Ultrastructural analysis of the morphology and function of the spermatheca of the pulmonate snail,Sonorella santaritana

The ultrastructure of the spermatheca of the reproductive tract in the pulmonate snail, Sonorella santaritana, was investigated. This organ has a debris-filled lumen and an outer wall which can be divided into three distinct layers. The cell layer adjacent to the lumen is comprised of two cell types, tall columnar epithelial cells with microvilli and cells lacking microvilli. The next layer also has two cell types, muscle cells and apparent pigment cells. The most distant layer is an adventitia of large glycogen-containing cells. The lumen of the spermatheca contains a core of partially digested sperm and related materials. The luminal contents and the cellular morphology of this organ suggest that the spermathecal functions are both digestive and absorptive. It is proposed that excess sperm and related materials are transported to the spermatheca, digested, and the usable products are reabsorbed.     

Molecular analysis of predation by carabid beetles (Carabidae) on the invasive Iberian slug Arion lusitanicus

The invasive Iberian slug, Arion lusitanicus, is spreading through Europe and poses a major threat to horticulture and agriculture. Natural enemies, capable of killing A. lusitanicus, may be important to our understanding of its population dynamics in recently invaded regions. We used polymerase chain reaction (PCR) to study predation on A. lusitanicus by carabid beetles in the field. A first multiplex PCR was developed, incorporating species-specific primers, and optimised in order to amplify parts of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of large Arion slugs, including A. lusitanicus from the gut contents of the predators. A second multiplex PCR, targeting 12S rRNA mtDNA, detected predation on smaller Arion species and the field slug Deroceras reticulatum. Feeding trials were conducted to measure the effects of digestion time on amplicon detectability. The median detection times (the time at which 50% of samples tested positive) for A. lusitanicus and D. reticulatum DNA in the foreguts of Carabus nemoralis were 22 h and 20 h, respectively. Beetle activity-densities were monitored using pitfall traps, and slug densities were estimated using quadrats. Predation rates on slugs in the field by C. nemoralis in spring ranged from 16-39% (beetles positive for slug DNA) and were density dependent, with numbers of beetles testing positive being positively correlated with densities of the respective slug species. Carabus nemoralis was shown to be a potentially important predator of the alien A. lusitanicus in spring and may contribute to conservation biological control.     

The involvement of opioid peptides in stress-induced analgesia in the slug Arion ater

Application of tail-pinch stress to the terrestrial slug, Arion ater, produced a significant increase in the response time when tested on the hot-plate for foot-lifting response. The analgesia was completely reversed by injections of the opiate antagonists, naltrexone and ICI 174864, in a dose-dependent manner. Analgesia could also be elicited by the injection into the foot of beta-endorphin and the enkephalin analogues, DAGO and DADLE. No effect was seen with dynorphin A (1-8) or dynorphin A (1-17). The stress-induced analgesia disappeared after 30 minutes but could be maintained for 100 min following the injection of a mixture of bestatin and the enkephalinase inhibitor, N-carboxymethyl-L-phenylalanyl-L-leucine. This work suggests that in the slug, a physical stressor produces an analgesia which may be due to the release of endogenous opiates.     

Low temperature survival in different life stages of the Iberian slug, Arion lusitanicus

The slug Arion lusitanicus Mabille (Gastropoda: Pulmonata: Arionidae) is an invasive species which has spread to most parts of Europe. The area of origin is unknown, but A. lusitanicus seems to cope well with the local conditions in the countries to which it has migrated. It spreads rapidly, occurs often in high densities and has become a serious pest in most European countries. Therefore there is an urgent need for better knowledge of the ecophysiology of A. lusitanicus, such as the influence of climatic conditions, in order to develop prognostic models and strategies for novel pest management practises.The aim of our study was to investigate the influence of subzero temperatures in relation to winter survival. A. lusitanicus is shown to be freeze-tolerant in some life stages. Most juveniles and some adult slugs survived being frozen at −1.3 °C for 3 days, but none of the slugs survived freezing at −3 °C. The eggs survived subzero temperatures (down to −2 °C) probably by supercooling. Juveniles and adults may also survive in a supercooled state (down to −3 °C) but are generally poor supercoolers. Therefore, the winter survival of A. lusitanicus depends to a high degree on migration to habitats protected from low winter temperatures, e.g. under plant litter, buried in the soil or in compost heaps.     

