Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies

Summary Specific stress treatments applied to isolated tobacco (Nicotiana tabacum L.) microspores efficiently induced haploid embryo formation in vitro. A heat shock at 33 or 37°C in the presence of sugar, as well as sucrose-starvation at 25°C, resulted in the formation of embryogenic microspores. A combination of both treatments had an additive effect. Under optimal induction conditions all viable microspores in the culture were embryogenic and developed subsequently into pollen embryos by culture at 25°C in a sugar-containing medium, with induction frequencies of more than 70% with respect to the initial microspore population. A high fraction of the early pollen embryos continued their development in vitro, giving rise to haploid plants. In contrast to other available systems for microspore/pollen embryogenesis, the new protocol allows the production of homogeneous populations of embryogenic microspores and early globular embryos in large-scale cultures, without any purification step, and is therefore well suited for biochemical and molecular work.Abbreviations EDTA ethylenediaminetetraacetate - DAPI 4,6-diamidino-2-phenylindole     

Lead toxicity at different life stages of the giant prawn (Macrobrachium rosenbergii,de Man): considerations of osmoregulatory capacity and histological changes in adult gills

This study evaluated the acute toxicity of lead in different life stages of the freshwater prawn Macrobrachium rosenbergii, determined the effect of sublethal Pb concentrations on osmoregulatory capacity (OC), measured the Pb level in gills, and investigated the effect of Pb on the structure of the gills of adult prawns. The 24-, 48-, and 96-h LC₅₀ values for Pb to M. rosenbergii increased progressively with increasing life stage, from post-larvae (PL), juvenile to adult. The 24- and 48-h LC₅₀ values for post-larvae ranged from 0.01- to 0.09-mg Pb/L. The 24-, 48-, and 96-h LC₅₀ values for Pb were lower at 12 ppt than those at 0 ppt for either the juveniles or the adults. At 12 ppt, the 96-h LC₅₀ values in PL11, juvenile and adult were 0.47-, 0.58-, and 2.03-mg Pb/L, respectively. Meanwhile, at 12 ppt, the 96-h LC₅₀ values in PL11, juvenile and adult were 0.63-, 4.44-, and 7.98-mg Pb/L, respectively. In adults, the OC values of controls and prawns exposed to 2- and 4-mg Pb/L at 0 ppt were not significantly different. The OC of prawns exposed to and 2-mg Pb/L at 12 ppt increased by 72 and 109% from the OC of the control prawns. At media 12 ppt, the OC value of prawns exposed to 1-mg Pb/L was significantly different from that of prawns exposed to 2-mg Pb/L. The concentrations of Pb in gill tissues increased significantly in Pb exposed prawns both at 0 and 12 ppt. The level of Pb in gills of prawns exposed to 2-mg Pb/L at 12 ppt was not significantly different from those exposed at 0 ppt. The severe toxic actions of Pb were noted in gills of prawns exposed in media 12 ppt. Hyperplasia and necrosis were observed in gill lamellae, resulting in abnormal gill tips after Pb exposure at media 12 ppt. Since the effect of Pb is more pronounced in higher salinity (12 ppt) than in freshwater (0 ppt) it is clear that aquaculture of M. rosenbergii should be conducted in freshwater ponds.     

Large‐scale coral reef rehabilitation after blast fishing in Indonesia

The severely degraded condition of many coral reefs worldwide calls for active interventions to rehabilitate their physical and biological structure and function, in addition to effective management of fisheries and no‐take reserves. Rehabilitation efforts to stabilize reef substratum sufficiently to support coral growth have been limited in size. We documented a large coral reef rehabilitation in Indonesia aiming to restore ecosystem functions by increasing live coral cover on a reef severely damaged by blast fishing and coral mining. The project deployed small, modular, open structures to stabilize rubble and to support transplanted coral fragments. Between 2013 to 2015, approximately 11,000 structures covering 7,000 m² were deployed over 2 ha of a reef at a cost of US$174,000. Live coral cover on the structures increased from less than 10% initially to greater than 60% depending on depth, deployment date and location, and disturbances. The mean live coral cover in the rehabilitation area in October 2017 was higher than reported for reefs in many other areas in the Coral Triangle, including marine protected areas, but lower than in the no‐take reference reef. At least 42 coral species were observed growing on the structures. Surprisingly, during the massive coral bleaching in other regions during the 2014–2016 El Niño–Southern Oscillation event, bleaching in the rehabilitation area was less than 5% cover despite warm water (≥30°C). This project demonstrates that coral rehabilitation is achievable over large scales where coral reefs have been severely damaged and are under continuous anthropogenic disturbances in warming waters.     

