Mannan-degrading enzymes purified from the crop of the brown garden snail Helix aspersa Müller (Gastropoda Pulmonata)

Two mannan-degrading enzymes were purified from the crop of the terrestrial snail Helix aspersa Müller. The crude extracts were taken from dormant (for 4 months) snails. The enzymes were a betaD-mannanase of 37.4 +/- 0.3 kDa (EC 3.2.1.78) and a betaD-mannosidase of 77.8 +/- 1.9 kDa (EC 3.2.1.25). Both enzymes degraded insoluble mannan, releasing either mannose only (beta-mannosidase) or oligosaccharides, possibly mannotriose and mannopentaose (beta-mannanase). The beta-mannanase had a typical endo-activity pattern, while the beta-mannosidase was an exoenzyme. The incubation of both enzymes with mannan increased the catalysis by 83%. The best synergy was found with 75% mannosidase combined with 25% mannanase. The beta-mannanase also hydrolysed beta-linked heteromannans and its affinity for different galactomannans was studied. The Km values, varying from 2.89 +/- 0.47 mg x ml(-1) to 0.26 +/- 0.01 mg x ml(-1), revealed the inhibitory effect of the alphaD-galactosyl residues released. The beta-mannosidase was acidic (optimum pH = 3.3) and heat-sensitive (50% residual activity at 42 degrees C after 5 min of pre-incubation), while the beta-mannanase remained stable until 48.5 degrees C (50% residual activity) and over a pH range of 4.3-7.5. The properties of these mannanolytic enzymes are discussed in this paper compared with those purified in other gastropods and in a bacterium, Enterococcus casseliflavus, a quite similar strain previously isolated from this snail intestine. The occurrence of thermostable enzymes in H. aspersa digestive tract could be a zootechnic parameter of great importance for snail farming. J. Exp. Zool. 290:125-135, 2001.     

Biological studies and phenology of the slug Deroceras reticulatum (Müller, 1774) (Pulmonata: Stylommatophora)

Summary

In the southeast of Buenos Aires Province (Argentina), slugs cause reductions in crop yield by killing seeds and seedlings, by destroying stems and growing points, and by reducing leaf area. Deroceras reticulatum (Müller, 1774) was introduced into Argentina a long time ago, but the biology and ecology of this species in this country are still unknown. The aim of this study was to determine how temperature affects growth rate, reproduction and survival of D. reticulatum and also to assess the seasonal phenology of this species. The life cycle of D. reticulatum was investigated at 12°C, 20°C and at a temperature which alternated between 12°C for 16 h and 20°C for 8 h. The light/dark regime during the experiment was LD: 8/16 h. Slug populations were sampled from grassland located at the Experimental Station of INTA Balcarce. Eggs hatched after 16.3 to 39.8 days and fertility varied between 83.9% and 91.1%. The mean time from hatching to oviposition was longer and fecundity lower at 20°C than 12°C and 12/20°C (p <0.05). The net reproductive rate was 1.49 to 70.53 and the mean generation time varied between the 27 and 30 weeks. In natural conditions, eggs of D. reticulatum were found from early winter until the end of the spring, when soil temperature was 8–15°C and humidity was above 15%. These results indicate that D. reticulatum has one generation per year, univoltine phenology, with peak densities of slugs from the beginning of winter to the end of spring, which coincides with the sowing and emergence of winter and summer crops.     

General morphology and ultrastructure of the radula of Testudinalia testudinalis (O. F. Müller, 1776) (Patellogastropoda,Gastropoda)

The radular morphology of the patellid species Testudinalia testudinalis (O. F. Müller, 1776) from the White Sea was studied using light, electron, and confocal microscopy. The radula is of the docoglossan type with four teeth per row and consisting of six zones. We characterize teeth formation in T. testidinalis as follows: one tooth is formed by numerous and extremely narrow odontoblasts through apocrine secretion; this initially formed tooth consists of numerous vesicles; the synthetic apparatus of the odontoblasts is localized in the apical and central parts of the cells throughout the cytoplasm and is penetrated by microtubules which are involved in the transport of the synthesized products to the apical part of the odontoblast; the newly formed teeth consist of unpolymerized chitin. Mitotic activity is located in the lateral parts of the formation zone. The first four rows contain an irregular arrangement of teeth, but the radular teeth are regularly arranged after the fifth row. The irregularly arranged teeth early on could be a consequence of the asynchronous formation of teeth and the distance between the odontoblasts and the membranoblasts. The morphological data obtained significantly expands our knowledge of the morphological diversity of the radula formation in Gastropoda.     