The modulation of rhythmic behaviour in the pulmonate slug Limax pseudoflavus by season and photoperiod

Freshly caught samples of slugs were maintained at constant temperature and humidity but at day lengths (from 5 h 50 min to 16 h darkness) appropriate to the time of year at which they were collected. The duration of their feeding and crawling activities as well the total time spent active was recorded by time-lapse cinematography.
Total activity was significantly higher in autumn and spring than in summer and winter. The activity period was symmetrically disposed about the midpoint of the dark period except for two samples in autumn. At scotoperiods longer than about 12 h, activity onset was delayed relative to the start of the dark period and activity cessation was advanced relative to the end of the period, resulting in the short duration of activity seen in winter. At scotoperiods of less than 12 h the duration of activity was relatively independent of scotoperiod.
In spring the total time spent crawling was composed of relatively few bouts of long duration whereas in the autumn the number of bouts was greater and their duration shorter. The winter was characterized by slugs making single journeys to and from the food. While emerged these slugs stayed in close association with the food but not necessarily feeding on it.
These data are discussed in terms of comparisons with homoiothermic nocturnal animals. It is concluded that seasonal responses in poikilothermic animals are likely to be limited by the hazards of winter even when such animals are maintained in apparently non-limiting conditions.     

Functional morphology of the neuropeptidergic light-yellow-cell system in pulmonate snails

The light yellow neuropeptidergic cell system of the basommatophoran snail Lymnaea stagnalis is homologous to the R3-R14 system of the opisthobranch Aplysia californica, and produces three different neuropeptides. Systems homologous to the light yellow cells of Lymnaea stagnalis have been investigated morphologically in two Basommatophora (Lymnaea ovata, Bulinus truncatus) and three Stylommatophora (Helix aspersa, Cepaea nemoralis, Deroceras reticulatum). To this end, an antibody to synthetic light-yellow-cell peptide-II and oligonucleotides to mRNAs encoding parts of peptide-I and peptide-III, were used. The in situ hybridization probes gave negative results. On the other hand, neuronal cell clusters were observed in the central nervous system of all specias studied by immunocytochemistry. These clusters were located in the ganglia of the visceral complex. The neurons project axons into all nerves of these ganglia, especially into the pallial nerves, into the connective tissue of the central nervous system, and into the neuropile of various ganglia. The morphology of the systems is similar to that of the light-yellow-cell system of Lymnaea stagnalis. In all species, the wall of the aorta was innervated by immunoreactive axons. Peripheral innervation by the light-yellow-cell system was investigated in Helix aspersa and Deroceras reticulatum. Serial and alternate sections of whole snails were studied. Reconstructions were made of the heart-kidney-lung complex of these animals. In both species, the muscular vessels of the pulmonary system at the right side of the body were strongly innervated by immunoreactive axons. Furthermore, immunopositive innervation was observed to muscles in the secondary ureter-pneumostome area. The light-yellow-cell system of pulmonates is thus probably involved in the regulation of blood pressure and urine release.     

Effect of chronic and acute lead treatment in the slug Arion ater on calcium and delta-aminolaevulinic acid dehydratase activity

Accumulation of lead in the tissues was greater after acute exposure. Most of the metal was deposited in the intestine and least in foot. After chronic exposure to lead the calcium content of the intestine decreased. After chronic and acute exposure to lead the calcium content of the digestive gland and foot was lower than control values. Lead in the digestive gland granules was higher after acute lead treatment. Less than 45% of the lead was associated with the granules. These results are discussed in view of the difference in metabolism and chemical nature of the organs investigated. ALAD activity was located in the digestive gland and it was reduced after acute treatment with lead.     

Ultrastructural morphology of the genusKineosporia

The ultrastructure ofKineosporia aurantiaca was investigated with phosphotungstic acid negative staining and thin sectioning techniques. Motile spores were observed, each having a polar tuft of flagella. Thick-walled somatic hyphae displayed prokaryotic morphologies. Sporangia were seen having a single spore with no evidence of spore segmentation or branching. The findings were consistent with the light microscopic observations of Pagani and Parenti.     

Comparison of spermatheca morphology between reproductive and non-reproductive females in social wasps

Social wasps show an obvious evolution of the differentiation in behavior and external size between reproductive and non-reproductive females, with no clear differences in the Stenogastrinae, via overlap in the Polistinae, to clear differences in the Vespinae. In this study, we examined the morphological appearance of the spermatheca in representative species of these three subfamilies. The general anatomical organization of the spermatheca comprises a reservoir, a duct and two spermathecal glands, and is in line with its common structure in other social Hymenoptera. All examined wasp species have a spermathecal reservoir with uniform wall thickness, which is similar to the situation in the bees, but differentiates them from the ants. Within the wasps, the shape of the reservoir, the shape of the spermathecal glands and their attachment site to the spermatheca differs among the Stenogastrinae, Polistinae and Vespinae. The reservoir wall is thick in the Polistinae and Vespinae, while in the Stenogastrinae, it varies from thin in Parischnogaster to thick in Eustenogaster, with an intermediate situation in Liostenogaster. In all examined species, we found no differences in the spermathecal development between reproductive and non-reproductive wasps.     

