Elaboration and characterization of bioplastic films based on bitter cassava starch (Manihot esculenta) reinforced by chitosan extracted from crab (Shylla seratta) shells

Bioplastics are polymer plastics which are derived from renewable biomass resources. In this study, bioplastic films based on two different polysaccharides such as bitter cassava starch and chitosan extracted from crab shells were produced by casting technique, using glycerol as plasticizer. The purposes of this research are to characterize and to figure out the effect of additional chitosan concentrations (0; 10; 20; 30; 50% by weight of starch) on the physicochemical, mechanical and water barrier properties of bioplastic films. The film's solubility in water (S), water absorption capacity (WAP), water vapor permeability (WVP), tensile strength (TS), elongation at break (E), Young's modulus (YM) and biodegradability were investigated. The possible interactions between starch and chitosan molecules were evaluated by Fourier transform infrared spectroscopy (FTIR). From the analysis, the incorporation of the chitosan shows improved results on the water barrier properties of the bioplastic films. Optimum solubility in water, water absorption capacity, and water vapor permeability are obtained on the composition of starch/chitosan was 50/50. Actually, the addition of chitosan increased tensile strength, and elongation at break. The characterization of optimum mechanical proprieties also occurred on the 50/50 composition of cassava-starch and chitosan. At this ratio, tensile strength obtained were 6,3000 MPa; and the elongation at break were 62,8571%. It was found that cassava-starch/chitosan-based films have a stable structure compared to native cassava-starch films.Comment: 14 pages, 15 figure

Evaluation of transgenic Prunus domestica L., clone C5 resistance to Plum pox virus

Plum pox virus (PPV) is one of the most devastating diseases of Prunus species. Since few sources of resistance to PPV have been identified, transgene-based resistance offers a complementary approach to developing PPV-resistant stone fruit cultivars. C5, a transgenic clone of Prunus domestica L., containing the PPV coat protein (CP) gene, has been described as highly resistant to PPV in greenhouse tests, displaying characteristics typical of post-transcriptional gene silencing (PTGS). Moreover, C5 trees exposed to natural aphid vectors in the field remained uninfected after 4 years while susceptible transgenic and untransformed trees developed severe symptoms within the first year. In our study, a high and permanent infection pressure of PPV was provided by bud grafting of inoculum in the field trial of clone C5 conducted in the Czech Republic, in which PPV-infected and healthy control trees were used. Moreover, trees with combined inoculations by PPV, ACLSV and PDV were also used in the trial. The presence of the viruses throughout the tree tissues, the relative titre of the viruses and symptoms on C5 trees have been monitored over the years. The resistance stability of C5 clones under permanent infection pressure is discussed.Keywords: PPV, C5, resistance, real-time PC

Hairpin Plum pox virus coat protein (hpPPV-CP) structure in ‘HoneySweet’ C5 plum provides PPV resistance when genetically engineered into plum (Prunus domestica) seedlings

The genetically engineered plum ‘HoneySweet’ (aka C5) has proven to be highly resistant to Plum pox virus (PPV) for over 10 years in field trials. The original vector used for transformation to develop ‘HoneySweet’ carried a single sense sequence of the full length PPV coat protein (ppv-cp) gene, yet DNA blot analyses indicated that there was an inserted copy of the ppv-cp that appeared to be an inverted repeat structure. Since the resistance mechanism of ‘HoneySweet’ was found to be based on post-transcriptional gene silencing (PTGS), it was hypothesized that the inverted repeat structure conferred the resistance to PPV in ‘HoneySweet’. Sequencing of the transgene insertions confirmed the presence of an inverted repeat of the PPV-CP sequence. We hypothesized that transcription from this structure produced a hairpin (hp) RNA that was responsible for PTGS of the transgene and the destruction of PPV viral RNA resulting in the high level of resistance to PPV infection. In order to confirm this hypothesis the hpPPV-CP insert was cloned from ‘HoneySweet’ and transferred into ‘Bluebyrd’ plum seedlings through Agrobacterium tumefaciens transformation of hypocotyl slices. The introduced DNA contained the CP inverted repeat flanked by 35S promoters on either end. Transgenic plum plants containing single or multiple copies of this hp insert were inoculated with PPV D isolated from Pennsylvania, USA. PPV infection was evaluated through three cycles of cold-induced dormancy (CID) by symptom expression and by two or more ELISA and PCR tests. Of the 18 plants evaluated, eight were always virusfree, five occasionally had weak or moderate infections, and five plants were clearly infected in multiple tests. While all plants of some clones were virus-free others had a mix of uninfected and mildly infected plants of the same clone. Most of the resistant plants contained a single copy of the hp construct. These data strongly support the hypothesis that the hp structure of the ppv-cp insert in ‘HoneySweet’ plum can confer PPV resistance.Keywords: Breeding, gene silencing, Rosaceae, shark

Bacterial diversity of field-caught mosquitoes, Aedes albopictus and Aedes aegypti, from different geographic regions of Madagascar.

