type III cells populate the dorsal midline and secrete azocarmine-staining granules that build the cocoon wall. Both cell types occupy spaces between deep muscle layers and extend long-neck tubules to the surface epithelium as they fill with granules a few days prior to egg laying. Other cell types appear to make minor contributions to the cocoon (e.g., Type I, Type IV) or have supporting or signaling roles (e.g., Type V). Our observations suggest that post-translational modification (i.e., glycosylation) of the same core protein(s) distinguishes the granules of Type II/III cells, and that the default state of the Type II/III precursor may be evolutionarily linked to secretory cells in basal polychaetes.     

A comparative survey of the Erpobdella octoculata (L.) populations in the Afon Crafnant,N. Wales,above and below an input of zinc from mine-waste

The life-cycle, growth, and reproductive success of a stream-dwelling Erpobdella octoculata (L.) were studied over a 19-month period at two sites, one above and one below an input of zinc pollution from mine-waste. Samples were collected monthly using quadrats. The blotted wet weight of all leeches was measured. An histological technique was employed to determine sexual maturity. Reproductive success was measured by estimating the numbers of cocoons and number of eggs per cocoon at each site. A wet ashing technique, followed by analysis using flame atomic absorption spectrometry, was used to determine the extent of bioaccumulation of zinc. Total zinc and other metals in the waters were similarly analysed.Differences were found between the populations at the two sites. The life-history of the Erpobdella above the contamination was comparable to that found for other stream-dwelling populations previously studied. Observed differences in densities of leeches between sites in the present and in previous studies by other workers may be due to recorded differences in the abundance of available food. At the polluted site, there was evidence that (a) there was a delay in cocoon deposition and hence hatching of young, (b) the adult leeches produced more misshapened and empty cocoons, (c) no relationship existed between the area of cocoons with eggs and number of eggs present, and (d) the proportion of young to adult leech was smaller at the polluted site. At the polluted site, higher levels of zinc were recorded in the tissues of Erpobdella and there was some evidence for the active intake of zinc by the leech.     

Intra- and interspecific predation on the cocoons of Erpobdella octoculata (L.) (Annelida: Hirudinea)

In laboratory experiments, eleven out of twenty-one invertebrate species and all of the three fish species tested fed on freshly laid flaccid cocoons of Erpobdella octoculata, though predation was only severe from a dytiscid beetle, E. octoculata and the fish species. There was a tendency for more cocoons to be eaten by starved than fed predators. Only snail species damaged one-week-old tougher cocoons.Only one out of 471 E. octoculata collected during their breeding season from the stony shores of two eutrophic lakes had a cocoon in its gut.The role of cannibalism of and interspecific predation on cocoons in the control and regulation of lake-dwelling populations of the erpobdellid is discussed, and assessed to be relatively unimportant. A more likely regulatory mechanism may involve high juvenile mortality from as yet unknown causes.     

Operculum regeneration following failed predation in the Silurian gastropod Oriostoma

Regeneration of the calcareous rigiclaudent operculum following severe, non‐lethal, predatory attacks is described in two specimens of the characteristic Silurian gastropod Oriostoma from the English Midlands. This is the first record of the regeneration of opercula in Palaeozoic gastropods and of opercula conjoined with shells that have been repaired after significant, failed, durophagous attacks. In one specimen, a series of repaired injuries culminates in a repaired aperture closed by the operculum. In the second specimen, new shell growth has failed to recover the full extent of the original broken whorl, and the new aperture and operculum, though well developed, are smaller than the originals. Both opercula are unusually thin centrally when compared to other, strongly domed, Oriostoma opercula.     

