Establishment and characterization of a new continuous cell line from Lutzomyia longipalpis (Diptera: Psychodidae) and its susceptibility to infections with arboviruses and Leishmania chagasi

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28ºC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

C-banding and FISH in chromosomes of the blow flies Chrysomya megacephala and Chrysomya putoria (Diptera, Calliphoridae)

The blow flies Chrysomya putoria and C. megacephala have 2n=12 chromosomes, five metacentric pairs of autosomes and an XX/XY sex chromosome pair. There are no substantial differences in the karyotype morphology of these two species, except for the X chromosome which is subtelocentric in C. megacephala and metacentric in C. putoria and is about 1.4 times longer in C. putoria. All autosomes were characterized by the presence of a C band in the pericentromeric region; C. putoria also has an interstitial band in pair III. The sex chromosomes of both species were heterochromatic, except for a small region at the end of the long arm of the X chromosome. Ribosomal genes were detected in meiotic chromosomes by FISH and in both species the NOR was located on the sex chromosomes. These results confirm that C. putoria was the species introduced into Brazil in 1970s, and not C. chloropyga as formerly described.

A new continuous cell line from the mosquito Psorophora confinnis (Diptera: Culicidae) and its susceptibility to infections with some arboviruses

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma.

Phlebotomine sand flies (Diptera: Psychodidae) associated with changing patterns in the transmission of the human cutaneous leishmaniasis in French Guiana

Between March 2000 and December 2001 a survey of the sand flies (Diptera: Phlebotominae) of French Guiana was carried out during 14 nights of captures with CDC light-traps and Malaise traps, and resulted in the collection of 2245 individuals of 38 species. The most abundant species were Lutzomyia (Trichophoromyia) ininii Floch & Abonnenc, Lu.(Psychodopygus) squamiventris maripaensis Floch & Abonnenc, and Lu .(Nyssomyia) flaviscutellata Mangabeira. Half of the collected sand flies females were dissected under field conditions and five species were found harboring Leishmania-like parasites. The Leishmania (Kinetoplastidae: Trypanosomatidae) species were identified by molecular typing, and for the first time Lu. (Nys.) flaviscutellata was found harboring Leishmania (Viannia) guyanensis and Lu. (Tri) ininii harboring unknown Leishmania. The first record for French Guiana of Lu. (Psy.) squamiventris maripaensis harboring L. (V.) naiffi, was also reported. The patterns of diversification of the human cutaneous leishmaniasis transmission in French Guiana are discussed.

Chromosome variability in the Chagas disease vector Rhodnius pallescens (Hemiptera, Reduviidae, Rhodniini)

Rhodnius pallescens is the main vector of Trypanosoma cruzi in Panama and one of the most relevant secondary vectors in Colombia. Despite the importance of this species, there is limited knowledge about the genetic variability along its geographical distribution. In order to evaluate the degree of karyotype variability we analyzed the meiotic behavior and banding pattern of the chromosomes of 112 males of R. pallescens coming from different regions of Colombia and Panama. Using the C-banding technique we identified two chromosomal patterns or cytotypes characterized by differences in the amount, size and distribution of constitutive heterochromatic regions in the chromosome complement (2n = 20 autosomes plus XY in males). The individuals can be easily classified in each cytotype by the analysis of the chromosomes during first meiotic prophase. The frequencies of the cytotypes are variable according to the geographic origin of the populations. This chromosomal divergence together with morphological data supports the existence of three genetically different populations of R. pallescens and provides new information to understand the distribution dynamics of this species.

Karyotypic variation and geographic distribution of Anopheles campestris-like (Diptera: Culicidae) in Thailand

Seventy-one isolines of Anopheles campestris-like were established from wild-caught females collected from human-biting and animal-biting traps at 12 locations in Thailand. All isolines had an average branch summation of seta 2-VI pupal skins ranging from 20.3-30.0 branches, which is in the range of An. campestris (17-58 branches). They showed three different karyotypes based on the amount of extra heterochromatin in the sex chromosomes, namely Forms B (X2, Y2), E (X1, X2, X3, Y5) and a new karyotypic Form F (X2, X3, Y6). Form B has been found only in Chaing Mai and Kamphaeng Phet populations, while Forms E and F are widely distributed throughout the species range. Genetic crosses between the 12 isolines, which were arbitrarily selected as representatives of An. campestris-like Forms B, E and F, revealed genetic compatibility that provided viable progeny through F2 generations, suggesting a conspecific nature of these karyotypic forms. These results are supported by the very low intraspecies variation (genetic distance < 0.005) of ITS2, COI and COII from genomic DNA of the three karyotypic forms.

