Microhabitats within Venomous Cone Snails Contain Diverse Actinobacteria

Actinomycetes can be symbionts in diverse organisms, including both plants and animals. Some actinomycetes benefit their host by producing small molecule secondary metabolites; the resulting symbioses are often developmentally complex. Actinomycetes associated with three cone snails were studied. Cone snails are venomous tropical marine gastropods which have been extensively examined because of their production of peptide-based neurological toxins, but no microbiological studies have been reported on these organisms. A microhabitat approach was used in which dissected tissue from each snail was treated as an individual sample in order to explore bacteria in the tissues separately. Our results revealed a diverse, novel, and highly culturable cone snail-associated actinomycete community, with some isolates showing promising bioactivity in a neurological assay. This suggests that cone snails may represent an underexplored reservoir of novel actinomycetes of potential interest for drug discovery.Interest in natural products and drug discovery has been a major driving force for the study of microbial communities associated with marine invertebrates. Sponges, which have provided more bioactive metabolites than any other marine invertebrate group (see reference 4 and previous references in that series) have been the main focus of these investigations, yielding numerous reports of associated bacteria and complex microbial communities (17, 43). Other examples of marine invertebrate-associated microbes explored for their involvement in natural products include bryozoans (9, 41), ascidians (37), and shipworms (46). Thus far, there are literature reports of diverse bacterial taxa involved in natural product biosynthesis in marine animals (9, 10). Notably less well studied are the symbiotic actinomycetes, for which the biology of host-actinomycete associations is just beginning to be explored in a methodical way. Actinomycetes are known to be important symbionts in a number of biological systems, such as plants, insects, and marine invertebrates, contributing as nitrogen fixers in plants (39) or utilizing their chemical arsenal for defense purposes (8, 22, 38).An initial investigation of a cone snail yielded a surprisingly high number of actinomycetes (data not shown), prompting this follow-up study on additional samples. Cone snail mollusks belong to the genus Conus, which contains about 500 closely related species (11). These mollusks are well known for their complex neurologically active venoms that they use to immobilize their prey, including fish, worms, and other mollusks. The venom of cone snails has been extensively studied, but to our knowledge no study of cone snail microbial communities has been reported. Cone snails are part of the larger superfamily Conoidea, comprising ∼20,000 species, making it an extremely diverse group (25, 34). Given the initial observation of cultivable actinomycetes from cone snails, this large group seemed like a potentially excellent source of new bacterial natural products and new models of actinomycete symbiosis.The goal of this study was to examine the actinobacteria community associated with tropical marine snails of the genus Conus, using a microhabitat approach by which individual organs are treated as independent samples, and to assess the bioactivities of the isolates obtained by using a neurological assay. Although similar approaches have previously been used in microbial ecology (3, 42), this is a novel approach in drug discovery for the identification of bioactive bacteria. We report here the association between three cone snails, Conus pulicarius, Conus rolani, and Conus tribblei, and their actinobacteria as well as the bioactivities of some of these actinobacteria.     

The 5-Choice Continuous Performance Test: Evidence for a Translational Test of Vigilance for Mice

Background

Attentional dysfunction is related to functional disability in patients with neuropsychiatric disorders such as schizophrenia, bipolar disorder, and Alzheimer''s disease. Indeed, sustained attention/vigilance is among the leading targets for new medications designed to improve cognition in schizophrenia. Although vigilance is assessed frequently using the continuous performance test (CPT) in humans, few tests specifically assess vigilance in rodents.

Methods

We describe the 5-choice CPT (5C-CPT), an elaboration of the 5-choice serial reaction (5CSR) task that includes non-signal trials, thus mimicking task parameters of human CPTs that use signal and non-signal events to assess vigilance. The performances of C57BL/6J and DBA/2J mice were assessed in the 5C-CPT to determine whether this task could differentiate between strains. C57BL/6J mice were also trained in the 5CSR task and a simple reaction-time (RT) task involving only one choice (1CRT task). We hypothesized that: 1) C57BL/6J performance would be superior to DBA/2J mice in the 5C-CPT as measured by the sensitivity index measure from signal detection theory; 2) a vigilance decrement would be observed in both strains; and 3) RTs would increase across tasks with increased attentional load (1CRT task<5CSR task<5C-CPT).

