Correia Repeat Enclosed Elements and Non-Coding RNAs in the Neisseria Species.

Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Neisseria meningitidis is a closely related pathogen that shares many of the same genomic features and virulence factors, but causes the life threatening diseases meningococcal meningitis and septicaemia. The importance of non-coding RNAs in gene regulation has become increasingly evident having been demonstrated to be involved in regulons responsible for iron acquisition, antigenic variation, and virulence. Neisseria spp. contain an IS-like element, the Correia Repeat Enclosed Element, which has been predicted to be mobile within the genomes or to have been in the past. This repeat, present in over 100 copies in the genome, has the ability to alter gene expression and regulation in several ways. We reveal here that Correia Repeat Enclosed Elements tend to be near non-coding RNAs in the Neisseria spp., especially N. gonorrhoeae. These results suggest that Correia Repeat Enclosed Elements may have disrupted ancestral regulatory networks not just through their influence on regulatory proteins but also for non-coding RNAs.

Multiple roles for protein kinase C in gastropod embryogenesis.

Protein kinase C (PKC) contributes to the correct development of organisms, but its importance to the embryogenesis of molluscs is not yet known. We report here that PKC activation is cyclic within early developing embryos of the gastropod snail Lymnaea stagnalis, and that activation with phorbol myristate acetate (PMA) results in disorganised and developmentally arrested embryos within 24 h. Moreover, chronic modulation of PKC activation by PMA or by the PKC inhibitor GF109203X in early embryos results in altered rotation and gliding behaviours and heartbeat during development. Finally, dis-regulation of PKC activity during early development significantly increased the duration to hatching. Our findings thus support novel roles for PKC in L. stagnalis embryos, in several physiological contexts, providing further insights into the importance of protein kinases for gastropod development in general.

Unravelling the riddle of Radix: DNA barcoding for species identification of freshwater snail intermediate hosts of zoonotic digeneans and estimating their inter-population evolutionary relationships.

Radix spp. are intermediate host snails for digenean parasites of medical and veterinary importance. Within this genus, species differentiation using shell and internal organ morphology can result in erroneous species identification, causing problems when trying to understand the population biology of Radix. In the present study, DNA barcoding, using cox1 and ITS2 sequences, identified populations of Radix auricularia and Radix balthica from specimens originally morphologically identified as Radix peregra from the UK. Assessment of cox1 and ITS2 as species identification markers showed that, although both markers differentiated species, cox1 possessed greater molecular diversity and higher phylogenetic resolution. Cox1 also proved useful for gaining insights into the evolutionary relationships of Radix species populations. Phylogenetic analysis and haplotype networks of cox1 indicated that R. auricularia appeared to have invaded the UK several times; some haplotypes forming a distinct UK specific clade, whilst others are more akin to those found on mainland Europe. This was in contrast to relationships between R. balthica populations, which had low molecular diversity and no distinct UK specific haplotypes, suggesting recent and multiple invasions from mainland Europe. Molecular techniques therefore appear to be crucial for distinguishing Radix spp., particularly using cox1. This barcoding marker also enables the population biology of Radix spp. to be explored, and is invaluable for monitoring the epidemiology of fluke diseases especially in the light of emerging diseases and food security.

Identification of a major causative agent of human cercarial dermatitis, Trichobilharzia franki (Müller and Kimmig 1994), in southern England and its evolutionary relationships with other European populations.

BACKGROUND: Trichobilharzia is the most species rich and widely distributed genus of schistosomes and is known throughout Europe and North America as an agent of human cercarial dermatitis. The disease is caused by an acute allergic reaction in the skin that develops as a consequence of repeated contact with water containing schistosomatid cercariae. However, despite historical outbreaks of the disease, there are no published records of accurately identified Trichobilharzia species from the UK. METHODS: Two hundred Radix auricularia (L.) were sampled from a recreational fishing lake in Hampshire and emerging schistosomatid cercariae were collected for microscopy and DNA extraction. General morphological description of the cercariae was performed, alongside sequencing and phylogenetic analysis of the 28S ribosomal DNA for accurate species identification as well as comparisons of ITS1 in order to identify evolutionary affinities with other European populations. All molecular comparisons were performed using published sequences. RESULTS: The phylogenetic analysis of 28S sequences identified the cercariae as Trichobilharzia franki. Two unique British ITS1 haplotypes were identified which were most closely related to haplotypes of T. franki populations from France. Haplotype network analysis indicated the mixing of T. franki populations throughout Europe. It is suggested that parasite distribution is the probable result of the movement of migratory waterfowl. CONCLUSIONS: This is the first accurate record of T. franki in the UK. The movement of T. franki with waterfowl could pose a considerable human health risk, as in mainland Europe, and signifies T. franki-associated human cercarial dermatitis as a re-emerging disease in the UK.

Potential environmental and host gender influences on prevalence of Haemogregarina platessae (Adeleorina:Haemogregarinidae) and suspected Haemohormidium terraenovae (incertae sedis) in Brazilian flounder from the Patos Lagoon Estuary, southern Brazil.

