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.     

Susceptibility and immune response of Deroceras reticulatum, Milax gagates and Limax pseudoflavus exposed to the slug parasitic nematode Phasmarhabditis hermaphrodita

We exposed three slug species (Deroceras reticulatum (Müller), Milax gagates (Draparnaud) and Limax pseudoflavus L.) to the parasitic nematode Phasmarhabditis hermaphrodita Schneider. P. hermaphrodita was able to cause mortality and feeding inhibition to both D. reticulatum and M. gagates but did not negatively affect L. pseudoflavus. On dissection of surviving L. pseudoflavus large numbers of P. hermaphrodita were found encapsulated in the shell of the slug. We found that by increasing shell size, the slug was able to trap invading nematodes, which could be an immune response to P. hermaphrodita invasion. This is the first report of a slug defense mechanism to inhibit P. hermaphrodita.     

Immunohistochemical demonstration of cholinergic structures in central ganglia of the slug (Limax maximus, Limax valentianus)

Immunohistochemical techniques were used to study the distribution of cholinergic neurons containing choline acetyltransferase of the common type (cChAT), the synthetic enzyme of acetylcholine, in the central nervous system of the slug Limax maximus and Limax valentianus. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, three methods were used in order to validate antibody specificity for the Limax counterpart enzyme. Western blot combined with ChAT activity assay following native gel electrophoresis and immunoprecipitation analysis both indicated that immunoreactive Limax brain molecules were capable of synthesizing acetylcholine. Western blot after denatured gel electrophoresis of Limax brain extracts revealed a single band of about 67kDa. All findings obtained with these three methods clearly indicated that the antiserum effectively recognized Limax cChAT. 1400 neuronal cell bodies positive for cChAT, mainly small to medium-sized, were found in various brain regions in the buccal, cerebral, pleural, parietal, visceral and pedal ganglia. cChAT immunoreactive nerve fibers were distributed extensively in the neuropil, connectives and commissures of these central ganglia. The map of cChAT-positive cells provided here are valuable for understanding the cholinergic mechanism in the slug brain, as well as giving an important hint to clarifying the mechanisms of learning and memory in higher vertebrates including humans.     

Responses of slug numbers and slug damage to crops in a silvoarable agroforestry landscape

1. Alternative farming practices such as set-aside and agroforestry are likely to be of continuing interest to European agriculture but may have associated problems, such as increased populations of crop pests such as slugs.
2. A silvoarable agroforestry experiment has been in progress since 1987 at Leeds University Farms at Bramham, West Yorkshire, UK. It consists of four replicate blocks, each with rows of trees separating alleys of arable crops; all four blocks have their own arable control areas in adjacent fields.
3. Pitfall trap catches within the experiment indicate that the slug population increased over the period 1991–94. The increase was greatest, and most consistent, within the tree rows in the agroforestry blocks. The increase was slower and less consistent in the arable controls and the arable areas within the agroforestry blocks.
4. In spring 1994, the slugs in each of the treatments in the agroforestry experimental area were sampled using pipe traps, refuge traps and pitfall traps. The number and diversity of slugs were highest in the grassed understorey beneath the rows of trees and significantly higher in the alleys between the rows of trees than in the arable control areas.
5. The levels of slug damage to a pea crop were assessed by surveys that recorded the number of emerging plants and the number of damaged leaves per plant. There were significant correlations between the number of slugs caught and the damage to the crop by slugs. It is concluded that slugs have the potential to be important pests of some crops in silvoarable agroforestry landscapes and that this could influence the choice of crops for this type of farming.
6. Major conclusions are emboldened in the Discussion.     