Conclusions

C57BL/6J mice exhibited superior SI levels compared to DBA/2J mice, but with no difference in accuracy. A vigilance decrement was observed in both strains, which was more pronounced in DBA/2J mice and unaffected by response bias. Finally, we observed increased RTs with increased attentional load, such that 1CRT task<5CSR task<5C-CPT, consistent with human performance in simple RT, choice RT, and CPT tasks. Thus we have demonstrated construct validity for the 5C-CPT as a measure of vigilance that is analogous to human CPT studies.     

The fire ephemeral Tersonia cyathiflora (Gyrostemonaceae) germinates in response to smoke but not the butenolide 3-methyl-2H-furo[2,3-c]pyran-2-one

Background and Aims

Tersonia cyathiflora (Gyrostemonaceae) is a fire ephemeral with an obligate requirement for smoke to germinate. Whether it is stimulated to germinate by 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikinolide, KAR₁), the butenolide isolated from smoke that stimulates the germination of many other smoke-responsive species, is tested.

Methods

Seeds of T. cyathiflora were buried in autumn following collection and were exhumed 1 year later, as this alleviates dormancy and enables seeds to germinate in response to smoke-water. Exhumed seeds were tested with smoke-water and KAR₁. Fresh preparations of these solutions were again tested on seeds exhumed 2 months later under a broader range of conditions. They were also tested on Grevillea eriostachya (Proteaceae) and Stylidium affine (Stylidiaceae) to confirm the activity of KAR₁.

Key Results

T. cyathiflora seeds germinated in response to smoke-water but not to KAR₁. In contrast, G. eriostachya and S. affine germinated in response to both smoke-water and KAR₁.

Conclusions

Although many smoke-responsive seeds germinate in the presence of KAR₁, this does not apply universally. This suggests that other chemical(s) in smoke-water may play an important role in stimulating the germination of certain species.     

Molecular and Structural Insight into proNGF Engagement of p75NTR and Sortilin

Nerve growth factor (NGF) is initially synthesized as a precursor, proNGF, that is cleaved to release its C-terminal mature form. Recent studies suggested that proNGF is not an inactive precursor but acts as a signaling ligand distinct from its mature counterpart. proNGF and mature NGF initiate opposing biological responses by utilizing both distinct and shared receptor components. In this study, we carried out structural and biochemical characterization of proNGF interactions with p75NTR and sortilin. We crystallized proNGF complexed to p75NTR and present the structure at 3.75-Å resolution. The structure reveals a 2:2 symmetric binding mode, as compared with the asymmetric structure of a previously reported crystal structure of mature NGF complexed to p75NTR and the 2:2 symmetric complex of neurotrophin-3 (NT-3) and p75NTR. Here, we discuss the possible origins and implications of the different stoichiometries. In the proNGF-p75NTR complex, the pro regions of proNGF are mostly disordered and two hairpin loops (loop 2) at the top of the NGF dimer have undergone conformational changes in comparison with mature NT structures, suggesting possible interactions with the propeptide. We further explored the binding characteristics of proNGF to sortilin using surface plasmon resonance and cell-based assays and determined that calcium ions promote the formation of a stable ternary complex of proNGF-sortilin-p75NTR. These results, together with those of previous structural and mechanistic studies of NT-receptor interactions, suggest the potential for distinct signaling activities through p75NTR mediated by different NT-induced conformational changes.     

14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity

We have investigated the role that S259 phosphorylation, S621 phosphorylation, and 14-3-3 binding play in regulating Raf-1 activity. We show that 14-3-3 binding, rather than Raf-1 phosphorylation, is required for the correct regulation of kinase activity. Phosphorylation of S621 is not required for activity, but 14-3-3 binding is essential. When 14-3-3 binding to conserved region 2 (CR2) was disrupted, Raf-1 basal kinase activity was elevated and it could be further activated by (V12,G37)Ras, (V23)TC21, and (V38)R-Ras. Disruption of 14-3-3 binding at CR2 did not recover binding of Raf-1 to (V12,G37)Ras but allowed more efficient recruitment of Raf-1 to the plasma membrane and stimulated its phosphorylation on S338. Finally, (V12)Ras, but not (V12,G37)Ras, displaced 14-3-3 from full-length Raf-1 and the Raf-1 bound to Ras. GTP was still phosphorylated on S259. Our data suggest that stable association of Raf-1 with the plasma membrane requires Ras-mediated displacement of 14-3-3 from CR2. Small G proteins that cannot displace 14-3-3 fail to recruit Raf-1 to the membrane efficiently and so fail to stimulate kinase activity.     