Flounder, Paralichthys orbignyanus (Valenciennes), were captured in polluted and non-polluted sites within the Patos Lagoon Estuary, southern Brazil, over four seasons. Blood films showed a high prevalence of infection with a haemogregarine, or mixed parasitaemias of this and an organism resembling Haemohormidium terraenovae So, 1972. Haemogregarine gamont stages conformed to existing descriptions of Desseria platessae (Lebailly, 1904) Siddall, 1995 from flatfishes, but intraerythrocytic division of meronts was observed, leading to the recommendation for nomenclatural correction, placing the haemogregarine in the genus Haemogregarina (sensu lato) Danilewsky, 1885. Statistical analyses suggested that although sample sizes were small, infections with meront stages, immature and mature gamonts were all influenced by site, and possibly therefore, by pollution. Season also appeared to determine likelihood of infection with meronts and immature gamonts, but not mature gamonts, while adult fish gender apparently affected infection with immature and mature gamonts, but not meronts. The H. terraenovae-like organism exhibited unusual extracellular forms and did not match closely with the type description of H. terraenovae; precise identification was therefore difficult. Data analyses suggested that parasitism by this organism was influenced by site and fish gender, since females and males from non-polluted water were infected, but only females from the polluted site. Season was also important and significantly more adult fish of both sexes were infected with this parasite in the Brazilian summer and autumn, compared with winter and spring. Finally, these appeared to be the first observations of Haemogregarina platessae, and possibly H. terraenovae, from the southern hemisphere.

Integrin engagement modulates the phosphorylation of focal adhesion kinase, phagocytosis, and cell spreading in molluscan defence cells.

Integrins play a key role in cellular immune responses in a variety of organisms; however, knowledge of integrins and their effects on cell signalling and functional responses in molluscan defence reactions is poor. Using integrin-mediated cell adhesion kits, alphaVbeta3 and beta1 integrin-like subunits were identified on the surface of Lymnaea stagnalis haemocytes. Haemocyte binding via these integrins was found to be dependent on Ca2+/Mg2+. Western blotting with an anti-phospho (anti-active) focal adhesion kinase (FAK) antibody revealed a 120-125 kDa FAK-like protein in these cells; this protein was transiently phosphorylated upon haemocyte adhesion over 90 min, with maximal phosphorylation occurring after 30 min binding. Also, integrin engagement with the tetrapeptide Arg-Gly-Asp-Ser (RGDS) resulted in a rapid increase in phosphorylation of the FAK-like protein; however, RGDS did not affect the phosphorylation of extracellular signal-regulated kinase. Treatment of haemocytes with RGDS (2 mM) inhibited phagocytosis of E. coli bioparticles by 88%. Moreover, at this concentration, RGDS reduced cell spreading by 61%; stress fiber formation was also impaired. Taken together, these results demonstrate a role for integrins in L. stagnalis haemocyte adhesion and defence reactions and, for the first time, link integrin engagement to FAK activation in molluscs.

The use of an alternative food source (red clover) as a means of reducing slug pest damage to winter wheat: towards field implementation.

Slugs are major pests of many crops in the UK, including winter wheat, yet current methods of control are often unreliable. The aim of this study was to investigate three issues key to the successful field implementation of a control strategy that uses red clover as an alternative food source to reduce the amount of damage caused to winter wheat by the field slug, Deroceras reticulatum (Müller). A series of three experiments was designed to assess this aim. Firstly, under laboratory conditions, red clover was consumed in greater quantities than wheat, even when wheat was presented as a novel food. Secondly, red clover had no significant effects on the emergence and early growth of wheat in a polytunnel experiment. Both these results are crucial to the successful implementation of a strategy that uses red clover as an alternative food source. Lastly, the results of a field experiment were consistent with the results of the polytunnel experiment, in that red clover did not significantly affect wheat emergence. However, plots in which red clover was left to grow until the time of wheat harvest resulted in significantly lower (43%) wheat yields than plots without red clover. These results suggest that red clover must be removed from the field after the wheat has passed its vulnerable seedling stage. Recommendations for the potential use of red clover as an alternative food source for reducing damage to winter wheat in field conditions are discussed and opportunities for further work are suggested.

Phagocytosis by Lymnaea stagnalis haemocytes: a potential role for phosphatidylinositol 3-kinase but not protein kinase A.

The molecular events that regulate phagocytosis, an important innate immune response, in invertebrate defence cells (haemocytes) are poorly understood. Lymnaea stagnalis haemocytes were used as a model to elucidate the role of cell signalling pathways in phagocytosis by molluscan defence cells. The phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, significantly impaired haemocyte phagocytic activity in a dose-responsive manner with 10 microM LY294002 reducing internalization of fluorescent-conjugated Escherichia coli by 62% (P < or = 0.001). In contrast, the protein kinase A (PKA) inhibitor KT5720 was without effect. Therefore, PI3-K, but not PKA, appears to control phagocytosis by haemocytes in these gastropod molluscs.