Evolutionary histories of expanded peptidase families in Schistosoma mansoni

Schistosoma mansoni is one of the three main causative agents of human schistosomiasis, a major health problem with a vast socio-economic impact. Recent advances in the proteomic analysis of schistosomes have revealed that peptidases are the main virulence factors involved in the pathogenesis of this disease. In this context, evolutionary studies can be applied to identify peptidase families that have been expanded in genomes over time in response to different selection pressures. Using a phylogenomic approach, we searched for expanded endopeptidase families in the S. mansoni predicted proteome with the aim of contributing to the knowledge of such enzymes as potential therapeutic targets. We found three endopeptidase families that comprise leishmanolysins (metallopeptidase M8 family), cercarial elastases (serine peptidase S1 family) and cathepsin D proteins (aspartic peptidase A1 family). Our results suggest that the Schistosoma members of these families originated from successive gene duplication events in the parasite lineage after its diversification from other metazoans. Overall, critical residues are conserved among the duplicated genes/proteins. Furthermore, each protein family displays a distinct evolutionary history. Altogether, this work provides an evolutionary view of three S. mansoni peptidase families, which allows for a deeper understanding of the genomic complexity and lineage-specific adaptations potentially related to the parasitic lifestyle.

Establishment and characterisation of a new cell line derived from Culex quinquefasciatus (Diptera: Culicidae)

Insect cell cultures are an important biotechnological tool for basic and applied studies. The objective of this work was to establish and characterise a new cell line from Culex quinquefasciatus embryonic tissues. Embryonated eggs were taken as a source of tissue to make explants that were seeded in L-15, Grace's, Grace's/L-15, MM/VP12, Schneider's and DMEM culture media with a pH range from 6.7-6.9 and incubated at 28ºC. The morphological, cytogenetic, biochemical and molecular characteristics of the cell cultures were examined by observing the cell shapes, obtaining the karyotypes, using a cellulose-acetate electrophoretic system and performing random amplified polymorphic DNA-polymerase chain reaction analysis, respectively. The Grace's/L-15 medium provided the optimal nutritional conditions for cell adhesion and proliferation. Approximately 40-60 days following the explant procedure, a confluent monolayer was formed. Cellular morphology in the primary cultures and the subcultures was heterogeneous, but in the monolayer the epithelioid morphology type predominated. A karyotype with a diploid number of six chromosomes (2n = 6) was observed. Isoenzymatic and molecular patterns of the mosquito cell cultures matched those obtained from the immature and adult forms of the same species. Eighteen subcultures were generated. These cell cultures potentially constitute a useful tool for use in biomedical applications.

The level of ascorbate peroxidase is enhanced in benznidazole-resistant populations of Trypanosoma cruzi and its expression is modulated by stress generated by hydrogen peroxide

Ascorbate peroxidases (APX) are class I heme-containing enzymes that convert hydrogen peroxide into water molecules. The gene encoding APX has been characterized in 11 strains of Trypanosoma cruzi that are sensitive or resistant to benznidazole (BZ). Bioinformatic analysis revealed the presence of two complete copies of the T. cruzi APX (TcAPX) gene in the genome of the parasite, while karyotype analysis showed that the gene was present in the 2.000-kb chromosome of all of the strains analyzed. The sequence of TcAPX exhibited greater levels of similarity to those of orthologous enzymes from Leishmania spp than to APXs from the higher plant Arabidopsis thaliana. Northern blot and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no significant differences in TcAPX mRNA levels between the T. cruzi strains analyzed. On the other hand, Western blots showed that the expression levels of TcAPX protein were, respectively, two and three-fold higher in T. cruzi populations with in vitro induced (17 LER) and in vivo selected (BZR) resistance to BZ, in comparison with their corresponding susceptible counterparts. Moreover, the two BZ-resistant populations exhibited higher tolerances to exogenous hydrogen peroxide than their susceptible counterparts and showed TcAPX levels that increased in a dose-dependent manner following exposure to 100 and 200 µM hydrogen peroxide.

