Lipids in Insect Oocytes: From the Storage Pathways to Their Multiple Functions.

In insect physiology, the mechanisms involved in the buildup and regulation of yolk proteins in developing oocytes have been thoroughly researched during the last three decades. Comparatively, the study of lipid metabolism in oocytes had received less attention. The importance of this issue lies in the fact that lipids make up to 40% of the dry weight of an insect egg, being the most important supply of energy for the developing embryo. Since the oocyte has a very limited capacity to synthesize lipids de novo, most of the lipids in the mature eggs arise from the circulation. The main lipid carriers in the insect circulatory system are the lipoproteins lipophorin and vitellogenin. In some species, the endocytosis of lipophorin and vitellogenin may account for about 10% of the lipids present in mature eggs. Thus, most of the lipids are transferred by a lipophorin-mediated pathway, in which the lipoprotein unloads its lipid cargo at the surface of oocytes without internalization. This chapter recapitulates the current status on lipid storage and its utilization in insect oocytes and discusses the participation of key factors including lipoproteins, transfer proteins, lipolytic enzymes, and dynamic organelles such as lipid droplets. The new findings in the field of lipophorin receptors are presented in the context of lipid accumulation during egg maturation, and the roles of lipids beyond energy source are summarized from the perspective of oogenesis and embryogenesis. Finally, prospective and fruitful areas of future research are suggested.

Comparative morphology of spermatozoa and reproductive systems of zorapteran species from different world regions (Insecta, Zoraptera).

The male and female reproductive apparatus of Zorotypus magnicaudelli (Malaysia), Zorotypus huxleyi (Ecuador) and Zorotypus weidneri (Brazil) were examined and documented in detail. The genital apparatus and sperm of the three species show only minor differences. The testes are larger in Z. magnicaudelli. Z. huxleyi lacks the helical appendage in the accessory glands. A long cuticular flagellum is present in Z. magnicaudelli and in the previously studied Zorotypus caudelli like in several other species, whereas it is absent in Z. weidneri, Z. huxleyi, Zorotypus hubbardi, Zorotypus impolitus and Zorotypus guineensis. Characteristic features of the very similar sperm are the presence of: a) two dense arches above the axoneme; b) a 9 + 9+2 axoneme with detached subtubules A and B of doublets 1 and 6; c) the axonemal end degenerating with enlarging accessory tubules; d) accessory tubules with 17 protofilaments; e) three accessory bodies beneath the axoneme; and f) two mitochondrial derivatives of equal shape. The first characteristic (a) is unknown outside of Zoraptera and possibly autapomorphic. The sperm structure differs distinctly in Z. impolitus and Z. hubbardi, which produce giant sperm and possess a huge spermatheca. The presence of the same sperm type in species either provided with a sclerotized coiled flagellum in males or lacking this structure indicates that a different organization of the genital apparatus does not necessarily affect the sperm structure. The flagellum and its pouch has probably evolved within Zoraptera, but it cannot be excluded that it is a groundplan feature and was reduced several times. The fossil evidence and our findings suggest that distinct modifications in the genital apparatus occurred before the fragmentation of the Gondwanan landmass in the middle Cretaceous.

Determination of diflubenzuron residues in milk and cattle tissues / Determinação de resíduos de diflubenzuron em leite e tecidos bovinos

Diflubenzuron (DFB) is used to control ectoparasitic infestation by inhibiting larvae development in the manure and feces of treated animals. It is also currently been used to control tick infestations. In this study, milk and tissues from cattle treated orally with DFB for a 77-120 day period with a commercial product containing the compound were analyzed for the presence of residues. DFB residues were determined by using extraction with acetonitrile, cleanup with C18 SPE and chromatographic analysis by HPLC with UV detection (254nm). DFB was not detected in any of the analysed samples (<0.006mg kg-1 for fat, <0.014mg kg-1 for muscle, <0.015mg kg-1 for kidney, <0.016mg kg-1 for liver and <0.0006mg kg-1 for milk). In this manner, the use of this compound, according to the manufacturer's suggested doses may result in cattle milk, liver, kidneys, fat and muscles being considered safe regarding the presence of DFB residues.

