Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix.

The cuticle of the silkworm Bombyx mori was demonstrated to contain pro-phenol oxidase [zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] and its activating cascade. The activating cascade contained at least one serine proteinase zymogen (latent form of pro-phenol oxidase activating enzyme). When the extracted cascade components were incubated with Ca2+, the latent form of pro-phenol oxidase activating enzyme was itself activated and, in turn, converted through a limited proteolysis of pro-phenol oxidase to phenol oxidase. Immuno-gold localization of prophenol oxidase in the cuticle using a cross-reactive hemolymph anti-pro-phenol oxidase antibody revealed a random distribution of this enzyme in the nonlamellate endocuticle and a specific orderly arrayed pattern along the basal border of the laminae in the lamellate endocuticle of the body wall. Furthermore, prophenol oxidase was randomly distributed in the taenidial cushion of the tracheal cuticle. At the time of pro-phenol oxidase accumulation in the body wall cuticle, no pro-phenol oxidase mRNA could be detected in the epidermal tissue, whereas free-circulating hemocytes contained numerous transcripts of pro-phenol oxidase. Our results suggest that the pro-phenol oxidase is synthesized in the hemocytes and actively transported into the cuticle via the epidermis.

Molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin.

Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes. A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%. Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins. Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins. The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence-Asn-49-Arg-50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both subunits were indicated to be N-acetylated. The cDNAs of both pro-PO subunits lacked signal peptide sequences. This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.

The role of insect cell lines in an artificial diet for the parasitold wasp, Trichogramma pretiosum

Trichogramma species are mass-produced for biological control using host eggs. Artificial diets have been developed to reduce production costs, however, most include insect haemolymph as a major component, which still results in a significant expense. Medium conditioned with insect cell lines has produced some success as a haemolymph replacement in artificial diets for several parasitoid wasp species. Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae) was the first species to develop successfully to the adult stage on diets containing concentrated HeliothiS zea (Boddie) (Lepidoptera: Noctuidae) cells. Tricho-gramma pretiosum Riley (Hymenoptera: Trichogrammatidae) was subsequently grown to the adult stage on a similar cell line diet. This success encouraged a systematic investigation into the use of insect cell lines in Trichogramma artificial diets. We compared the effect of diets containing insect cells with diets containing conditioned cell line media. Diets containing insect cells produced significantly more pupae than diets containing conditioned medium and, although not significant, produced a higher number of adults. Second, we compared the effect of diets containing cell lines established from ovary-associated tissue of H. zea and embryo tissue of Aedes albopictus (Skuse) (Diptera: Culicidae) on T pretiosum development. Trichogramma pretiosum development was not significantly different on diets containing cells from the two origins and tissue types. Third, the effect of cell storage on T pretiosum development was observed. HeliothiS zea cells in medium were stored at 4 degrees C and room temperature (22 degrees C for one, two, four and seven days before addition to artificial diets. Cell viability was calculated for these storage treatments. HeliothiS zea cells could be stored at 4 degrees C for up to seven days with no detrimental effect on T pretiosum development. Tricho-gramma pretiosum development did not depend on cell viability. The use of insect cell lines as a haemolymph replacement has the potential to significantly reduce production costs and simplify Trichogramma artificial diets with the eventual aim of replacing host production in mass rearing facilities. (c) 2005 Elsevier Inc. All rights reserved.

Are insect immune suppressors driving cellular uptake reactions?

Many insect parasitoids that deposit their eggs inside immature stages of other insect species inactivate the cellular host defence to protect the growing embryo from encapsulation. Suppression of encapsulation by polydnavirus-encoded immune-suppressors correlates with specific alterations in hemocytes, mainly cytoskeletal rearrangements and actin-cytoskeleton breakdown. We have previously shown that the Cotesia rubecula polydnavirus gene product CrV1 causes immune suppression when injected into the host hemocoel. CrV1 is taken up by hemocytes although no receptors have been found to bind the protein. Instead CrV1 uptake depends on dimer formation, which is required for interacting with lipophorin, suggesting a CrV1-lipophorin complex internalisation by hemocytes. Since treatment of hemocytes with oligomeric lectins and cytochalasin D can mimic the effects of CrV1, we propose that some dimeric and oligomeric adhesion molecules are able to cross-link receptors on the cell surface and depolymerise actin by leverage-mediated clearance reactions in the hemolymph.