Structural model of an antistasin/notch-like fusion protein from the cocoon wall of the aquatic leech, Theromyzon tessulatum

The aquatic leech, Theromyzon tessulatum, secretes a proteinaceous cocoon with extraordinary physical properties (e.g., proteolytic, thermal resiliency). The deduced amino acid sequence of a major protein (Tcp—Theromyzon cocoon protein) from the T. tessulatum cocoon wall has been used to model the endogenous structure of the Tcp protein. The Tcp protein sequence comprises six internal repeats, each containing 12 ordered Cys residues. Amino acid alignments suggest that the region Cys1→6 is homologous to antistasin, a leech anticoagulant, and Cys7→12 is homologous to an epidermal growth factor-like domain found in notch-class proteins, which play critical roles in development, signaling, and adhesion throughout the Animalia. Modeling of individual domains (i.e., antistasin and notch) positions multiple hydrophobic and charged residues on the surface. When the antistasin and notch domains were fused, hydrophobic pockets appeared that may facilitate a polymerization mechanism. Collectively, the predicted features of our Tcp model are consistent with the physical properties of the leech cocoon wall.      

Operculum closing as a defence against predatory leeches in four British freshwater prosobranch snails

The defence reaction of operculum closing in response to the presence of the molluscivorous leech Glossiphonia complanata (L.) and the non-molluscivorous Erpobdella octoculata (L.) was studied in four species of freshwater prosobranch gastropod. Bithynia tentaculata (L.) and Valvata piscinalis (Müller) can distinguish between the leeches, reacting only to G. complanata. V. piscinalis is capable of a greater degree of distance chemoreception of the leech ‘scent’. Valvata cristata Müller and Potamopyrgus jenkinsi (Smith) did not react to either leech. V. cristata may not be a potential prey item for G. complanata, while P. jenkinsi is fed on by the leech, but is a relative newcomer to the freshwater fauna. Animal Ecology Research Group, Department of Zoology, University of Oxford Commonwealth Forestry Institute, Department of Plant Sciences, University of Oxford     

体重、年龄和应激刺激对菲牛蛭繁殖性能的影响

采用单因素方差分析等方法探讨人工养殖条件下体重、年龄和环境应激对菲牛蛭繁殖性能的影响。结果表明,体重对卵茧大小和湿质量及幼蛭体长、体宽和湿体质量无显著影响(P>0.05),而亲蛭性腺系数、平均产茧数、孵化率和平均孵化数在各组间差异显著(P<0.05),其中体重10～15g组明显高于其他组(P<0.05);年龄对亲蛭平均产茧数、孵化率、平均孵化数和卵茧大小影响显著(P<0.05),其中2～3年龄亲本具有最高的平均产茧数、孵化率和平均孵化数,8月龄亲本所产卵茧个体明显大于其他年龄组(P<0.05),而卵茧湿质量及幼蛭体长、体宽和湿体质量在各年龄组间均无显著差异(P>0.05)。环境应激对亲蛭的平均产茧数、孵化率和平均孵化数影响显著(P<0.01),其中1～2次操作应激下的平均产茧数和孵化数最高,无操作刺激下的孵化率略高于1～2次操作刺激(P>0.05),而卵茧大小和湿质量及幼蛭体长、体宽和湿体质量在各应激处理组间无显著差异(P>0.05)。本研究获得了菲牛蛭繁殖的最佳体重和年龄,建议繁育生产中挑选10～15g和2～3年龄的个体作为亲本,期间进行1～2次操作刺激可提高其繁殖性能。     

The role of spider cocoons in controlling desiccation

Summary The abilities of the cocoons of the spiders Mecynogea lemniscata and Argiope aurantia to protect the enclosed egg and spiderling stages from desiccation were investigated in the laboratory under controlled humidities, and in the field under ambient conditions. For M. lemniscata, which has a relatively small clutch (8–30 eggs) and remains in the cocoon for approximately 9–10 months, removal of the cocoon had no effect on water loss from the egg stage, nor did it adversely affect hatching or molting success. Cocoon removal did, however, significantly affect water loss and, consequently, survival in the spiderling stage at all humidities in the laboratory and in the field. The importance of the cocoon for survival is probably related to the unusually long time M. lemniscata spiderlings spend in the cocoon overwintering. For A. aurantia, which has a substantially larger clutch size (300–1400 eggs) and remains in the cocoon for a shorter 6–7 months, cocoon removal had no effect on water loss, egg hatching success, molting success, nor spiderling survival. The lack of an effect suggests that other factors (e.g., relative humidity at the oviposition site, or a large clutch size) may be more important in controlling water loss for A. aurantia.     