peer reviewedSymbiotic bacteria are known to play important roles in the biology of insects, but the current knowledge of bacterial communities associated with mosquitoes is very limited and consequently their contribution to host behaviors is mostly unknown. In this study, we explored the composition and diversity of mosquito-associated bacteria in relation with mosquitoes’ habitats. Wild Aedes albopictus and Aedes aegypti were collected in three different geographic regions of Madagascar. Culturing methods and denaturing gradient gel electrophoresis (DGGE) and sequencing of the rrs amplicons revealed that Proteobacteria and Firmicutes were the major phyla. Isolated bacterial genera were dominated by Bacillus, followed by Acinetobacter, Agrobacterium and Enterobacter. Common DGGE bands belonged to Acinetobacter, Asaia, Delftia, Pseudomonas, Enterobacteriaceae and an uncultured Gammaproteobacterium. Double infection by maternally inherited Wolbachia pipientis prevailed in 98% of males (n = 272) and 99% of females (n = 413); few individuals were found to be monoinfected withWolbachia wAlbB strain. Bacterial diversity (Shannon–Weaver and Simpson indices) differed significantly per habitat whereas evenness (Pielou index) was similar. Overall, the bacterial composition and diversity were influenced both by the sex of individuals and by the environment inhabited by the mosquitoes; the latter might be related to both the vegetation and the animal host populations that Aedes used as food sources.‘Arthropod Symbioses: from fundamental to pest disease management’

Improvement of the growth of Arthrospira (Spirulina) platensis from Toliara (Madagascar): Effect of agitation, salinity and CO2 addition

Arthrospira (Spirulina) platensis Toliara isolated from alkaline and salt lakes in the south-western area of Madagascar is a potential source of proteins that could efficiently fight against food deficiency in developing countries like Madagascar. Up to now, productivity in this country has been low, so a better understanding of the growth conditions of this species is needed to improve its production. Growth experiments were undertaken in bubble columns at laboratory scale. The influence of agitation of the culture, medium salinity (ranging from 13 to 35gL−1) and CO2 addition (ranging from 0 to 2%, v/v) on growth and protein content was examined. Because Arthrospira cells are fragile, a bubble column without additional mixing gave the best growth. Arthrospira (Spirulina) platensis showed higher specific growth rate (μmax) and protein content for lower salinity. Addition of 1% of CO2 improved the productivity by near 60%. The feasability of semi-continuous culture was demonstrated and optimal culture conditions led to a mean productivity of 0.22±0.03gL−1d−1, a mean specific growth rate of 0.015±0.002h−1 and a protein content of 53±2% of total dry weight

Field trials of plum clones transformed with the Plum pox virus coat protein (PPV-CP) gene

Transgenic clones C2, C3, C4, C5, C6, and PT-6, of plum (Prunus domestica L.) transformed with the coat protein (CP) gene of Plum pox virus (PPV), PT-23 transformed with marker genes only, and nontransgenic B70146 were evaluated for sharka resistance under high infection pressure in field trials in Poland and Spain. These sites differed in climatic conditions and virus isolates. Transgenic clone C5 showed high resistance to PPV at both sites. None of the C5 trees became naturally infected by aphids during seven (Spain) or eight (Poland) years of the test, although up to 100% of other plum trees (transgenic clones and nontransgenic control plants) grown in the same conditions showed disease symptoms and tested positively for PPV. Although highly resistant, C5 trees could be infected artificially by chip budding or via susceptible rootstock. Infected C5 trees showed only a few mild symptoms on single, isolated shoots, even up to 8 years post inoculation. These results clearly indicate the long-term nature and high level of resistance to PPV obtained through genetically engineered resistance

Biogeography of the two major arbovirus mosquito vectors, Aedes aegypti and Aedes albopictus (Diptera, Culicidae), in Madagascar

Background: In the past ten years, the Indian Ocean region has been the theatre of severe epidemics of chikungunya and dengue. These outbreaks coincided with a high increase in populations of Aedes albopictus that outcompete its sister taxon Aedes aegypti in most islands sampled. The objective of this work was to update the entomological survey of the two Aedes species in the island of Madagascar which has to face these arboviroses. Methods: The sampling of Aedes mosquitoes was conducted during two years, from October 2007 to October 2009, in fifteen localities from eight regions of contrasting climates. Captured adults were identified immediately whereas immature stages were bred until adult stage for determination. Phylogenetic analysis was performed using two mtDNA genes, COI and ND5 and trees were constructed by the maximum likelihood (ML) method with the gene time reversible (GTR) model. Experimental infections with the chikungunya virus strain 06.21 at a titer of 107.5 pfu/mL were performed to evaluate the vector competence of field-collected mosquitoes. Disseminated infection rates were measured fourteen days after infection by immunofluorescence assay performed on head squashes. Results: The species Aedes aegypti was detected in only six sites in native forests and natural reserves. In contrast, the species Aedes albopictus was found in 13 out of the 15 sites sampled. Breeding sites were mostly found inman-made environments such as discarded containers, used tires, abandoned buckets, coconuts, and bamboo cuts. Linear regression models showed that the abundance of Ae. albopictus was significantly influenced by the sampling region (F = 62.00, p < 2.2 × 10-16) and period (F = 36.22, p = 2.548 × 10-13), that are associated with ecological and climate variations. Phylogenetic analysis of the invasive Ae. albopictus distinguished haplotypes from South Asia and South America from those of Madagascar, but the markers used were not discriminant enough to discern Malagasy populations. The experimental oral infection method showed that six Ae. albopictus populations exhibited high dissemination infection rates for chikungunya virus ranging from 98 to 100%. Conclusion: In Madagascar, Ae. albopictus has extended its geographical distribution whereas, Ae. aegypti has become rare, contrasting with what was previously observed. Changes are predominantly driven by human activities and the rainfall regime that provide suitable breeding sites for the highly anthropophilic mosquito Ae. albopictus. Moreover, these populations were found to be highly susceptible to chikungunya virus. In the light of this study, Ae. albopictus may have been involved in the recent outbreaks of chikungunya and dengue epidemics in Madagascar, and consequently, control measures should be promoted to limit its current expansion.Rôle des moustiques Culicidae, de leurs communautés microbiennes et des réservoirs vertébrés, dans la transmission des arbovirus à Madagasca