Effects of tissue extract of adults on metamorphosis in Ascidia mentula O.F. Müller and Ascidiella scabra (O.F. Müller)

Unhatched, but fully developed larvae of two solitary ascidians Ascidia mentula O.F. Müller and Ascidiella scabra (O.F. Müller) were incubated in a variety of adult tissue extracts, which an earlier study had shown to accelerate metamorphosis. Our study confirmed this observation and showed, furthermore, that tissue extracts induce tail resorption in unhatched larvae of Ascidiella. For both species, the most active induction of metamorphosis was obtained with tunic tissue extracts. The results indicate that chemical stimulation without the presence of tactile cues may initiate metamorphosis. We suggest that the ability of tunic extracts to induce metamorphosis may explain juvenile establishment on adult ascidians and their aggregated distribution found in nature.     

Habitat preference of freshwater snails in relation to environmental factors and the presence of the competitor snail Melanoides tuberculatus (Müller, 1774)

Our objective is to evaluate the habitat preference of freshwater snails in relation to environmental factors and the presence of the competitor snail Melanoides tuberculatus. In the first phase, snails was collected at 12 sites. This sampling sites presented a degree of organic input. In the second phase 33 sampling sites were chosen, covering a variety of lotic and lentic environments. The snail species found at Guapimirim, state of Rio de Janeiro, displayed a marked habitat preference, specially in relation to the physical characteristics of each environment. Other limiting factors for snail distribution at the studied lotic environments were the water current velocity and the amount of organic matter, mainly to Physa marmorata, M. tuberculatus, and Biomphalaria tenagophila. The absence of interactions between M. tuberculatus and another snails could be associated to the distinct spatial distribution of those species and the instability of habitats. This later factor may favor the coexistence of M. tuberculatus with B. glabrata by reduction of population density. In areas of schistosomiasis transmission some habitat modification may add to the instability of the environment, which would make room for the coexistence of M. tuberculatus and Biomphalaria spp. In this way, some of the usual measures for the control of snail hosts would prevent the extinction of populations of Biomphalaria spp. by M. tuberculatus in particular habitats.     

Some characteristics of genetic variants of Tubifex tubifex (Müller, 1774) (Oligochaeta: Tubificidae) in laboratory cultures

Genetic variants of the oligochaete Tubifex tubifex were identified with enzyme electrophoresis and subsequently reared in laboratory cultures. Three types are abundant in field populations. Individuals that show homozygotic bands of glucosephosphate-isomerase (GPI) 22 together with isocitrate-dehydrogenase (IDH) 35 were labeled Type A. Type B is characterized by GPI 23 together with IDH 11 and Type C is characterized by GPI 11 with either IDH 34 or IDH 33. Initial results on freshweights of adults and cocoon production revealed differences between the two main types, A and B. In the same period, Type B reached higher weights and produced five times as many cocoons as Type A, whereas number of eggs per cocoon were not different between these Types. Type B also had the lowest mortality in 16-week experiments with changing temperatures.     

Ecological and morphological differentiation among cryptic evolutionary lineages in freshwater limpets of the nominal form-group Ancylus fluviatilis (O.F. Müller, 1774)

The phylogeny and potential mode of speciation of the river limpet Ancylus fluviatilis (Basommatophora) was examined using mitochondrial DNA sequences from 16S ribosomal RNA, cytochrome c oxidase subunit I (COI) and nuclear DNA from internal transcribed spacer (ITS-1) regions from 103 populations across Europe. Four highly divergent lineages were observed within Ancylus. Clade 1, representing the nominal taxon Ancylus fluviatilis (O.F. Müller, 1774), is mainly found in central and northern Europe, Clade 2 is present in a single Portuguese population, Clade 3 is distributed on the Canary islands, North Africa and the eastern Mediterranean region, whereas Clade 4 inhabits the Northern Mediterranean coasts. Phylogenetic analyses revealed an overall consistent topology of nuclear and mitochondrial gene trees. Based on a molecular clock, we estimated that the basic radiation occurred in the late Pliocene. Although clades differ significantly in size independent shell shape, morphological differentiation of lineages is not feasible without genetic data. Environmental data related to climate (precipitation, temperature, etc.) showed a significant differentiation of clades. Clade 1 dwells in relatively colder and more stable habitats than Clades 3 and 4, whose habitats in turn differ in a low or high amount of precipitation during spring and autumn, respectively. Based on the combined data sets on mitochondrial DNA, nuclear DNA, morphological and ecological differentiation, we conclude that Ancylus represents a cryptic species complex of reproductively and genetically isolated lineages. In addition, the joint analysis suggests that ecological speciation is probable to explain current patterns.     