The ultrastructure of malpighian tubules and the chemical composition of the cocoon of Aeolothrips intermedius Bagnall (Thysanoptera)

The secretory activity of the two branched malpighian tubules (MTs) of the second‐instar larva in Aeolothrips intermedius is described. MTs of adult thrips have the typical ultrastructure of excretory epithelium with apical microvilli containing long mitochondria and a rich system of basal membrane infoldings. In the second‐instar larva just before pupation, the ultrastructure of MT epithelial cells is dramatically different, and there are numerous huge Golgi systems in the cytoplasm. These cells are involved in an intense secretory activity to produce an electron‐dense product which is released into the MTs lumen. This secretion is extruded from the hindgut and used by the second‐instar larva to build an elaborate protective cocoon for pupation. Electron‐spray‐ionization mass spectrometry analysis of the cocoon revealed the presence of a β‐N‐acetyl‐glucosamine, the main component of chitin, which is also present in the cocoons of Neuroptera and some Coleoptera. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Irregular helicoids in leech cocoon membranes

Helicoids in the cocoon membrane of leeches Theromyzon tessulatum and Erpobdella punctata comprise a twisted superposition of layers, each containing a variable number of planes formed by unidirectional fibrils. Straight fibrils intersecting at different angles were displayed in tangential sections through the cocoon wall of each species. When the sectioning angle was below a certain value (i.e., the critical angle), bow-shaped lines apparent in oblique sections were replaced by a succession of layers containing straight fibrils, permitting a direct measurement of step-angle change between successive layers in a helicoid. By this methodology, we determined that no regularities exist in the succession of step-angles or in layer thicknesses within the cocoon membranes, but that the distribution of step-angles between layers was unique for each cocoon type.     

Physiology of cold hardiness in cocoons of five earthworm taxa (Lumbricidae: Oligochaeta)

Earthworm cocoons are mostly found in the uppermost soil layers and are therefore often exposed to low temperatures during winter. In the present study, cocoons of five taxa of earthworms were investigated for their tolerance to freezing, melting points of cocoon fluids and dehydration of cocoons when exposed to a frozen environment. Embryos of the taxa investigated were freeze intolerant. The melting points of fully hydrated cocoon fluids were high (above –0.3°C) and thermal hysteresis factors were absent. Exposure to a frozen environment caused the cocoons to dehydrate drastically and dehydrated cocoons showed significantly lower super-cooling points than fully hydrated cocoons, reducing the risk of freezing for dehydrated cocoons. It is proposed therefore that the cold-hardiness strategy of the earthworm cocoons is based on dehydration upon exposure to subzero temperatures in the frozen environment. Cocoons of three surface-dwelling taxa, Dendrobaena octaedra, Dendrodrilus rubidus tenuis and Dendrodrilus rubidus norvegicus had lower supercooling points and survived frost exposure better than cocoons of two deeper-dwelling taxa, Aporrectodea caliginosa and Allolobophora chlorotica. One of the investigated taxa, D. r. norvegicus, was collected from a cold alpine habitat. However, it was not more cold hardy than the closely related D. r. tenuis collected from a lowland temperate habitat. D. octaedra was the most cold hardy taxon, its cocoons being able to withstand –8°C for 3 months and –13.5°C for 2 weeks in frozen soil.Abbreviations dw dry weight - fw fresh weight - SCP supercooling point     

Morphology,egg cocoons,and transmission paths of the Antarctic leech Glyptonotobdella antarctica Sawyer and White, 1969 (Hirudinea: Rhynchobdelliformes: Piscicolidae)