O diflubenzuron (DFB) é um inibidor de desenvolvimento de insetos que inibe a síntese de quitina com atividade ovicida e larvicida e está sendo utilizado na pecuária para o controle do carrapato. Leite e tecidos provenientes de bovinos tratados por um período de 77 a 120 dias com um produto comercial contendo DFB foram analisados quanto à presença de resíduos. Os resíduos de DFB foram determinados utilizando-se extração com acetonitrila, limpeza por SPE C18 e cromatografia líquida de alta eficiência com detecção por UV (254nm). DFB não foi detectado em nenhuma das amostras analisadas (<0.006mg kg-1 para gordura, <0,014mg kg-1 para músculo, <0,015mg kg-1 para rim, <0,016mg kg-1 para fígado e <0.0006mg kg-1 para leite). Dessa forma, a utilização do princípio ativo conforme recomendado pelo fabricante e em níveis suficientes para se obter o efeito larvicida desejado deve resultar em leite, fígado, rins, gordura e músculos que podem ser considerados seguros para o consumo em termos da presença DFB.

Determination of diflubenzuron residues in milk and cattle tissues / Determinação de resíduos de diflubenzuron em leite e tecidos bovinos

Diflubenzuron (DFB) is used to control ectoparasitic infestation by inhibiting larvae development in the manure and feces of treated animals. It is also currently been used to control tick infestations. In this study, milk and tissues from cattle treated orally with DFB for a 77-120 day period with a commercial product containing the compound were analyzed for the presence of residues. DFB residues were determined by using extraction with acetonitrile, cleanup with C18 SPE and chromatographic analysis by HPLC with UV detection (254nm). DFB was not detected in any of the analysed samples (<0.006mg kg-1 for fat, <0.014mg kg-1 for muscle, <0.015mg kg-1 for kidney, <0.016mg kg-1 for liver and <0.0006mg kg-1 for milk). In this manner, the use of this compound, according to the manufacturer's suggested doses may result in cattle milk, liver, kidneys, fat and muscles being considered safe regarding the presence of DFB residues.(AU)

O diflubenzuron (DFB) é um inibidor de desenvolvimento de insetos que inibe a síntese de quitina com atividade ovicida e larvicida e está sendo utilizado na pecuária para o controle do carrapato. Leite e tecidos provenientes de bovinos tratados por um período de 77 a 120 dias com um produto comercial contendo DFB foram analisados quanto à presença de resíduos. Os resíduos de DFB foram determinados utilizando-se extração com acetonitrila, limpeza por SPE C18 e cromatografia líquida de alta eficiência com detecção por UV (254nm). DFB não foi detectado em nenhuma das amostras analisadas (<0.006mg kg-1 para gordura, <0,014mg kg-1 para músculo, <0,015mg kg-1 para rim, <0,016mg kg-1 para fígado e <0.0006mg kg-1 para leite). Dessa forma, a utilização do princípio ativo conforme recomendado pelo fabricante e em níveis suficientes para se obter o efeito larvicida desejado deve resultar em leite, fígado, rins, gordura e músculos que podem ser considerados seguros para o consumo em termos da presença DFB.(AU)

Kinetic characterization of the group II Helicoverpa armigera nucleopolyhedrovirus propagated in suspension cell cultures: implications for development of a biopesticides production process.

Large-scale commercialization of baculovirus biopesticides for the control of insect pests requires a cell culture production process, and knowledge of the infection kinetics is a vital prerequisite for process optimization. Well-characterized kinetic parameters have so far only been reported for the commercially established recombinant Autographa californica nucleopolyhedrovirus (AcMNPV), a Group I NPV. In this work, key infection kinetic parameters of the Group II NPV Helicoverpa armigera nucleopolyhedrovirus (HaSNPV), and its Few Polyhedra (FP) mutant, were well characterized for the first time, in suspension HzAM1 insect cell cultures, to facilitate the scale-up of an HaSNPV-based biopesticide. The FP mutant had a selective advantage over wild-type HaSNPV in cell cultures, and the kinetic analysis showed that this was due to a superior budding rate, rather than a faster binding rate (BR) or longer budding duration. Another finding was that wild-type HaSNPV had very poor infection kinetics when compared with AcMNPV, exhibiting an 18-fold lower BR, a more than 50-fold lower budding rate, and a 60-fold lower extracellular/total progeny virus ratio. Such poor infection kinetics have serious implications during scale-up of an HaSNPV biopesticide production process, including the requirement for large volumes of virus inocula and the difficulty of achieving synchronous infections. Groups I and II NPVs may have very different infection kinetics because of their different envelope fusion proteins. This study is the first to compare the two groups of NPVs in terms of well-characterized cell-specific infection kinetics, and the findings may indicate a phylogenetic basis for kinetic differences.

Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells?