Death assemblages of molluscs on St Martin's Flats, Isles of Scilly: a surrogate for regional biodiversity?

We test the hypothesis that the biodiversity of the death assemblage of shelled molluscs (gastropods and bivalves) on a single sandflat is representative of the biodiversity of the regional living fauna from the complete spectrum of habitat types, and can therefore be used as a surrogate for the purposes of rapid biodiversity assessment. Two biodiversity indices considered appropriate for such an assessment are average taxonomic distinctness (⁺) and the variation in taxonomic distinctness (⁺), which are, respectively, measures of the degree to which the species are taxonomically related to each other (i.e. the average taxonomic spread), and the degree to which taxa are over or under represented (i.e. the evenness of the distribution across the taxonomic tree). In each case, measured values were compared with the regional living species pool using a randomisation test. Collections and identifications of shells were made by both a non-expert in mollusc taxonomy and by taxonomic experts, the latter using microscopic examination of sediment samples as well as simple surface searches for macro-species. In all studies, ⁺ for the gastropods in the death assemblage was fully representative of the regional living fauna, but for bivalves it was significantly lower. This is because most of the bivalves were characteristic of the sandflat habitat, which introduced a bias, whereas post-mortem transport of gastropods, characterising exotic habitats, is more random. For gastropods, in all studies but one, ⁺ was significantly higher than expected from a random sample of the regional species pool, and for bivalves this was the case for all studies, suggesting the over-representation of some taxa and the under-representation of others. For the total shelled molluscan fauna (gastropods + bivalves) all ⁺ values were below, and all ⁺ values above, expectation. Results obtained by experts and a non-expert in mollusc taxonomy did not differ. In general, death assemblages at a single location are only likely to be representative of a wider region if there is randomisation as a result of post-mortem transport processes.     

Electroantennogram responses of male Sphinx perelegans hawkmoths to floral and ‘green-leaf volatiles’

The effects of sublethal dosages of the chloronicotinyl insecticide imidacloprid on different strains of the tobacco whitefly, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae), have been studied after leaf dip and systemic application. All bioassays were performed with the insecticide susceptible strain, SUD-S, and two Spanish biotypes, ALM-2 and LMPA-2, both resistant to conventional insecticides and with a lower susceptibility towards imidacloprid. Honeydew, excreted by all strains feeding on treated and untreated cotton leaf discs was quantified by photometric analysis of its carbohydrate content. EC50-values for the depression of honeydew excretion in female adults after systemic application of imidacloprid were calculated at 0.037 ppm, 0.027 ppm and 0.048 ppm for strains SUD-S, ALM-2 and LMPA-2, respectively, indicating no significant differences between strains in feeding behaviour throughout an 48 h testing period. Depending on the strain these EC50-values were 150- to 850-times lower than LC50-values calculated for mortality in the same bioassay. Starvation tests revealed mean survival times of >48 h for female adults placed on agar without leaf discs, indicating that sublethal dosages of imidacloprid which caused antifeedant responses, were probably not covered in common 48 h systemic bioassays, used to monitor resistance to imidacloprid. Effects of sublethal dosages on honeydew excretion after leaf dip application seem to be minor. In choice situations with systemically treated and untreated leaf discs in a single container, female adults of B. tabaci showed a clear preference for the untreated leaf discs. However, when using leaf discs treated by painting the surface with imidacloprid in the same bioassay, feeding activities on treated and untreated leaf discs were not significantly different. The results of the present study demonstrate the antifeedant properties of imidacloprid on B. tabaci, which might play an essential role after soil application or seed treatment under field conditions.