Sharing competences in strategic alliances: a case study of the Cosan and Shell biofuel venture

In a competitive world, the way a firm establishes its organizational arrangements may determine the enhancement of its core competences and the possibility of reaching new markets. Firms that find their skills to be applicable in just one type of market encounter constraints in expanding their markets, and through alliances may find a competitive form of value capture. Hybrid forms of organization appear primarily as an alternative to capturing value and managing joint assets when the market and hierarchy modes do not present any yields for the firm's competitiveness. As a result, this form may present other challenging issues, such as the allocation of rights and principal-agent problems. The biofuel market has presented a strong pattern of changes over the last 10 years. New intra-firm arrangements have appeared as a path to participate or survive among global competition. Given the need for capital to achieve better results, there has been a consistent movement of mergers and acquisitions in the Biofuel sector, especially since the 2008 financial crisis. In 2011 there were five major groups in Brazil with a grinding capacity of more than 15 million tons per year: Raízen (joint venture formed by Cosan and Shell), Louis Dreyfus, Tereos Petrobras, ETH, and Bunge. Major oil companies have implemented the strategy of diversification as a hedge against the rising cost of oil. Using the alliance of Cosan and Shell in the Brazilian biofuel market as a case study, this paper analyses the governance mode and challenging issues raised by strategic alliances when firms aim to reach new markets through the sharing of core competences with local firms. The article is based on documentary research and interviews with Cosan's Investor Relations staff, and examines the main questions involving hybrid forms through the lens of the Transaction Cost Economics (TCE), Agency Theory, Resource Based View (RBV), and dynamic capabilities theoretical approaches. One focal point is knowledge "appropriability" and the specific assets originated by the joint venture. Once the alliance is formed, it is expected that competences will be shared and new capabilities will expand the limits of the firm. In the case studied, Cosan and Shell shared a number of strategic assets related to their competences. Raízen was formed with economizing incentives, as well to continue marshalling internal resources to enhance the company's presence in the world energy sector. Therefore, some challenges might be related to the control and monitoring agents' behavior, considering the two-part organism formed by distinctive organizational culture, tacit knowledge, and long-term incentives. The case study analyzed illustrates the hybrid arrangement as a middle form for organizing the transaction: neither in the market nor in the hierarchy mode, but rather a more flexible commitment agreement with a strategic central authority. The corporate governance devices are also a challenge, since the alignment between the parent companies in the joint ventures is far more complex. These characteristics have led to an organism with bilateral dependence, offering favorable conditions for developing dynamic capabilities. However, these conditions might rely on the partners' long-term interest in the joint venture.

Biogeographic analysis of Crocidiinae (Diptera, Bombyliidae): finding congruence among morphological, molecular, fossil and paleogeographical data

Biogeographic studies dealing with Bombyliidae are rare in the literature and no information is available on its origin and early diversification. In this study, we found evidence from molecular phylogeny and from fossil record supporting a Middle Jurassic origin of the Bombylioidea, taken as a starting point to discuss the biogeography and diversification of Crocidiinae. Based on a previously published phylogenetic hypothesis, we performed a Brooks Parsimony Analysis (BPA) to discuss the biogeographical history of Crocidiinae lineages. This subfamily is mostly distributed over arid areas of the early components of the Gondwanaland: Chile and southern Africa, but also in southwestern Palaearctic and southwestern Nearctic. The vicariant events affecting the Crocidiinae biogeography at the generic level seems to be related to the sequential separation of a Laurasian clade from a Gondwanan clade followed by the splitting of the latter into smaller components. This also leads to a hypothesis of origin of the Crocidiinae in the Middle Jurassic, the same period in which other bombyliid lineages are supposed to have arisen and irradiated.

Biogeographical aspects of drosophilids (Diptera, Drosophilidae) of the Brazilian savanna

Some species of drosophilids have been intensely studied, especially in the areas of genetics and molecular biology. However, little is known about their ecology and biogeography. Here, we revise the records of Drosophilidae species in the Cerrado domain, in order to organize the available information and to recognize patterns of geographic distribution and endemism. The 43 sampled sites in this domain reveal 100 species of drosophilids (10 of them exotic to the Neotropical region), distributed in eight genera. The collections were concentrated mainly in the States of Goiás (including the Federal District) and Minas Gerais. Serra do Cipó is the site with the highest richness of these insects. Of the 90 neotropical species found in the Cerrado, 77 also occur outside this domain, most of which are in the Atlantic Forest. Since extensive areas of this domain were poorly sampled for drosophilids and most studies used only baited traps to collect the flies, this absence of endemism should be carefully considered. It is recommended new inventories in poorly sampled regions, especially in the northeastern part of the domain. Also diversification of collection methods and improvement in the taxonomy of these flies are proposed. These directions should provide a significant increase in the list of species of drosophilids of the Cerrado; and thus, increase our knowledge about its biodiversity.