Cry proteins, produced by Bacillus thuringiensis (Bt), are widely used for the control of insect pests in agriculture as spray products or expressed in transgenic crops, such as maize and cotton. Little was known regarding the mechanism of action of these toxins when the first commercial Bt product was introduced fifty years ago. However, research on the mechanism of action over the last two decades has enhanced our knowledge of toxin interaction with membrane receptors and their effects in insect midgut cells. All this information allowed for the rational design of improved toxins with higher toxicity or toxins that overcome insect resistance, which could compromise Bt use and effectiveness in the field. In this review we discuss and evaluate the different models of the mode of action of Cry toxins, including a discussion about the role of various receptors in toxin action.

HgeTx1, the first K+-channel specific toxin characterized from the venom of the scorpion Hadrurus gertschi Soleglad.

A novel toxin was identified, purified and characterized from the venom of the Mexican scorpion Hadrurus gertschi (abbreviated HgeTx1). It has a molecular mass of 3950 atomic mass units (a.m.u.) and contains 36 amino acids with four disulfide bridges established between Cys1-Cys5, Cys2-Cys6, Cys3-Cys7 and Cys4-Cys8. It blocks reversibly the Shaker B K(+)-channels with a Kd of 52nM. HgeTx1 shares 60%, 45% and 40% sequence identity, respectively, with Heterometrus spinnifer toxin1 (HsTX1), Scorpio maurus K(+)-toxin (maurotoxin) and Pandinus imperator toxin1 (Pi1), all four-disulfide bridged toxins. It is 57-58% identical with the other scorpion K(+)-channel toxins that contain only three disulfide bridges. Sequence comparison, chain length and number of disulfide bridges analysis classify HgeTx1 into subfamily 6 of the alpha-KTx scorpion toxins (systematic name: alpha-KTx 6.14).

Proteins in a DNA world: expression systems for their study.

Every day, new proteins are discovered and the need to understand its function arises. Human proteins have a special interest, because to know its role in the cell may lead to the design of a cure for a disease. In order to obtain such information, we need enough protein with a high degree of purity, and in the case of the human proteins, it is almost impossible to achieve this by working on human tissues. For that reason, the use of expression systems is needed. Bacteria, yeast, animals and plants have been genetically modified to produce proteins from different species. Even "cell-free" systems have been developed for that purpose. Here, we briefly review the options with their advantages and drawback, and the purification systems and analysis that can be done to gain understanding on the function and structure of the protein of interest.

Rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems.

Virus-like particles have a wide range of applications, including vaccination, gene therapy, and even as nanomaterials. Their successful utilization depends on the availability of selective and scalable methods of product recovery and purification that integrate effectively with upstream operations. In this work, a strategy based on aqueous two phase system (ATPS) was developed for the recovery of double-layered rotavirus-like particles (dlRLP) produced by the insect cell-baculovirus expression system. Polyethylene glycol (PEG) molecular mass, PEG and salt concentrations, and volume ratio (Vr, volume of top phase/volume of bottom phase) were evaluated in order to determine the conditions where dlRLP and contaminants concentrated to opposite phases. Two-stage ATPS consisting of PEG 400-phosphate with a Vr of 13.0 and a tie-line length (TLL) of 35% (w/w) at pH 7.0 provided the best conditions for processing highly concentrated crude extract from disrupted cells (dlRLP concentration of 5 microg/mL). In such conditions intracellular dlRLP accumulated in the top phase (recovery of 90%), whereas cell debris remained in the interface. Furthermore, dlRLP from culture supernatants accumulated preferentially in the interface (recovery of 82%) using ATPS with a Vr of 1.0, pH of 7.0, PEG 3350 (10.1%, w/w) and phosphate (10.9%, w/w). The purity of dlRLP from culture supernatant increased up to 55 times after ATPS. The use of ATPS resulted in a recovery process that produced dlRLP with a purity between 6 and 11% and an overall product yield of 85% (w/w), considering purification from intracellular and extracellular dlRLP. Overall, the strategy proposed in this study is simpler than traditional methods for recovering dlRLP, and represents a scalable and economically viable alternative for production processes of vaccines against rotavirus infection with significant scope for generic commercial application.