Insect mortality in Spathodea campanulata Beauv. (Bignoniaceae) flowers

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Insect feeding preferences on Piperaceae species observed in São Paulo city, Brazil

Piperaceae species have been placed among the basal angiosperm and are adapted to a variety of habitats including moist forests, secondary vegetation and dry high lands. The major anatomical/morphology features are of small trees, vines, and shrubs for Piper species, while the epiphytic and succulent characteristics are predominant forms among Peperomia species. Their secondary chemistry can be mostly represented by amides, phenylpropanoids/lignoids, and chromenes in addition to a phletoria of biosynthetically mixed-origin secondary compounds. Although several amides and lignans are known as insecticides, several phytophagous insects, among which some considered pests of economic importance, have been observed feeding vigorously on Piperaceae species. Herein we describe the feeding preferences of fourteen phytophagous species of Coleoptera, Lepidoptera and Hemiptera over approximately fifty Piperaceae species observed in São Paulo, SP, Brazil, in a long-term basis.

Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

Background: Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS). Methods: In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results: The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions: Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.

Africanized honey bees more efficiently convert protein diets into hemolymph protein than do Carniolan bees (Apis mellifera carnica)

The superiority of Africanized over European honey bees in tropical and subtropical regions of the New World is both well documented and poorly understood. As part of an effort to try to understand the process by which the displacement of European bees occurred, we examined the ability of these two types of bees and of hybrids between the two to convert natural and artificial diets into usable protein. Newly emerged bees from colonies of tropically adapted Africanized and temperate-origin Carniolan bees and first-generation hybrids between the two were caged and fed artificial and natural protein diets for six days to determine whether there were differences in their ability to use these diets. The Africanized bees developed significantly higher protein levels in the hemolymph than did the Carniolan bees. The difference was 31% when the bees were fed bee bread (37.5 and 28.56 mu g protein/mu L hemolymph, respectively). The hybrids developed protein levels intermediate between the two parental types. These were approximately 10 times the levels found in bees fed with sucrose alone. Superior food conversion efficiency of the Africanized bees may be one of the reasons for their superiority both in the wild and for beekeeping in tropical and subtropical Latin America.

A New Generation of X Ray Irradiators for Insect Sterilization

Recent fears of terrorism have provoked an increase in delays and denials of transboundary shipments of radioisotopes. This represents a serious constraint to sterile insect technique (SIT) programs around the world as they rely on the use of ionizing radiation from radioisotopes for insect sterilization. To validate a novel X ray irradiator, a series of studies on Ceratitis capitata (Wiedemann) and Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) were carried out, comparing the relative biological effectiveness (RBE) between X rays and traditional gamma radiation from (60)Co. Male C. capitata pupae and pupae of both sexes of A. fraterculus, both 24 - 48 h before adult emergence, were irradiated with doses ranging from 15 to 120 Gy and 10-70 Gy, respectively. Estimated mean doses of 91.2 Gy of X and 124.9 Gy of gamma radiation induced 99% sterility in C. capitata males, Irradiated A. fraterculus were 99% sterile at approximate to 40-60 Gy for both radiation treatments. Standard quality control parameters and mating indices were not significantly affected by the two types of radiation. The RBE did not differ significantly between the tested X and gamma radiation, and X rays are as biologically effective for SIT purposes as gamma rays are. This work confirms the suitability of this new generation of X ray irradiators for pest control programs that integrate the SIT.