Distribution of biogenic amines in the slug,Limax maximus

Summary The distribution of monoamines inLimax maximus was studied by the histochemical fluorescent method of Falck and Hillarp. The number of 5-HT-containing and catecholamine-containing perikarya in the central nervous system is small compared with the non-fluorescent perikarya. However, all the ganglia except the proto-cerebral ganglia have some amine-containing neurons. There are relatively larger numbers of fluorescent cells in the cerebral, visceral, pedal and right parietal ganglia than in the other ganglia. A single, giant 5-HT-containing neuron was observed in each meta-cerebral ganglion.Monoamine neurons are localised in a number of peripheral tissues (heart, integument, tentacles, penis retractor muscle, sole of foot, kidney, alimentary canal, reproductive organs and tentacular, pharyngeal and cephalic retractor muscles). Neurons containing catecholamine are mostly associated with sensory structures such as the statocysts, the retina of the eye and the integument of the tentacles, whereas 5-HT-containing nerve fibres are mainly observed in muscle tissues.We wish to thank the Wellcome Trust for financial support.     

Distribution of GABA-like immunoreactive neurons in the slug Limax maximus

Summary Immunohistochemical techniques were used to study the distribution of gamma-amino butyric acid (GABA)-like immunoreactive neurons in the nervous system of the slug Limax maximus. Approximately 170 GABA-like immunoreactive cell bodies were found in the central nervous system. These were located in the cerebral, buccal and pedal ganglia. Most GABA-like immunoreactive neurons had small cell bodies, which were aggregated into discrete clusters within the cerebral and pedal ganglia. Three pairs of longer, uniquely identifiable, GABA-like immunoreactive cells were found in the cerebral ganglion. GABA-like immunoreactive nerve fibres were also found in all of the central ganglia but were absent from peripheral nerves. These results suggest that GABA acts as a central neurotransmitter in the slug. The possible roles of GABA-ergic neurotransmission in the slug are discussed.     

Modification of cardiac activity in response to dehydration in the terrestrial slug, Limax maximus

The level of body hydration in the terrestrial slug Limax maximus modifies several aspects of behavior such as pneumostome activity, feeding responsiveness, huddling, and contact-rehydration. The relationship between water balance and pneumostome activity and respiratory function suggested that cardiac activity might also be affected. To pursue this possibility, intact slugs and isolated heart-central nervous system (CNS) preparations were used to investigate cardiac responses to the increase in hemolymph osmolality which occurs during dehydration. In intact animals, heart rate increased in response to progressive air-dehydration and to increases in hemolymph osmolality resulting from injections of hyperosmotic solutions of mannitol or NaCl. In isolated preparations, the heart or CNS were separately exposed to hyperosmotic saline. Exposure of the heart alone to hyperosmotic saline resulted in decreased heart rate while exposure of only the CNS resulted in an increase in heart rate. These observations suggest that the increase in heart rate that is observed in intact air-dehydrated slugs is primarily mediated by the CNS.     

Brain-gonad axis and photoperiodically-stimulated sexual maturation in the slug,Limax maximus

Summary Physiological and endocrine mechanisms mediating long-day (LD 168) triggered sexual maturation were studied in the terrestrial slug,Limax maximus. Our findings were: (1) Maturation was induced in immature slugs seeing only short days (LD 816) after implantation of whole brains from maturing donors, but no development was found in sham-operated control slugs or in animals receiving implants of muscle from mature donors (Table 1). (2) Removal of the optic tentacles did not block maturation in LD 168 or promote maturation in LD 816 (Fig. 1). (3) Gonadectomy (castration) abolished penis development in 9 of 11 slugs exposed to LD 168 for periods of up to 31 weeks (Table 2). The results are consistent with a model forLimax reproductive tract development in which the perception of long days by extraocular receptors results in the secretion of a maturation hormone by the brain followed by the production of a separate male-phase sex hormone by the developing gonad.Abbreviations ASO accessory sex organs - DB dorsal bodies - FPSH female-phase sex hormone - MH maturation hormone - MPSH male-phase sex hormone This work was supported in part by grants from NSF (BNS 78-01408) and NIH (MH 27948) to P.G.S. The assistance of Mr. T.M. Gordon, Mr. J.L. Broyles, and Mr. M. Bullock is gratefully acknowledged.     