Cell death in the Germline: mechanisms and consequences for reproductive plasticity in social bees

Its serial architecture makes the insect ovary an interesting playground to study the regulation of cell death and identify critical check points along the apical-basal axis of the ovarioles. In Drosophila melanogaster, cell death is observed at two points: (1) in the germarium, where entire germ cell clusters may die in response to environmental conditions, and (2) as an obligatory event at the end of oogenesis, when nurse cells dump their cytoplasm into the oocyte and, subsequently, when the follicle epithelial cells form a chorion. The social organization of bees, wasps and ants depends on the monopolization of reproduction by a queen. This has marked consequences on the ovary phenotype of queens and workers. The role of programmed cell death in larval ovary development and in adult ovary function is best studied in honey bees. During larval development, workers loose over 90% of the ovariole primordia. This cell death is induced by a low juvenile hormone titer causing breakdown of the actin cytoskeleton in germ cell clusters. The actin cytoskeleton also plays a major role in the control of cell death in the ovary of adult bees, where many TUNEL-labeled and pycnotic nuclei are detected in a germarial region rich in actin agglomerates. This suggests that common mechanisms may regulate cell death in the ovaries of bees, both during the shaping of the caste-specific ovary phenotypes during larval development, and during the tuning of reproductive activity in adult bees.

Proteins in a DNA world: expression systems for their study / Proteínas en el mundo del DNA: Sistemas de expresión para su estudio

Every day, new proteins are discovered and the need to understand its function arises. Human proteins have a special interest, because to know its role in the cell may lead to the design of a cure for a disease. In order to obtain such information, we need enough protein with a high degree of purity, and in the case of the human proteins, it is almost impossible to achieve this by working on human tissues. For that reason, the use of expression systems is needed. Bacteria, yeast, animals and plants have been genetically modified to produce proteins from different species. Even "cell-free" systems have been developed for that purpose. Here, we briefly review the options with their advantages and drawback, and the purification systems and analysis that can be done to gain understanding on the function and structure of the protein of interest.

Cada día, nuevas proteínas son descubiertas y surge la necesidad de caracterizarlas, siendo las de origen humano las que presentan un mayor interés. Conocer su función nos ayudará a entender padecimientos y diseñar una posible cura. Sin embargo, obtener suficiente cantidad de proteínas humanas en cantidad para llevar a cabo los análisis pertinentes, presenta una gran dificultad. Por tal razón, es necesario el uso de sistemas de expresión de proteínas heterólogas. Bacterias, levaduras, animales y plantas han sido modificados genéticamente para expresar proteínas de otras especies, e incluso sistemas in vitro han sido desarrollados para producir proteínas. En este artículo se revisan brevemente las opciones con sus ventajas y desventajas, así como las estrategias de purificación y los análisis que se pueden llevar a cabo para avanzar en el conocimiento de la función y estructura de la proteína de interés.

Intracellular distribution of rotavirus structural proteins and virus-like particles expressed in the insect cell-baculovirus system.

The production of virus-like particles (VLP) is of interest to several fields. However, little is known about their assembly when they are expressed in insect cells, as it occurs in conditions different to those of native virus. Knowledge of the localization of recombinant proteins and of the site of accumulation of VLP can increase the understanding of VLP assembly and be useful for proposing production strategies. In this work, the rotavirus proteins VP6 and the fusion protein GFPVP2 were expressed in High Five insect cells. Recombinant proteins and rotavirus-like particles (RLP) were located and visualized by confocal, epifluorescence and electron microscopy. Single-layered (sl) RLP (conformed by GFPVP2) accumulated in the cytoplasm as highly ordered aggregates. In contrast, VP6 formed fibrillar structures composed of various tubes of VP6 that were not associated to microtubules. Coexpression of GFPVP2 and VP6 altered the distribution of both proteins. VP6 formed aggregates, even when all other conditions of individual protein expression remained unchanged. Double-layered (dl) RLP were observed in dense zones of the cytoplasm, but were not in ordered aggregates. It was determined that the assembly of both slRLP and dlRLP occurs intracellularly. Accordingly, strategies for the optimum assembly of dlRLP should guarantee that each cell produces both recombinant proteins.

Production of viral progeny in insect cells undergoing apoptosis induced by a mutant Anticarsia gemmatalis nucleopolyhedrovirus.

The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is the most successful viral biopesticide in use worldwide. We have demonstrated that despite widespread apoptosis and no protein synthesis at 48 h p.i., UFL-AG-286 cells infected with a mutant of AgMNPV (vApAg), produced significant amounts of budded virus (BVs) and viral DNA late in infection. However, a different susceptible cell line (BTI-Tn5B 1-4) showed no signs of apoptosis and produced 3.5 times more budded virus when infected with vApAg. A comparison of DNA from AgMNPV and vApAg digested with the same restriction enzymes showed differences in the restriction pattern, indicating that the vApAg phenotype might be due to a mutation in a gene or genes responsible for directly or indirectly inhibiting apoptosis in UFL-AG-286 cells.