Inorganic elements in the fat bodies of Diatraea saccharalis (Lepidoptera: Crambidae) larvae parasitized by Cotesia flavipes (Hymenoptera: Braconidae)

Koinobiont parasitoids use several strategies to regulate the host`s physiological processes during parasitism. Although many aspects of host-parasitoid interactions have been explored, studies that attempted to assess the effects of parasitism on the availability of inorganic elements in the host are virtually nonexistent. Therefore, we aimed to evaluate the effects of parasitism on the concentrations of inorganic elements in the fat bodies of larvae of Diatraea saccharalis (Lepidoptera: Crambidae) during the development of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae), by using total reflection X-ray fluorescence (TXRF). TXRF analysis allowed comparisons of the changes in the availability of the elements P. S. K, Ca, Cr, Fe, Ni, Cu, and Zn in the fat body tissues of D. saccharalis larvae parasitized by C. flavipes. Overall, the concentration of inorganic elements was higher early in parasitoid development (1 and 3 days after parasitism) compared to non-parasitized larvae, but much lower towards the end of parasitoid development (7 and 9 days after parasitism). Ca, K, and S were reduced after the fifth day of parasitism, which affected the total abundance of inorganic elements observed in the fat bodies of the parasitized hosts. The regulatory mechanisms or pathological effects related to the observed variation of the host inorganic elements induced by the parasitoid remain unknown, but there might be a strategy to make these elements available to the parasitoid larvae at the end of their development, when higher metabolic activity of the host fat body is required to sustain parasitoid growth. The observed variation of the host`s inorganic elements could also be related to the known effects of parasitism on the host`s immune response. (C) 2010 Elsevier Inc. All rights reserved.

Citrus Sudden Death Is Transmitted by Graft-Inoculation and Natural Transmission Is Prevented by Individual Insect-Proof Cages

Citrus sudden death (CSD) transmission was studied by graft-inoculation and under natural conditions. Young sweet orange trees on Rangpur rootstock were used as indicator plants. They were examined regularly for one or two characteristic markers of CSD: (i) presence of a yellow-stained layer of thickened bark on the Rangpur rootstock, and (ii) infection with the CSD-associated marafivirus. Based on these two markers, transmission of CSD was obtained, not only when budwood for graft-inoculation was taken from symptomatic, sweet orange trees on Rangpur, but also when the budwood sources were asymptomatic sweet orange trees on Cleopatra mandarin, indicating that the latter trees are symptomless carriers of the CSD agent. For natural transmission, 80 young indicator plants were planted within a citrus plot severely affected by CSD. Individual insect-proof cages were built around 40 indicator plants, and the other 40 indicator plants remained uncaged. Only two of the 40 caged indicator plants were affected by CSD, whereas 17 uncaged indicator plants showed CSD symptoms and were infected with the marafivirus. An additional 12 uncaged indicator plants became severely affected with citrus variegated chlorosis and were removed. These results strongly suggest that under natural conditions, CSD is transmitted by an aerial vector, such as an insect, and that the cages protected the trees against infection by the vector.

Modifying insect population age structure to control vector-borne disease

Age is a critical determinant of the ability of most arthropod vectors to transmit a range of human pathogens. This is due to the fact that most pathogens require a period of extrinsic incubation in the arthropod host before pathogen transmission can occur. This developmental period for the pathogen often comprises a significant proportion of the expected lifespan of the vector. As such, only a small proportion of the population that is oldest contributes to pathogen transmission. Given this, strategies that target vector age would be expected to obtain the most significant reductions in the capacity of a vector population to transmit disease. The recent identification of biological agents that shorten vector lifespan, such as Wolbachia, entomopathogenic fungi and densoviruses, offer new tools for the control of vector-borne diseases. Evaluation of the efficacy of these strategies under field conditions will be possible due to recent advances in insect age-grading techniques. Implementation of all of these strategies will require extensive field evaluation and consideration of the selective pressures that reductions in vector longevity may induce on both vector and pathogen.

Semliki Forest virus as an expression vector in insect cell lines

Studies were undertaken to determine if replication-deficient Semliki Forest virus expression vectors could be successfully used to express foreign gene constructs in insect cell lines. Using green fluorescent protein (GFP) as a marker we recorded infection levels of nearly 100% in the Aedes albopictus cell lines C6/36 and Aa23T, as well as in the Ae. aegypti cell line MOS20. The virus was capable of infecting an Anopheles gambiae cell line MOS55. The amount of GFP protein produced in each cell line was quantified. Northern analysis of viral transcription revealed the presence of novel transcripts in Aa23T, C6/36, and MOS55 cell lines, but not in the BHK or MOS20. The initial characterization of these transcripts is described.