Phenotypic and molecular methods used for identification of oral streptococci and related microorganisms

The oral cavity contains the greatest biodiversity, over 70 species being isolated from mouth mucosa, saliva, denture surfaces and/or dental-plaque. The oral streptococci, representing over 80% of the mouth micro flora, are able to synthesize glucosyl-transferases, enzymes involved in glucans production. Glucans are involved in production of an extracellular slime layer promoting adhesion and formation of a dental plaque biofilm. The 43 isolates studied obtained from partially and/or totally edentulous, were identified by VITEK system using gram-positive identification cards. Species-specific regions within the genes coding for glucosyl-transferases (gtf genes) were targeted for PCR identification of isolates. Sequencing of 16S rRNA was used as gold standard for strain confirmation. VITEK system identified a number of 11 strains as S. mitis/oralis, 12 strains as S. anginosus/gordonii, 12 strains as S. sanguinis/parasanguinis, 3 strains as S. salivarius, 3 strains as S. plurianimalium, 1 strain as S. cristatus and 1 strain as S. alactolyticus, respectively. The PCR system targeting gtf genes was able to identify S. oralis, S. salivarius and S. gordonii strains. Sequence of 16S rRNA discriminated among streptococci species and revealed 16 strains of Leuconostoc mesenteroides. Many studies are needed in order to select the most reliable phenotypic and genotypic methods in order to improve the identification algorithm for oral streptococci used by clinical laboratories. Their accurate identification is mandatory for better understanding their role in human infections.     

Kinetic mechanism of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae

Kinetic data have been collected suggesting a preferred sequential ordered kinetic mechanism for the histidine-tagged homocitrate synthase (HCS) from Saccharomyces cerevisiae with alpha-ketoglutarate binding before AcCoA and CoA released before homocitrate. Oxaloacetate is also a substrate for HCS, but with lower affinity than alpha-ketoglutarate. In agreement with the ordered kinetic mechanism desulfo-CoA is uncompetitive and citrate is competitive vs alpha-ketoglutarate. Varying AcCoA, citrate is a noncompetitive inhibitor as predicted, but CoA is noncompetitive vs AcCoA suggesting binding of CoA to E:homocitrate and E:alpha-ketoglutarate. The product CoA behaves in a manner identical to the dead-end analogue desulfo-CoA, suggesting an E:alpha-ketoglutarate:CoA dead-end complex. Data further suggest an irreversible reaction overall, in agreement with the downhill nature of the reaction as a result of homocitryl-CoA hydrolysis. Fluorescence titration data generally agree with the steady state data, but show finite binding of CoA and AcCoA to free enzyme, suggesting that the mechanism may be random with a high degree of synergism of binding between the reactants.     

Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.     

A molluscan model system in the search for the engram.

A 3-neuron central pattern generator, whose sufficiency and necessity has been directly demonstrated, mediates aerial respiratory behaviour in the pond snail, Lymnaea stagnalis. This behaviour can be operantly conditioned, and this associative learning is consolidated into long-lasting memory. Depending on the operant conditioning training procedure used the learning can be consolidated into intermediate term (ITM) or long-term memory (LTM). ITM persists for only 2-3 h, whilst LTM persists for days to weeks. LTM is dependent on both altered gene activity and new protein synthesis while ITM is only dependent on new protein synthesis. We have now directly established that one of the 3-CPG neurons, RPeD1, is a site of LTM formation and storage. We did this by ablating the soma of RPeD1 and leaving behind a functional primary neurite capable of mediating the necessary synaptic interactions to drive aerial respiratory behaviour by the 3-neuron CPG. However, following soma ablation the neuronal circuit is only capable of mediating learning and ITM. LTM can no longer be demonstrated. However, if RPeD1's soma is ablated after LTM consolidation memory is still present. Thus the soma is not needed for the retention of LTM. Using a similar strategy it may be possible to block forgetting.     

Establishment and characterization of an Ostrinia nubilalis cell line, and its response to ecdysone agonists

Summary A cell line derived from embryonic tissues of the European corn borer, Ostrinia nubilalis (UMC-OnE), was established in EX-CELL 401 medium containing 10% fetal bovine serum. The cells grew in suspension, and were mainly spherical in shape. The cell doubling times at the 17th and 79th passages were 56 and 36 h, respectively. DNA amplification fingerprinting showed that the DNA profile of the OnE cell line was different from that of the southwestern corn borer, Diatraea grandiosella (UMC-DgE), and that of the cotton bollworm, Helicoverpa zea (BCIRL-HZ-AM1). The OnE cell line was responsive to treatments of 20-hydroxyecdysone and the ecdysone agonists, methoxyfenozide (RH-2485) and tebufenozide (RH-5992). These compounds caused similar effects on the cells, which included cell clumping and decreased cell proliferation. The clumps were observed on the third day of incubation, and became larger after 7 d of incubation. After 168 h of incubation, methoxyfenozide and tebufenozide were 35 and 11 times more effective, respectively, in inhibiting proliferation of the OnE cells than was 20-hydroxyecdysone.