Distribution pattern of the dinoflagellateCeratium hirundinella (O. F. Müller) Bergh in a reservoir

Horizontal distribution of the dinoflagellateCeratium hirundinella (O. F. Müller) Bergh in the Ishitegawa Reservoir, Ehime Prefecture, Japan, was investigated. Water quality was also surveyed. It was observed that the population ofC. hirundinella exponentially decreased in number from the head of the reservoir to the dam site. Further investigation proved thatC. hirundinella initiated growth at the head of the reservoir, and later gradually expanded downstream. It was found during the period of increase in water temperature that the cell density ofC. hirundinella at the uppermost station exponentially increased.     

Observations on the clypeasteroid Echinocyamus pusillus (O. F. Müller)

The tiny echinoid Echinocyamus pusillus (O. F. Müller) is equipped with specialized external structures that suit it for a wide variety of environments. Special features include the ability to burrow in sediments of fine sand to shell gravel and to climb vertically.Specimens dredged off the west coast of Scotland were observed in aquaria and with the SEM. E. pusillus is characterized by three kinds of spines, and by two of pedicellariae. In contrast to sand dollars, the spines play a passive rôle in the feeding and burrowing operation, probably retaining a defensive nature as in the regular urchins. It is the podia that are chiefly involved in climbing, burrowing, righting, and probably feeding. Surface ciliary currents transport particles, but not to the mouth; they may have a respiratory or cleansing function. Experimental animals did not burrow in either very fine or very coarse sand, probably because a certain relationship exists between particle weight and podia size.E. pusillus shares behavioural and structural characteristics with regular and irregular urchins. It is not a true sand dollar, but may illustrate an evolutionary stage towards such a form.     

Physiological adaptations in the survival of rotifers (Brachionus plicatilis,O. F. Müller) at low temperatures

The biology of the rotifer Brachionus plicatilis has been studied extensively in recent years, due to its importance in aquaculture. Today, the culture of several marine fish species relies completely on the daily production of live rotifers.In the present paper we explore the conditions that facilitate maintaining live rotifers for extensive periods at low temperatures. In addition to its possible contribution in providing reserve stocks for commercial application, these studies may be of ecological importance. They could explain some of the physiological adaptive mechanisms that are involved in the survival of rotifers under adverse environmental conditions.Experimental results showed relatively high survival rates (82–85%) in rotifers that were cultured at 25 °C and exposed later to -1 °C for 12–14 days. During this period, rotifers were kept without food and their media were not changed. The survival was found to depend on the rotifer culture conditions, prior to exposure to -1 °C. These included the type of food fed to rotifers (yeast or algae), the salinity in which they were cultured, and an essential acclimation period of 2–6 day at 4 °C or 10 °C. The acclimation period was associated with the synthesis of at least one specific protein and accumulation of lipids. Profiles of protein synthesis in rotifers incubated at 10 °C revealed a 94 kD protein, which did not appear in rotifers cultured at 25 or 37 °C. Immunoisolation, using a polyclonal antibody that was prepared against HSP60, revealed that this protein was synthesized in rotifers kept at 10, 25 or 37 °C. However, this antibody did not react with the 94 kD peptide.In addition, rotifers kept at 10 °C accumulated substantial amounts of lipids, including eicosapentaenoic acid (EPA), which is found in the algae fed to them. These results support the hypothesis of specific adaptations to survival at low temperatures during an acclimation period.     

Population dynamics and estimation of production in Bosmina longirostris (O. F. Müller) in the River Danube (Danubialia Hungarica,CVIII)

Analysis of the population dynamics of Bosmina longirostris (O. F. Müller) was carried out in the River Danube in the years 1981 and 1982. An estimation method of secondary production elaborated far standing waters was adapted to river conditions. In the River Danube, compared to lakes, standing crop and production values were smaller, while fecundity, P/B values were larger and turnover time was shorter. Possible reasons for this are discussed.     

Short-term feeding of Temora longicornis Müller in the laboratory and the field

Short-term feeding (min to h) of the copepod Temora longicomis (Müller) was investigated in the laboratory and in the field by monitoring the increase in gut fluorescence of adult females. Laboratory feeding studies using the diatoms Thalassiosira pseudonana (Hust.), T. weissflogii (Hust.) and Ditylumbrightwellii (West) showed that ingestion rates increased with increased food concentration and cell size. The maximum ingestion rate was 9.5 ng chl. equiv. female ⁻¹. h ⁻¹ on a diet D. brightwellii. Clearance rates decreased with increased food concentration and decreased cell size. Maximum clearance rate was 1.9 ml. female⁻¹·h⁻¹ also on a diet of D. brightwellii. On two of three occasions, ingestion and clearance rates in the field were twice as high at night as during the day. Night rates were similar to laboratory rates in the chlorophyll range found in the field. Diel differences in feeding activity in the field appeared to have been behaviorally induced rather than being related to vertical migration of individuals into food rich layers.     

The population density effects on the reproductive biology of the snail Bradybaena similaris (Férussac, 1821) (Mollusca, Gastropoda)

The influence of population density on some aspects of the reproductive biology of the snail Bradybaena similaris was studied. Molluscs were maintained under 0.2 (isolated), 0.3, 0.6, 1.0, 1.3 and 1.7 snail/m(2) densities. The animals maintained under 0.3 and 0.6 snail/m(2) showed the lowest numbers of eggs laid/snail, being the highest value observed to the 1.7 snail/m(2). The hatching of the snails maintained under 0.3 snail/m(2) density, begun at the 21st day after laying, and the maximum time required to the hatching was 36 days was observed to the eggs came from snails maintained under the densities 0.6, 1.0, 1.3 snail/m(2), respectively. The highest percentage hatchability (55.56%) was observed to isolated snails. The galactogen content in the albumen gland did not seem to accompany the alterations occurred in the reproduction of B. similaris in response to the different population densities.     

Micro-structure and chemical composition of vateritic deformities occurring in the bivalve Corbicula fluminea (Müller, 1774)

Vateritic deformities occurring in the invasive heterodont bivalve Corbicula fluminea from several locations in the UK were characterised in detail for the first time using scanning electron microscopy, X-ray diffraction and different geochemical techniques (electron microprobe, ICP-AES, and mass spectrometry). Large volumes of vaterite are produced abnormally in the animals' shells in the form of yellow-green bulges. These are distinguished from the aragonitic parts of the shell by their characteristic micro-structures, content of organic material, trace elemental composition and carbon stable isotope signatures. The most commonly observed micro-structures include columnar vaterite, lamellar vaterite and different irregular structures occurring in all parts of the shell. There are indications that organic material is present largely as intracrystalline impurities or nano-scale phases and not as envelopes around microstructural units. These micro-structures are novel, nothing equivalent having yet been described for other vateritic systems. Euhedral vaterite crystals also occur occasionally. The vaterite has generally higher Mg/Ca and lower Na/Ca, K/Ca than the aragonite. In addition, δ13C is also always lower. Microstructural characteristics would suggest loss of biological control probably due to physiological stress(es) inducing the switch to vaterite production. The vaterite might be stabilised by its higher content of organic material and magnesium.     

The mitochondrial genome of the land snail Camaena cicatricosa (Müller, 1774) (Stylommatophora,Camaenidae): the first complete sequence in the family Camaenidae

The complete mitochondrial (mt) genome of the snail Camaena cicatricosa (Müller, 1774) has been sequenced and annotated in this study. The entire circular genome is 13,843 bp in size and represents the first camaenid mt genome, with content of 31.9%A, 37.9%T, 13.5%C and 16.7%G. Gene content, codon usage and base organization show similarity to a great extent to the sequenced mt genome from Stylommatophora, whereas, gene order is different from them, especially the positions of tRNA^Cys, tRNA^Phe, COII, tRNA^Asp, tRNA^Gly, tRNA^His and tRNA^Trp. All protein coding genes use standard initiation codons ATN except for COII with GTG as start signal. Conventional stop codons TAA and TAG have been assigned to all protein coding genes. All tRNA genes possess the typical clover leaf structure, but the TψC arm of tRNA^Asp and dihydrouridine arm of tRNA^Ser(AGN) only form a simple loop. Shorter intergenic spacers have been found in this mt genome. Phylogenetic study based on protein coding genes shows close relationship of Camaenidae and Bradybaenidae. The presented phylogeny is consistent with the monophyly of Stylommatophora.