The biology of piscicolids which move between different host species is poorly understood. Very little is known about leeches of Antarctic waters, though they are not uncommon in that region. This paper adds to the fundamental knowledge of the Antarctic piscicolid leech Glyptonotobdella antarctica Sawyer and White, 1969. According to the new findings the distribution of the species has to be extended to the geographical latitude of 76°08.1′S and to a depth of 665 m. Besides Glyptonotus antarcticus Eights 1853, sea urchins of the genus Sterechinus (Meissner 1900), and the benthic octopus Pareledone (charcoti?) (Joubin 1905) serve as hosts. Hitherto there were only two records on piscicolids from Octopus dofleini and none from sea urchins. Egg cocoons on the ventral side of the isopod Glyptonotus antarcticus were attributed to the leech. Most likely Glyptonotobdella antarctica moves between different hosts, i.e. Antarctic octopuses and their potential prey. Surface structures of the leech are studied by light and scanning electron microscope. The observations complement the basic knowledge on Antarctic leeches and provide arguments for phylogenetic discussions.     

Facts and myths of antibacterial properties of silk

Silk cocoons provide protection to silkworm from biotic and abiotic hazards during the immobile pupal phase of the lifecycle of silkworms. Protection is particularly important for the wild silk cocoons reared in an open and harsh environment. To understand whether some of the cocoon components resist growth of microorganisms, in vitro studies were performed using gram negative bacteria Escherichia coli (E. coli) to investigate antibacterial properties of silk fiber, silk gum, and calcium oxalate crystals embedded inside some cocoons. The results show that the previously reported antibacterial properties of silk cocoons are actually due to residues of chemicals used to isolate/purify cocoon elements, and properly isolated silk fiber, gum, and embedded crystals free from such residues do not have inherent resistance to E. coli. This study removes the uncertainty created by previous studies over the presence of antibacterial properties of silk cocoons, particularly the silk gum and sericin. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 237–245, 2014.     

Protease inhibitors in Bombyx mori silk might participate in protecting the pupating larva from microbial infection

Pupae inside cocoons rarely suffer from disease. It is apparent that some factors in the cocoon exert antimicrobial effects whereby the pupae inside can be protected from microbial infection. In the present study, we investigated the expression of cocoon protease inhibitors using immunoblotting and activity staining. Enzymatic hydrolysis of cocoon proteins in vitro was performed to characterize their roles in protecting the cocoon from microbial proteases. We found that some protease inhibitors, particularly trypsin inhibitor‐like (TIL)‐type protease inhibitors, can be secreted into the cocoon layer during the spinning process, thereby providing effective protection to the cocoon and pupa by inhibiting the extracellular proteases that can be secreted by pathogens.     

Cretaceous and living colloniidae of the redefined subfamily Petropomatinae,with two new genera and one new species,with notes on opercular evolution in turbinoideans,and the fossil record of Liotiidae (Vetigastropoda: Turbinoidea)

Opercular characters are of major importance to understanding the living and fossil groups in the vetigastropod superfamily TurbinoideaRafinesque. Here we evaluate current knowledge of fossil turbinoid opercula in the light of a newly discovered Recent colloniid with an unfamiliar opercular morphology. The operculum ofLiotipoma n. gen. has a multispiral and conical inner surface; the outer surface is rugose with a central pit. Similar opercula are known from the Late CretaceousSohlipoma n. gen. and the Early CretaceousPetropoma Gabb. These genera are assigned to the here redefined colloniid subfamily PetropomatinaeCox inKnight et al., which was based originally onGabb’s misinterpretation of the inner and outer sides of the operculum ofPetropoma peruana Gabb. In contrast, opercula of most living members of Colloniinae s.S., have a flat inner side with a multispiral pattern that becomes paucispiral. In addition, opercula typical for living Turbinidae are here traced to the Late Cretaceous. The new genusLiotipoma has lamellar sculpture like that of Liotiidae H. & A.Adams, showing that such sculpture is not restricted to the Liotiidae. We also reject previous allocations of the Permian DichostasiinaeYochelson and Jurassic Cross-ostomatinaeCox inKnight et al. to Liotiidae, and find an undisputed earliest known Jurassic occurrence for the Liotiidae. The recent suggestion that the change from corneous to calcareous opercula among turbinids occurred concurrently with a change in pattern from multispiral to paucispiral is not supported by our data.     