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.     

Expression of Asn-d-Trp-Phe-NH2 in the brain of the terrestrial slug Limax valentianus

The tripeptide Asn-d-Trp-Phe-NH(2) (NdWFamide) is a D-amino acid-containing cardioexcitatory peptide initially isolated from Aplysia. Previously we detected NdWFamide immunoreactivity in the visceral giant cells, the largest neurons in the brain of the terrestrial slug Limax located at the dorsal surface of the visceral ganglia. In the present study, we further analyzed the morphological features of these neurons by an intracellular injection of Lucifer yellow, and found that these neurons extend neurites out of the brain through at least 5 nerve bundles. We then isolated a gene and a cDNA clone potentially encoding a NdWFamide precursor, and investigated expression at the levels of mRNA and protein in Limax. The NdWFamide gene consists of 5 exons spanning at least 17 kb of the genome, and its open reading frame extends over 3 exons. The spatial expression pattern of NdWFamide mRNA was almost identical to that of the NdWFamide peptide, with some minor discrepancies in between. Although the most remarkable expression was evident in the visceral giant cells, we also found the expression of NdWFamide mRNA and peptide in the cerebral and pedal ganglia. These results suggest the involvement of NdWFamide in the regulation of a broad area of the slug's body.     

Distribution of FMRFamide-like immunoreactivity in the nervous system of the slug Limax maximus

Summary The distribution of FMRFamide-like immunoreactive neurons in the nervous system of the slug Limax maximus was studied using immunohistochemical methods. Approximately one thousand FMRFamide-like immunoreactive cell bodies were found in the central nervous system. Ranging between 15 m and 200 m in diameter, they were found in all 11 ganglia of the central nervous system. FMRFamide-like immunoreactive cell bodies were also found at peripheral locations on buccal nerve roots. FMRFamide-like immunoreactive nerve fibres were present in peripheral nerve roots and were distributed extensively throughout the neuropil and cell body regions of the central ganglia. They were also present in the connective tissue of the perineurium, forming an extensive network of varicose fibres. The large number, extensive distribution and great range in size of FMRFamide-like immunoreactive cell bodies and the wide distribution of immunoreactive fibres suggest that FMRFamide-like peptides might serve several different functions in the nervous system of the slug.     

Responses of Trachelospermum asiaticum (Apocynaceae) seedlings to growth in a light intensity gradient

We recorded the growth of 24 seedlings of Trachelospermum asiaticum, a root climber, placed between a light source and a wall. Shoot length of seedlings planted at the brightest points nearly matched the distance the shoot tips moved toward the wall surface. In contrast, although the seedlings planted at the darkest points did elongate, the tips moved an average distance of only 0.4 cm. Creeping shoots of T. asiaticum planted in brighter environments exhibit negative phototropism, which encourages them to grow rapidly toward dark places, allowing them to reach supporting hosts faster than those that germinate close to the host.     

Fine structure of the epidermis of the optic tentacle in a slug, Limax flavus L.

The epidermis at the tip of the optic tentacle in Limax flavus is constructed of columnar epithelial cells, distal processes of nerve cells, and scattered processes of the collar cells. The epithelial cells extend stout microvilli called plasmatic processes by Wright perpendicularly from the free surface. Each plasmic process branches into a few terminal twigs embedded in a fuzzy filamentous substance. Most nerve cells have their nuclei under the basal lamina. The distal processes of these nerve cells reach the free surface and send long microvilli to form the spongy layer under a filamentous covering. At the side surface of the tentacle the epithelial cells are cuboidal or squamous and the neural elements are fewer. Here, no spongy layer is formed; and the collar cell processes are replaced by the lateral cell processes. Peculiar secretion granules are contained in the lateral and collar cell processes as well as in their cell bodies situated beneath the basal lamina.     

