Preliminary selection study of potential probiotic bacteria from aquacultural area in Tunisia

In order to test the ability to produce antibacterial substances within marine bacteria, prior to select potential probiotics for use in shellfish farming, we targeted a large collection of bacterial isolates (132 strains), brought from the clamRuditapes decussatus and 37 reference strains. First, we proceeded to their biochemical identification and the screening of antibiotic resistance profiles. Else, we investigated their inhibitory activityin vitro against several fish and shellfish pathogens, using two methods: the double-layer agar and the direct simultaneous antagonism methods. The results showed high frequencies of inhibitory producing bacteria (IPB) within the isolates. These bacteria (25%) were aerobic mesophylic bacteria belonging to various bacterial groups: 33.7% oxidase-positive Gram-negative bacteria, 7.4%Enterobacteriaceae and 28% lactic acid bacteria. Besides this group, nine strains produced strong inhibition effect. These bacteria belonged to:Aeromonas hydrophila, Aeromonas sobria, Pseudomonas cepacia, Vibrio sp,Serratia liquefaciens andLactobacillus rhamnosus. They were active against pathogenic bacteria belonging to the genera:Aeromonas, Pseudomonas andVibrio. These potential probiotics were submitted to further investigations prior to their introduction in larval shellfish farming.     

Optimization of β-mannanase production by Bacillus subtilis US191 using economical agricultural substrates

The Bacillus subtilis US191 strain producing highly thermostable β-mannanase was previously selected as potential probiotic candidate for application as feed supplement in poultry industry. Initially, the level of extracellular β-mannanase production by this strain was 1.48 U ml⁻¹. To improve this enzyme titer, the present study was undertaken to optimize the fermentation conditions through experimental designs and valorization of agro-industrial byproducts. Using the Plackett–Burman design, in submerged fermentation, a set of 14 culture variables was evaluated in terms of their effects on β-mannanase production. Locust bean gum (LBG), soymeal, temperature, and inoculum size were subsequently optimized by response surface methodology using Box–Behnken design. Under optimized conditions (1 g L⁻¹ LBG, 8 g L⁻¹ soymeal, temperature of 30°C and inoculum size of 10¹⁰ CFU ml⁻¹), a 2.59-fold enhancement in β-mannanase titer was achieved. Next, to decrease the enzyme production cost, the effect of partial substitution of LBG (1 g L⁻¹) by agro-industrial byproducts was investigated, and a Taguchi design was applied. This allowed the attaining of a β-mannanase production level of 8.75 U ml⁻¹ in presence of 0.25 g L⁻¹ LBG, 5 g L⁻¹ of coffee residue powder, 5 g L⁻¹ of date seeds powder, and 5 g L⁻¹ of prickly pear seeds powder as mannans sources. Overall, a 5.91-fold improvement in β-mannanase production by B. subtilis US191 was achieved.     

Genetic evidence for species cohesion,substructure and hybrids in spruce

The origin and history of species are shaped by various evolutionary dynamics, including their persistence in the face of potential gene flow from related taxa. In this study, we use broad geographical and taxonomic sampling (2,219 individuals) to establish the distribution of species, hybrids and cryptic genetic variation within the conifer genus Picea (spruce) across western North America. We demonstrate that the six species of spruce in this region are distinguishable based on their genetic composition, and that the more closely related Engelmann spruce (P. engelmannii) and white spruce (P. glauca) have generated numerous and widespread hybrids. These hybrids occur in the central Rocky Mountains, well to the south of the well‐established region of admixture in Canada. Additionally, we provide evidence for subdivision within Engelmann spruce, manifested as a southern Rocky Mountains form, and a northern Rocky Mountain and Cascade mountains (western) form. In the intervening central Rocky Mountains region (forests in Wyoming and adjacent states) we found primarily individuals with admixed ancestry. Following their origin, these species of spruce have interacted repeatedly and in different geographical contexts. Multiple pairs of species have been shown to hybridize, yet the species persist and retain distinguishable compositions. At the same time, large geographical areas exist where hybrids are pervasive. Consequently, spruce provide a case study for the maintenance of species boundaries, particularly for how widespread hybridization need not lead to the collapse and loss of species.     

Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections

Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity.     

Population genetic structure of Picea engelmannii, P. glauca and their previously unrecognized hybrids in the central Rocky Mountains

Areas of geographic overlap between potentially hybridizing species provide the opportunity to study interspecific gene flow and reproductive barriers. Here we identified hybrids between Picea engelmannii and P. glauca by their genetic composition at 17 microsatellite markers, and determined the broad-scale geographic distribution of hybrids in the central Rocky Mountains of North America, a geographic region where hybrids and isolation between species had not previously been studied. Parameter estimates from admixture models revealed considerable variation in ancestry within and among collection sites, suggesting that within this area of geographic overlap, the interaction of the two species varies extensively. The results document a previously unrecognized patchy distribution of hybrids between P. engelmannii and P. glauca, including locations where hybrids were not known or expected to exist. Further, the ancestry of many hybrids was consistent with multiple generations of hybridization, with probable directional backcrossing to P. engelmannii, suggesting a relatively porous species boundary. The identification and characterization of hybridization between these spruce in this region raises the question of what factors maintain barriers to gene flow in these long-lived forest trees. The current research lays the groundwork for future study of the ecological and evolutionary contexts of their hybridization, as well as of differential introgression and permeability of species boundaries.