The Natural Feeding and Resource Partitioning of Erpobdella octoculata L. and Erpobdella testacea SAV. in Lake Esrom,Denmark

The natural feeding and competitive interactions between populations of Erpobdella ostoculata and Erpobdella testacea were investigated in Lake Esrom, Denmark, during 1979/80. Statistical analysis revealed that the size of the leeches was the main factor involved in resource utilization. Size-specific feeding included differences in the numerical composition of diets and the size of individual prey. Five weight classes of Erpobdella spp. were distinguished. The number of prey per leech increased from 1.63-2.84 for the first to the fifth weight class, whereas diversity in feeding averaged 1.71 prey per leech. Quantification of competitive interactions indicated that the temporal differences in weight class composition of the two species were sufficient to explain coexistence, although both species have an annual life cycle, and neither species showed taxonomic food refuges.     

The influence of body size on immediate reproductive success in Dugesia gonocephala (Tricladida,Paludicola)

The individual reproductive output of the stream-dwelling flatworm Dugesia gonocephala was investigated. Various measures of reproductive success were related to body size. (I) For the first 30 days in the laboratory small individuals produced no cocoons, individuals of intermediate size produced unfertilized cocoons and large individuals usually produced fertilized cocoons. (II) In individuals that produced a cocoon, no correlation was found between the number of cocoons produced in one month and body size. (III) Large individuals, however, produced larger cocoons. This was not due to the fact that unfertilized cocoons were smaller. (IV) Large cocoons tended to contain more young. (V) The average size of young hatching from large cocoons was larger. (VI) Large individuals produced their first cocoon soon after their arrival in the laboratory and seemed to have a higher chance of producing a fertilized first cocoon. (VII) A trade-off existed between producing many small versus few large young.     

Biological studies upon Enchytraeus variatus Bouguenec & Giani 1987 in breeding cultures

The life history of an enchytraeid worm, Enchytraeus variatus, was studied under laboratory conditions at 18–22 °C. This species can reproduce simultaneously by asexual (architomy) or sexual reproduction. The number of ova per cocoon varies from 5 to 20 (x = 10.9). The generation period (from cocoon to next cocoon) varies from 14 to 39 days (x = 26.1) according to the period of the year. The number of generations per year is between 7.3 and 26.1 (x = 14). A mature worm can lay between 23.7 and 25.8 cocoons during its life (254 days as maximum observed) at a mean rate of 0.12 cocoon worm^–1 day^–1. Experimental cultures were carried out to determine the structure, density and biomass of the populations. A maximal density of 1 396 314 worms was recorded after 85 days of culture. Net production reached 21.48 g m^–2 day^–1 after 26 days in a culture initiated from cocoons.     

The effects of feeding regime on the growth and reproduction of the medicinal leech Hirudo medicinalis

1. The feeding frequency, the size of meals, the number of meals required to attain reproductive maturity and the number of meals taken between iteroparous reproductive bouts were determined in the laboratory under optimal conditions for the medicinal leech Hirudo medicinalis fed exclusively on mammalian (bovine) blood. In addition the number of bouts of reproduction and the numbers of cocoons and hatchlings per cocoon produced were determined.
2. The average time for H. medicinalis to reach reproductive maturity at 20°C was 289 days, at an average wet biomass of 8143 mg with two–nine separate bouts of cocoon production. The number of meals to first reproduction was 8.9 (mean meal size of 3066.7 mg), with a significant correlation between total mass of blood ingested and the numbers of reproductive bouts and number of cocoons produced. Mean lifetime cocoon production per individual was 12.43, with 3.9 hatchlings per cocoon.
3. The significant positive relationships between ingestion, fecundity and developmental rate observed support the hypothesis that declining abundances of field populations of H. medicinalis are the result of lower available energy for growth, reflecting leeches now feeding predominantly on amphibian blood of lower energetic value than mammalian blood.