Rearing Tardigrades: Results and Problems

We report our first results of attempts to rear four species of eutardigrades inhabiting different substrates, feeding on different kinds of food and characterized by different sexual conditions and modes of reproduction. Attempts were carried out to follow individual terrestrial carnivorous (Macrobiotus richtersi, M. joannae) and limnic herbivorous (Diphascon cf. scoticum; Isohypsibius monoicus) species. Carnivorous leaf litter-dwelling species were reared in small dishes containing agar as substrate and bacteriophagous nematodes as food. Five generations were obtained with the triploid thelytokous strain of M. richtersi, whereas three generations were obtained with the hermaphrodite species M. joannae. Diphascon cf. scoticum and I. monoicus were reared in small dishes containing algae as food and substrate. Several generations were obtained for both species. Males were never found in D. cf. scoticum and I. monoicus was hermaphroditic. Specimens isolated from hatchings were maintained and reproduced in both species, demonstrating parthenogenesis in the first one and self-fertilization in the latter. Consideration of the problems and on the future applications of tardigrade rearing are discussed.     

Nitrifying activity monitoring and kinetic parameters determination in a biofilm airlift reactor by respirometry

The applicability of batch respirometry, as a simple technique for monitoring off-line nitrifying activity and kinetic parameters, was evaluated using two sets of ammonia and nitrite concentrations. The O₂ uptake rate (OUR) profiles obtained from the assays were adjusted to a substrate inhibition model. The maximum specific ammonia-oxidizing biomass activity (r_Smax) was 0.079 g N-NH₄ ⁺ g VSS^–1 d^–1 with a half saturation coefficient (K_S) of 11 mg N-NH₄ ⁺ l^–1 and an inhibition coefficient (K_i) of 3300 mg N-NH₄ ⁺ l^–1. Besides, the maximum specific value of nitrite-oxidizing activity was 0.082 g N-NO₂ ^– g VSS^–1 d^–1 with a K_S of 4.1 mg N-NO₂ ^– l^–1 and K_i of 1400 mg N-NO₂ ^– l^–1.     

Habitat loss of a rainforest specialist pollinator fly as an indicator of conservation status of the South American Temperate Rainforests

We estimate habitat loss and fragmentation in a hoverfly, Aneriophora aureorufa, used as a representative forest specialist species. This species is a pollinator specialist of two native trees, forming a triad endemic to the South American Temperate Rainforest (SATR). We combine species distribution models with species-specific requirements to estimate the habitat range of A. aureorufa over two non-overlapping time periods (before human settlement to 2000, and from 2000 to 2014). We analyzed the predicted distribution range of A. aureorufa in Chile, quantifying habitat loss in both periods and fragmentation in the latter. In addition, we evaluated the representativeness of the Chilean protected areas system in relation to the current habitat of the species. We found that the total habitat of A. aureorufa decreased by 68.3% compared to historic pre-settlement levels; in the period 2000–2014 the loss was 4.9%. The northern zone was the most affected by habitat loss and fragmentation, with an estimated total loss of 89.9% from the historic period to 2014, with the loss of 238.2 km² per year between 2000 and 2014. Eighteen percent of the habitat of A. aureorufa occurs within protected areas. We found an overrepresentation in the southern zone (24.79%) and an underrepresentation in the northern zone (3.44%). We propose that forest specialist species of the northern zone of the SATR could be threatened due to the high pressure of habitat loss and the underrepresentation of the Chilean protected areas systems.     