Population Genetics and Identity of an Introduced Terrestrial Slug: Arion subfuscus s.l. in the North-east USA (Gastropoda, Pulmonata, Arionidae)

Several European species of the terrestrial slug genus Arion have been introduced into North America. A case in point is the species complex A. subfuscus s.l. which has become one of the most abundant slug taxa in North America. In Europe this complex consists of at least two cryptic species, viz. A. fuscus and A. subfuscus s.s., the latter of which is further subdivided in five strongly divergent mtDNA lineages (A. subfuscus S1–S5). In order to determine which of these A. subfsucus s.l. taxa are present in the NE USA and in order to assess their population genetic structure, we compared mtDNA, nDNA and allozyme variation between populations from the NE USA and Europe. Our results show that (1) at least A. subfuscus S1 has become successfully established in the NE USA, (2) founder effects are the most likely explanation for the loss of a large amount of molecular genetic variation in populations from the NE USA (i.e. a loss of 96% of the 16S rDNA haplotypes, 67% of the ITS1 alleles and 46% of the alleles at polymorphic allozyme loci), and (3) part of the remaining genetic variation in NE USA populations was probably due to multiple introductions from the British Isles and the European mainland, and the hybrid structure of most of these source populations. Apparently, the extreme loss of molecular genetic variation in this introduced species has not prevented it from successfully establishing and spreading in novel environments.     

Ultrastructure of gametogenesis in the ovotestis of an estuarine pulmonate slug,Onchidium tigrinum (Stoliczka, 1869)

The pulmonate slug Onchidium tigrinum (Stoliczka, 1869) is an estuarine protandrous gastropod. Transmission electron microscopy of both the gonadal and somatic cell populations of the ovotestis of the slug is documented. The acini of smaller slugs are comprised of developing spermatogenic cells and three to four small ill-developed oocytes. Details of the microscopic structures of Sertoli cells, interacinar cells and acinar boundary are described in-depth, revealing their secretory function. Sertoli cells are more numerous in the ovotestes of smaller slugs than in those of larger slugs. Tunnelling nanotubes of 200–400?nm in diameter are described for the first time in the Sertoli cells of molluscan ovotestis. These nanotubes may help to supply various cellular materials into distantly developing spermatogenic cells. The acini of larger slugs possess 2–3 mature oocytes along with a few spermatogenic cells. Sertoli cells, interacinar cells and spermatogonial cells are fewer in number in the acini of the ovotestis of larger individuals establishing the predominance of oogenesis in this phase of life. The number of oocytes per acinus is analysed in relation to the habitat of the pulmonates.     

The ultrastructure of the epicardium epithelium of the slug, Arion ater (Linnaeus, 1758), (Pulmonata, Stylommatophora)

In the present paper we investigated the fine structure of terrestial slug Arion ater (L.) Interacisternal microtubules were formed in Arion ater, however they do not correspond to interacisternal polycylinders because they differ in size and they are not continuous with the membrane cistern. From our study it may be concluded that these intracisternal microtubules suggest an accumulation of protein.     

Applicability of the black slug Arion ater for monitoring exposure to polycyclic aromatic hydrocarbons and their subsequent bioactivation into DNA binding metabolites

The applicability of terrestrial black slugs Arion ater (Mollusca, Gastropoda) was studied for biomonitoring environmental exposure to polycyclic aromatic hydrocarbons (PAHs). In laboratory experiments, slugs were orally exposed to benzo[a]pyrene (BaP) for a short term (3 days) or a long term (119 days) period. Test animals were collected in the field, or were reared under laboratory conditions to ensure that they had no history of PAH-exposure. Benzo[a]pyrene hydroxylase (BPH) activity was measured in the digestive gland as a biomarker for BaP exposure. Bulky DNA adduct formation in kidney was measured as an effect biomarker for BaP bioactivation into DNA-binding metabolites. Although success of clutching was relatively low (5 out of 18 slugs produced egg packages), sufficient number of slugs were obtained to perform exposure experiments due to high hatching (89%) and survival rates (79%). After a short exposure to a relatively high BaP doses of 20 and 200 microg/g fresh feed, a dose-dependent and significant increase of BPH activity and bulky DNA adduct levels could be demonstrated in A. ater. Induction factors were low (two times control level), but optimization of the test conditions yielded a higher BPH induction factor of 4.8 times control level. BPH activity and bulky DNA adduct levels, however, did not increase after a long-term exposure to environmentally relevant BaP doses (upto 0.25 microg/g fresh feed). Based on this lack of response after realistic exposure it is concluded that A. ater is not sensitive to BaP exposure and, therefore, not suitable for monitoring environmental exposure to PAHs.