Perfusion culture of baculovirus-infected BTI-Tn-5B1-4 insect cells: a method to restore cell-specific beta-trace glycoprotein productivity at high cell density.

The impact of different cultivation-infection strategies on the productivity of baculovirus-infected BTI-Tn-5B1-4 (High Five) cells was investigated. Using beta-trace protein as the recombinant glycoprotein, the effects of multiplicity of infection (MOI) and time of infection (TOI) were studied on growth after infection as well as the degree of infection and recombinant protein productivity in batch culture. The highest productivities were found when infecting Tn5 cells at early exponential growth phase (EGP) (low cell density) using a high MOI. To increase the productive cell density of Tn5 cells after beta-trace-baculovirus infection, we performed studies infecting cells in the range of 1 to 5 x 10(6) cells/mL in fresh medium. Although the protein production was increased twofold, a strong negative cell density effect was still observed when maximal productive cell density exceeded 1 x 10(6) cells/mL. To verify whether the changing cell environment of the batch experiments was responsible for the decrease in protein productivity at increasing cell density at infection, several perfusion experiments were designed by infecting Tn5 cells at cell densities over 2 x 10(6) cells/mL under more steady-state conditions. The use of this experimental setup enabled successful infections at high cell densities with volumetric productivities of up to 1.2 g L(-1) day(-1) of beta-trace protein, which is very high for a glycoprotein expressed with the baculovirus expression vector system (BEVS). The cell specific protein productivity observed after infections at higher cell densities in perfusion mode was the same as in batch experiments at low cell concentrations, which clearly demonstrates that the cell density effect could be completely overcome with perfusion cultivation.

The effect of 1,10-phenanthroline on the chromosome damage and sister-chromatid exchanges induced by streptozotocin in mammalian and insect cells.

The effect of the metal chelating agent 1,10-Phenanthroline (PNT) on the streptozotocin (STZ)-induced chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) and mosquito (Aedes albopictus) cells was investigated. Treatment of CHO and mosquito cells with STZ produced a significant and dose-response increase in the yield of CAs as well as SCEs (p<0.05). The addition of PNT prevented the induction of CAs by STZ in both types of cells, causing a significant decrease in the frequency of STZ-induced CAs (46.5-72.5%) (p<0.05). This fact indicates that intracellular transition metals are implicated in STZ-induced CAs and that the Fenton reaction (Fe(2+)+H(2)O(2)-->OH degrees +OH(-)+ Fe(3+)) is partly responsible for the production of CAs by this compound. On the other hand, the addition of PNT to CHO and mosquito cell cultures did not prevent the induction of SCEs by STZ. Therefore, it is valid to assume that the induction of CAs and SCEs by STZ occurs by different mechanisms.

Hematoxylin: a simple, multiple-use dye for chromosome analysis

A staining mixture of hematoxylin-iron alum combined with a strong hydrochloric hydrolysis was successfully applied for chromosome observation of several kinds of plants and some animals. Slightly different procedures were developed for different materials and objectives. For plant cells, the most important technical aspect was the use of 5 N HCl hydrolysis, which resulted in a very transparent cytoplasm, combined with an intense, specific hematoxylin stain. This technique is recommended for cytogenetical analysis in general, and it is especially indicated for practical classes, due to its simplicity and high reproducibility of results. Moreover, the deep contrast observed makes this technique very useful for sequential staining of cells previously analyzed with other stains, as well as for materials with fixation problems.

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain.

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 10(9) infected cells in three liters of culture.

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain

A simple and inexpensive shaker/Erlenmeyer flask system for largescale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive biorector consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 10(9) infected cells in three liters of culture.

Trypanosoma cruzi flagellar repetitive antigen expression by recombinant baculovirus: towards an improved diagnostic reagent for Chagas' disease.

We constructed a recombinant baculovirus that expressed part of a Trypanosoma cruzi flagellar repetitive antigen (FRA). Both cell- associated and secreted forms of recombinant FRA were detected in cultures of virus-infected Spodoptera frugiperda (Sf9) cells. These forms show a complex pattern after polyacrylamide gel electrophoresis and Western blot analysis using either an anti-FRA rabbit serum or human Chagasic sera. Competitive Western-blot experiments revealed that all bands react with the same antibodies as a bacterially-derived FRA. Polymerase chain reaction and Southern blots of the recombinant viral DNA also showed a complex pattern, suggesting the presence of more than one repeat unit in the viral genome. When tested against a panel of human sera from an endemic area for Chagas' disease, FRA recombinant-Sf9 culture supernatant showed the same reactivity as purified FRA produced in bacteria.