Distributional patterns of the Neotropical genus Thecomyia Perty (Diptera, Sciomyzidae) and phylogenetic support

Distributional patterns of the Neotropical genus Thecomyia Perty (Diptera, Sciomyzidae) and phylogenetic support. The distributional pattern of the genus Thecomyia Perty, 1833 was defined using panbiogeographic tools, and analyzed based on the phylogeny of the group. This study sought to establish biogeographical homologies in the Neotropical region between different species of the genus, based on their distribution pattern and later corroboration through its phylogeny. Eight individual tracks and 16 generalized tracks were identified, established along nearly the entire swath of the Neotropics. Individual tracks are the basic units of a panbiogeographic study, and correspond to the hypothesis of minimum distribution of the organisms involved. The generalized tracks, obtained from the spatial congruence between two or more individual tracks, are important in the identification of smaller areas of endemism. Thus, we found evidence from the generalized tracks in support of previous classification for the Neotropical region. The Amazon domain is indicated as an area of outstanding importance in the diversification of the group, by the confluence of generalized tracks and biogeographic nodes in the region. Most of the generalized tracks and biogeographical nodes were congruent with the phylogenetic hypothesis of the genus, indicating support of the primary biogeographical homologies originally defined by the track analysis.

Carbon stock and its compartments in a subtropical oxisol under long-term tillage and crop rotation systems

Soil organic matter (SOM) plays a crucial role in soil quality and can act as an atmospheric C-CO2 sink under conservationist management systems. This study aimed to evaluate the long-term effects (19 years) of tillage (CT-conventional tillage and NT-no tillage) and crop rotations (R0-monoculture system, R1-winter crop rotation, and R2- intensive crop rotation) on total, particulate and mineral-associated organic carbon (C) stocks of an originally degraded Red Oxisol in Cruz Alta, RS, Southern Brazil. The climate is humid subtropical Cfa 2a (Köppen classification), the mean annual precipitation 1,774 mm and mean annual temperature 19.2 ºC. The plots were divided into four segments, of which each was sampled in the layers 0-0.05, 0.05-0.10, 0.10-0.20, and 0.20-0.30 m. Sampling was performed manually by opening small trenches. The SOM pools were determined by physical fractionation. Soil C stocks had a linear relationship with annual crop C inputs, regardless of the tillage systems. Thus, soil disturbance had a minor effect on SOM turnover. In the 0-0.30 m layer, soil C sequestration ranged from 0 to 0.51 Mg ha-1 yr-1, using the CT R0 treatment as base-line; crop rotation systems had more influence on soil stock C than tillage systems. The mean C sequestration rate of the cropping systems was 0.13 Mg ha-1 yr-1 higher in NT than CT. This result was associated to the higher C input by crops due to the improvement in soil quality under long-term no-tillage. The particulate C fraction was a sensitive indicator of soil management quality, while mineral-associated organic C was the main pool of atmospheric C fixed in this clayey Oxisol. The C retention in this stable SOM fraction accounts for 81 and 89 % of total C sequestration in the treatments NT R1 and NT R2, respectively, in relation to the same cropping systems under CT. The highest C management index was observed in NT R2, confirming the capacity of this soil management practice to improve the soil C stock qualitatively in relation to CT R0. The results highlighted the diversification of crop rotation with cover crops as a crucial strategy for atmospheric C-CO2 sequestration and SOM quality improvement in highly weathered subtropical Oxisols.

Soil uses in the sugarcane fallow period to improve chemical and physical properties of two latosols (oxisols)

Sugarcane production should be integrated with crop diversification with a view to competitive and sustainable results in economic, social and environmental aspects. The purpose of this study was to assess the influence of different soil uses during the sugarcane fallow period on the chemical and physical properties of eutroferric Red Latosol - LVef (Oxisol) and Acric Latosol - LVw (Acric Oxisol), in Jaboticabal, São Paulo State, Brazil (21º14'05'' S, 48º17'09'' W, 600 m asl). A randomized block design was used with five replications and four treatments, consisting of different soil uses (crops) in the sugarcane fallow period: soybean only, soybean/fallow/soybean, soybean/millet/soybean, and soybean/sunn hemp/soybean. After two soybean crops, the LVef chemical properties remained at intermediate to high levels; while those of the LVw, classified as intermediate to high in the beginning, increased to high levels. Thus, the different soil uses during the sugarcane fallow period allowed the maintenance of LVef fertility levels and the improvement of those of the LVw. Two soybean crops increased macroporosity in the 0.0-0.1 m layer of the LVef; reduced soil aggregates in the 0.0-0.1 and 0.1-0.2 m layers of both soils, and reduced aggregate stability in these two layers of the LVw. Planting pearl millet or sunn hemp between the two soybean growing seasons promoted the formation of larger soil aggregates in the surface layer (0.0-0.1 m) of the LVw.