Analysis of DNA endoreplication in the brain neurons in the terrestrial slug, Limax valentianus

DNA endoreplication is the DNA synthesis without cell division, resulting in the generation of a nucleus containing a larger amount of genomic DNA compared to a normal diploid genome. There are many such giant neurons in the molluscan brain that are generated as a result of repeated endoreplication. However, it has been controversial whether the endoreplication is the whole genome replication (polyploidy) or the local amplification of the genes that are necessary for the neuron's function (polyteny/polysomy). Here in this study, we investigated these two possibilities by (1) immunohistochemical analysis of the distribution of 5'-bromodeoxyuridine incorporated into the nuclei of the brain neurons, and by (2) quantitative genomic PCR directed to two different genes expressed in specific brain regions. Our data supported the view that the DNA endoreplication is the whole genome replication rather than the local amplification of a specific genomic region.     

Structure,crystallography, and morphogenesis of the cryptic shell of the terrestrial slug Limax maximus (Mollusca,gastropoda)

The structure and crystallography of the internal shell of the pulmonate gastropod slug Limax maximus were studied at the levels of light and scanning electron microscopy, revealing patterns of shell ontogeny and morphogenesis. The calcified portion of the slightly convex ovoid shell is composed of a single palisade layer of calcitic crystals. Numerous projections, 100 μm in width at the dorsal tip, are found on the dorsal surface of the shell and coincide with local nucleation sites of primordial calcium salt deposition onto the periostracum. With continued calcification these projections coalesce ventrally, forming the single crystalline shell layer. The organic portion of the shell includes the periostracum and an extensive PAS-staining conchiolin. In EDTA-etched preparations, conchiolin appears as a spongy network of fibers throughout the shell. Both horizontal and vertical components of the conchiolin are present, the former of variable thickness and occurring in an intercrystalline manner, the latter always occurring normal to the horizontal set. Macromorphogenic growth is characterized by three distinct temporal stages. Primary growth occurs radially from the umbonal region. Secondary growth is synonymous with shell thickening. Tertiary growth is characterized by both a lateral component, in which the shell extends beyond the primary growth boundaries, and a ventral component, in which the shell continues to grow in thickness. SEM of the ventral shell surface reveals a pattern of growth at the crystalmatrix interface. Proteinaceous fibers of the conchiolin occur unidirectionally in horizontal rows. Zones of incipient calcitic crystallization onto these hypostracal fiber bundles are contrasted by zones of increasing crystallization until the fibrous template (reduced hypostracum) is completely covered by crystals.     

Neurosecretory cells in the central nervous system of the giant garden slug, Limax maximus

The neurosecretory system of the giant garden slug Limax maximus was studied using the alcian blue/alcian yellow (AB/AY) staining technique for neurosecretion. Stainable cells could be identified in the paired cerebral, pleural, parietal, and buccal ganglia, and in the visceral ganglion. The cells occur as single cells or in groups of up to 100, with diameters ranging between 10 and 70 mu m. Axon tracts could only be traced for a small number of cells; neurohemal areas were not conclusively identified. The morphological similarities of the neurosecretory system of L. maximus is compared with that of other investigated stylommatophoran slugs.     

Responses of Odonata chromatophores to environmental stimuli

Responses to change in temperature and light intensity were studied in three species of Australian Odonata using time-lapse photography. In each species, responses to temperature are dependent on both the instantaneous temperature and the direction of temperature change. At temperatures below those which produce unstable colour phases, the change to dark phase takes about 9 hr and is constant in rate. The reverse change is directly temperature dependent and can be much more rapid. Responses to change in light intensity are attributable to the heating effect of light rather than to true light sensitivity. All colour changes show wide individual variation in both rate and amount. They are slightly affected by temperature acclimation but are unaffected by prevailing weather, time of year, geographic location, or age.