A laboratory-based comparison of a molluscicide and an alternative food source (red clover) as means of reducing slug damage to winter wheat.

Slugs are major pests of many crops in the UK, including winter wheat, yet current methods of control are often unreliable. This study investigates the potential use of red clover, as an alternative food source, to reduce the amount of damage caused to winter wheat by the field slug, Deroceras reticulatum (Müller). Two laboratory-based studies, each conducted over a 7-day period, investigated the effects of red clover seedlings and commercial metaldehyde pellets on damage to winter wheat seeds and seedlings. The results indicate that metaldehyde applications, in the form of commercially available pellets, resulted in significantly greater protection to wheat seeds compared with red clover, whereas metaldehyde and red clover were equally as effective in reducing damage to wheat seedlings. A further laboratory experiment investigated the effect of two slug population densities (48 and 16 adults m(-2)) and high and low red clover seed rates (125% and 75% of a standard rate) on damage to wheat seeds. Results showed that, at the highest slug population density, red clover sown at 125% of the standard rate gave 99% protection to wheat seeds, compared with the 75% seed rate which gave 55%. At the lower slug population density, both seed rates of red clover resulted in similar levels of protection. Implications for the potential use of red clover as an alternative food source for reducing damage to winter wheat in field conditions are discussed.

Carbohydrates that mimic schistosome surface coat components affect ERK and PKC signalling in Lymnaea stagnalis haemocytes.

Molluscs are intermediate hosts for helminth parasites such as Schistosoma spp. that possess an immunogenic surface coat of high carbohydrate content, with fucose as the predominant saccharide. More than a decade ago, it was postulated that such components could block receptors on snail haemocytes thus preventing recognition of intra-molluscan schistosome stages. Although more recent studies have shown that carbohydrates can suppress processes such as phagocytosis by haemocytes, interference of the haemocyte cell signalling pathways that regulate immunity by saccharides has not yet been investigated. We have recently reported the presence of extracellular-signal regulated kinase and protein kinase C in Lymnaea stagnalis haemocytes. Here we show that extracellular-signal regulated kinase and protein kinase C activities are down-regulated when haemocytes are exposed to albumin-linked fucose and galactose in the absence of haemolymph. Moreover, we demonstrate that phagocytosis is reduced under these conditions. Interestingly, in the presence of haemolymph, only protein kinase C activity is down-regulated and only galactose suppresses phagocytosis, implying a role for serum factors in the preservation of haemocyte function following exposure. We therefore propose that the establishment of a compatible relationship between a schistosome and its snail host is at least in part due to down-regulation of cell signalling events in haemocytes.

Activation of extracellular-signal regulated kinase is required for phagocytosis by Lymnaea stagnalis haemocytes.

Haemocytes are the primary defence cells of molluscs. In the present study, extracellular-signal regulated kinase (ERK) 1/2-like proteins were identified within Lymnaea stagnalis haemocytes, with apparent molecular weights of 44 and 43 kDa, respectively. Mitogen-activated protein kinase (MAPK) activity assays have confirmed that the L. stagnalis ERK possesses kinase activity towards Elk-1. Challenge of haemocytes with bacterial lipopolysaccharide (LPS) resulted in a transient activation of ERK, and immunocytochemistry revealed that phospho-ERK was present in both the perinuclear region and the nucleus following challenge. MAPK/ERK kinase (MEK) inhibitors blocked ERK activation confirming that MEK lies upstream of ERK in haemocytes. Moreover, phagocytosis assays, using various inhibitors, showed that ERK activity was vital for efficient phagocytosis and that ERK may be activated by both Ras-dependent and Ras-independent mechanisms. Overall, this study has furthered knowledge of ERK signalling in molluscan immunity and has shown that the ERK pathway regulates the phagocytic activity of molluscan haemocytes.

A laboratory evaluation of the palatability of legumes to the field slug, Deroceras reticulatum Müller.

Slugs are major pests of many crops, including winter wheat, in temperate climates, yet current methods of control are often unreliable. The aim of this study is to investigate the potential for common legume species to act as an alternative source of food, or trap crop, for the most damaging agricultural pest species, the grey field slug, Deroceras reticulatum Müller, thereby reducing damage to the wheat crop. A series of three controlled-environment experiments were designed to assess this aim. Individual slugs were fed leaves of one of ten legume species together with winter wheat leaves for a 72-h period. A clear hierarchy of acceptability was shown, with red clover, lucerne, lupin and white clover showing significantly higher Acceptability Indices than the other six species tested. Red clover produced the greatest reduction in mean wheat consumption (78%) from day 1 to day 3. When species were fed individually, red clover was consumed in significantly greater quantities than any of the other treatments: 40% more than white clover and 56% more than wheat. Furthermore, when fed with red clover the amount of wheat consumed was some 50% less than when the latter was fed alone. The results indicate that legumes vary greatly in their acceptability to D. reticulatum and it is essential that a legume with a high Acceptability Index is chosen, which results in the least amount of wheat consumed.