Genetic diversity of the brown marmorated stink bug <Emphasis Type="Italic">Halyomorpha halys</Emphasis> in the invaded territories of Europe and its patterns of diffusion in Italy

Halyomorpha halys is an invasive stink bug pest originating from East Asia. In Europe, it was first detected in Switzerland in 2004. It is now present in thirteen countries, and seems to be spreading throughout the continent. In Italy, where it has been recorded since 2012, other than being an urban nuisance, it is causing severe damage in commercial fruit orchards. An integrated approach, using current and previous observational data in space and time and molecular information, was used to identify the genetic diversity of this pest in Europe, its invasion history, and the potential pathways of entry and diffusion. The analysis of 1175 bp of mitochondrial DNA cytochrome c oxidase I and II genes (cox1, cox2) led to the identification of twenty previously unknown haplotypes. The European distribution of H. halys is the result of multiple invasions that are still in progress, and, in some cases, it was possible to identify the specific Asian areas of origin. Moreover, secondary invasions could have occurred among European countries by a bridgehead effect. In Italy, the data were more clearly related to their temporal occurrence, allowing for a clearer reading of the patterns of invasion and dispersion. After having successfully established in localized areas, H. halys further expanded its range by an active dispersion process and/or by jump dispersal events due to passive transport. The multiple introductions from different areas of the native range together with the different patterns of diffusion of H. halys, may hamper the pest management strategies for its containment.     

Cold storage of <Emphasis Type="Italic">Trichogramma nerudai</Emphasis> using an acclimation period

The availability of suitable storage methods for parasitoids is a valuable tool in biological control programs. Studies were conducted to investigate the effects of cold storage with acclimation period on the quality of Trichogramma nerudai Pintureau and Gerding. Prepupae were stored 50, 75 and 100 days at 5 °C with a previous acclimation period of 10 or 20 days at 12 °C. It was possible to arrest the development of T. nerudai. All the treatments with acclimation period of ten days had emergence values under 10% that were not useful to establish a cold storage protocol. Twenty days of acclimation had a positive impact on cold storage tolerance at 50 and 75 days. The adult emergence, the emergence time, the sex ratio, the parasitism and the progeny quality have not been affected by the storage of T. nerudai using an acclimation period of 20 days and until 50 days under cold temperature.     

Diversity of endophytic fungi in <Emphasis Type="Italic">Eucalyptus microcorys</Emphasis> assessed by complementary isolation methods

Brazil is the world’s largest producer country of eucalyptus. Although widely applied in the charcoal industry, no studies have focused on the microorganisms associated with Eucalyptus microcorys. Here, we evaluated the composition and structure of endophytic fungal communities in leaves of E. microcorys through two isolation techniques. A total of 120 fresh leaves were collected in a year-long survey at an eucalyptus plantation in the State of São Paulo (Brazil). Endophytic fungi were isolated by particle filtration (PF) and direct leaf fragment plating (LP) in two media: modified dicloran and synthetic nutrient agar, both supplemented with rose bengal and chloramphenicol. The isolates were grouped into morphospecies and identified by morphology and DNA sequencing. We recovered a total of 709 isolates, representing 59 taxa. All taxa found are reported as endophytic for the first time for E. microcorys. Castanediella eucalypticola and Neophaeomoniella eucalypti are new occurrences reported for Brazil. The LP technique recovered a higher number of taxa and isolates than the PF. However, the PF technique retrieved a higher species/isolate ratio than the LP method, 0.12 and 0.09, respectively. Fungal diversity assessed by diversity metrics did not significantly differ between isolation methods. Both techniques recovered a high number of unique taxa, demonstrating that neither method would individually represent the species richness from E. microcorys. The use of LP and PF provided a greater number of observed taxa and consequently new occurrence of species for Brazil.     

Improvement of the transfucosylation activity of α-l-fucosidase from Thermotoga maritima for the synthesis of fucosylated oligosaccharides in the presence of calcium and sodium

The influence of CaCl₂ and NaCl in the hydrolytic activity and the influence of CaCl₂ in the synthesis of fucosylated oligosaccharides using α-l-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-l-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (a_w) of 0.9672 and of 138% in the presence of 1.1 M CaCl₂ (a_w 0.9581). In addition, the hydrolytic activity was higher when using CaCl₂ compared to NaCl at a_w of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl₂ in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl₂ favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl₂ did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl₂, the decrement in a_w and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides.