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PubMed1997

Enhanced Bioactivity of Recombinant Baculoviruses Expressing Insect-Specific Spider Toxins in Lepidopteran Crop Pests

Two genetically enhanced isolates of the Autographa californica nuclear polyhedrosis virus (AcMNPV) expressing insect-specific neurotoxin genes from the spiders Diguetia canities and Tegenaria agrestis were evaluated for their commercial potential. Since prevention of feeding damage is of primary importance in assessing agronomic efficacy, a method for estimating the median time to cessation of feeding (FT50) was developed. Neonate droplet feeding assays with preoccluded virus samples were conducted to compare the FT50s and median survival times (ST50s) of larvae infected by the toxin-expressing recombinant viruses with those of larvae infected by wild-type AcMNPV and the appropriate polyhedrin-minus control viruses. Low dosages were used to minimize the effect of dosage on the response times, and the time to molting of noninfected larvae was used to audit variability among batches of larvae within and between tests. Appropriate statistics are discussed. To evaluate host spectrum, response times were compared in three lepidopteran insect pests, Trichoplusia ni Hubner, Spodoptera exigua (Hubner), and Heliothis virescens (Fabricius). The recombinant viruses expressing insect-specific toxin genes from T. agrestis and D. canities, designated vAcTalTX-1 and vAcDTX9.2, respectively, significantly reduced both FT50 and ST50 in all three lepidopteran pests. Reductions in feeding times compared to the wild-type virus ranged from 16 to 39% with vAcTalTX-1 and 30 to 40% with vAcDTX9.2. Reductions in survi val time were lower, ranging from 18 to 33% with vAcTalTX-1 and 9 to 24% with vAcDTX9.2. While vAcTalTX-1 tended to kill faster than vAcDTX9.2, vAcDTX9.2 stopped feeding faster than vAcTalTX-1, suggesting that it would be more effective in reducing crop damage.

Journal Article↗
PubMed1997

Replication of a Gonad-Specific Insect Virus in TN-368 Cells in Culture

A newly discovered, nonoccluded, insect virus, known as gonad-specific virus (GSV) was found to replicate in Trichoplusia ni (TN-368) tissue culture cells. Light-microscope observations indicated that 90% of the infected cells showed cytopathic effects by 2 days postinoculation. Electron-microscopic observations revealed the productive replication of this nonoccluded virus with enveloped virus particles clearly visible in the nucleus of infected cells. These particles had approximately the same size and shape reported for GSV recovered from the in vivo host, Helicoverpa zea (corn earworm). Southern blot analysis indicated that the EcoRI restriction enzyme profiles of viral DNA from GSV-infected TN-368 were nearly identical to that of viral DNA from insects. Inoculation of healthy female, H. zea adults with cell-culture-derived virus yielded progeny moths with the same symptoms as insects inoculated with GSV propagated in vivo. These studies clearly demonstrate the ability of GSV to replicate in TN-368 cells in culture.

Journal Article↗
PubMed1999

The role of destruxins in the pathogenicity of metarhizium anisopliae for three species of insect

The entomopathogenic fungus Metarhizium anisopliae var anisopliae produces a family of cyclic peptide toxins, destruxins (DTX), both in culture and in vivo in mycosed insects. The contribution of these insecticidal toxins to the disease process has been investigated in the tobacco hornworm, Manduca sexta (Lepidoptera), the desert locust, Schistocerca gregaria (Orthoptera), and the vine weevil, Otiorhynchus sulcatus (Coleoptera). A significant negative correlation was found between the titer of DTX production in vitro of isolates of M. anisopliae var anisopliae pathogenic for Otiorhynchus and the median lethal time, suggesting a role for the toxin in isolate virulence. The same was true for Manduca-active isolates. A key exception was isolate 703. This is highly virulent for M. sexta, yet does not produce DTX in vitro, grows largely as hyphal fragments in the hemolymph of infected insects, and does not cause host paralysis. These results are discussed in the light of the hypothesis that there are at least two possible virulence strategies among isolates of M. anisopliae var anisopliae pathogenic for Manduca viz the "toxin strategy" and the "growth strategy." For locusts, a strong positive correlation was found only between in vitro toxin production and percentage mortality of individuals in which sporulation did not occur on the cadaver. To account for this, it is suggested that if DTX kills locusts before the fungus has established itself, then the pathogen may not compete effectively with the saprophytic flora and, as a result, fails to sporulate. It is concluded that, in the pathogenesis of M. anisopliae var anisopliae for all three insects, there is a relationship between the titer of DTX production of isolates in vitro and the killing power. Copyright 1999 Academic Press.

Journal Article↗
PubMed2000

Characterization of a Bacillus thuringiensis delta-endotoxin which is toxic to insects in three orders.

We report here the first Bacillus thuringiensis (Bt) toxin which is toxic to insects from three insect orders (Diptera, Coleoptera, and Lepidoptera). An oligonucleotide probe based on the delta-endotoxin N-terminal sequence was used to detect the gene. A 23-kb BamHI fragment containing the intact gene was identified and cloned from Bt strain YBT-226 plasmid DNA into the vector pBluescript II. Through a series of DNA manipulations the size of this fragment was reduced and the gene sequenced. The deduced amino acid sequence gave a predicted molecular mass of 137 kDa and was identical to a cry1Ba protein from Bt subsp. thuringiensis HD-2, which is now designated as Cry1Ba1 under a new classification scheme. This protein also showed 81.6% similarity with the Cry1B protein (Cry1Bb1) from Bt strain EG 5847. When the YBT-226 cry1Ba1 gene was expressed in an acrystalliferous Bt subsp. israelensis strain it produced irregular bipyramidal crystals during sporulation, which reacted specifically with anti-Cry1Ba antiserum. Bioassays using these crystals after purification resulted in significant mortality at low to moderate concentrations to larvae of the house fly (Musca domestica, Diptera), cottonwood leaf beetle (Chrysomela scripta, Coleoptera), and tobacco hornworm (Manduca sexta, Lepidoptera). This broad-spectrum toxicity was not dependent on presolubilization. In assays with insect cell lines not derived from midgut cells, the soluble toxin killed CH1t (Manduca sexta cells) but was inactive against CF1 (Choristoneura fumiferana cells), Aa(s) (Aedes aegypti), and C2 (Culex quinquefasciatus) mosquito cells.

Bacillus thuringiensis↗
PubMed1995

Reflection-polarization patterns at flat water surfaces and their relevance for insect polarization vision

It has recently been shown that horizontally polarized ultraviolet light reflected from the surface of water is the main optical cue for habitat finding by insects living in, on, or near water. What are the polarization properties that make the skylight reflected by water attractive to flying water insects in nature? In this paper, as an approach to this problem, the patterns of the degree and direction of polarization of skylight visible over a flat water surface are computed for unpolarized light from an overcast sky and for partially polarized skylight as a function of the zenith distance of the sun. These patterns are compared with the corresponding celestial polarization patterns. The effect of depolarizing clouds on these reflection-polarization patterns is demonstrated. Reflectivity patterns of a flat water surface are also calculated for clear and overcast skies. The polarization of the blue sky is described by the semi-empirical Rayleigh model. It is assumed that the reflection polarization of skylight at the water surface is governed by the Fresnel formulae. The effect of some modifying factors on the reflection-polarization field is briefly discussed. The adaptations of the visual system of insects living in, on, or near water to reflection-polarization patterns at water surfaces are briefly reviewed and discussed by means of three representative species: the waterstrider (Gerris lacustris), the backswimmer (Notonecta glauca), and the dragonfly (Hemicordulia tau). Copyright 1995 Academic Press Limited

Journal Article↗
PubMed2002

Insects could exploit UV-green contrast for Landmark navigation.

Illumination-invariant detection of landmark features is a prerequisite for landmark navigation in insects. It is suggested that a contrast mechanism involving the UV and green receptors of insect eyes could guarantee a robust separation between natural objects as foreground and sky as background. Using a sensor with a UV and a green channel that in their spectral characteristics are close to the corresponding insect photoreceptors, data of natural objects and sky were collected. The data show that the two classes can be separated by a fixed threshold in the UV-green color space, offering an advantage over a purely UV-based separation that would require a dynamic threshold. Based on a numerical method, UV-green antagonism is shown to guarantee a more reliable discrimination than UV-blue antagonism.

Animals↗
PubMed1996

Immunologic and Ultrastructural Characterization of HIV Pseudovirions Containing Gag and Env Precursor Proteins Engineered in Insect Cells

Expression of human immunodeficiency virus (HIV) Gag precursor protein (Pr55) by recombinant baculoviruses in insect cells results in the assembly and budding of Pr55 as virus-like particles, or Gag pseudovirions. The ultrastructural morphology, size, and sucrose sedimentation rate of Gag pseudovirions are indistinguishable from immature lentivirus particles produced by HIV-infected human cells. Recombinant baculoviruses were engineered to express individually Pr55 and HIV Env glycoprotein precursor (gp160). These recombinant baculoviruses were used to co-infect insect cells to produce chimeric HIV Gag pseudovirions containing gp160 in experiments to develop methodologies for producing complex noninfectious particulate vaccines for HIV. Coexpression of HIV Pr55 and gp160 resulted in the apparent incorporation of gp160 into Gag pseudovirions as determined by immunoblotting with envelope-specific monoclonal antibodies. Furthermore, results from indirect immunogold electron microscopy using monoclonal antibodies to HIV gp120, a component of the Env glycoprotein precursor, suggested that HIV gp160 was specifically incorporated during the budding process into the outer surface of chimeric Gag pseudovirions. Parallel labeling experiments to localize gp120 and Pr55 epitopes on HIV-infected H9 lymphocytes provided results similar to those obtained with chimeric Gag pseudovirions producing recombinant baculovirus-infected insect cells. Parameters influencing immunoelectron microscopy results in cell-surface and postembedding labeling experiments are discussed.

Journal Article↗
PubMed2000

Nuclear genes resolve mesozoic-aged divergences in the insect order Lepidoptera.

Compared to the number of genes available for study of both younger and older divergences, few genes have yet been identified that can strongly resolve phylogenetic splits of Mesozoic age ( approximately 65-250 mya). Thus, reconstruction of Mesozoic-age phylogenies, exemplified by basal divergences within the major orders of holometabolous insects, is likely to be especially dependent on combining multiple lines of evidence. This study tests the potential of the 18S ribosomal RNA gene for reconstructing Mesozoic-aged divergences within the insect order Lepidoptera and its ability when combined with a second, previously analyzed nuclear gene (phosphoenolpyruvate carboxykinase, PEPCK) to strongly resolve these relationships. 18S sequences were obtained for 21 taxa, representing major clades of Lepidoptera plus outgroups from the other "panorpoid orders. A well-corroborated morphology-based "test phylogeny was used to evaluate the effects of partitioning the 18S gene according to variable versus conserved domains, paired versus unpaired sites in the secondary structure, and transition versus transversion substitutions. Likelihood and unweighted parsimony analyses of the 18S data recover the "test phylogeny" almost completely, with no improvement of agreement or support provided by any form of weighting or partitioning. No conflict in signal between 18S and PEPCK was detected by the partition homogeneity test. Combined parsimony analysis yielded strong bootstrap support for nearly all relationships, much higher than for either gene alone, thereby also providing strong evidence on several hypotheses about the early evolution of lepidopteran-plant interactions. These genes in combination may be widely useful for resolving insect divergences of comparable age.

Animals↗
PubMed1993

Expression and functional characterization of recombinant human vascular cell adhesion molecule-1 (VCAM1) synthesized by baculovirus-infected insect cells.

Vascular cell adhesion molecule-1 (VCAM1) is a cell surface glycoprotein produced by the vascular endothelium, as well as on macrophage-like and dendritic cell types, in response to certain inflammatory stimuli. VCAM1 interacts with the integrin VLA4 present on mononuclear leukocytes. We have isolated the cDNA for VCAM1 using RT-PCR by screening a cDNA library from IL-1 beta-activated human endothelial cells. To obtain large quantities of VCAM1 for structural and functional studies, we have produced this protein in insect cells using a baculovirus expression system. Insect cells infected with recombinant virus synthesized human VCAM1 at levels exceeding 3% of total cellular protein following 72 h postinfection. VCAM1-expressing insect cells were shown to bind specifically to a variety of VLA4 expressing cell lines (Jurkat, THP-1, U937). Thus, recombinant VCAM1 protein produced in the baculovirus expression system was localized to the cell surface and was biologically active. Large-scale availability of this adhesion protein should enhance efforts toward the discovery of new antiadhesive (anti-inflammatory and antiatherogenic) therapeutics.

Animals↗
PubMed1995

Expression of rat alpha 1-microglobulin-bikunin in baculovirus-transformed insect cells.

cDNA encoding rat alpha 1-microglobulin-bikunin was ligated into the transfer vector pVL 1392 and recombined with a wild-type baculovirus. The resulting alpha 1-microglobulin-bikunin-encoding baculovirus was used to infect Trichoplusia ni (Hi-5) insect cells. The infected cells secreted alpha 1-microglobulin with maximal concentrations of 15 mg/liter 5 days after infection. The secreted proteins migrated upon SDS-PAGE as two major protein bands, 40 and 26 kDa, corresponding to alpha 1-microglobulin-bikunin and free alpha 1-microglobulin. The results suggested that the cells secreted mostly alpha 1-microglobulin-bikunin, which subsequently was cleaved in the medium, yielding free alpha 1-microglobulin. Both forms were isolated by monoclonal anti-alpha 1-microglobulin affinity chromatography, and alpha 1-microglobulin-bikunin separated from free alpha 1-microglobulin by gel chromatography. The yields of purified alpha 1-microglobulin-bikunin and free alpha 1-microglobulin were approximately 1 and 5 mg, respectively, per liter medium. Insect cell alpha 1-microglobulin displayed a size, shape, and charge heterogeneity similar to alpha 1-microglobulin isolated from rat urine. A panel of monoclonal antibodies raised against urinary alpha 1-microglobulin from several different species bound to rat urinary alpha 1-microglobulin and insect cell secreted alpha 1-microglobulin-bikunin and free alpha 1-microglobulin with approximately the same strength, indicating that the three proteins are folded in similar ways. The results of glycosidase treatments and lectin blotting indicate the absence of neuraminic acid but the presence of one N-linked oligosaccharide and an unspecified number of O-linked oligosaccharides in alpha 1-microglobulin-bikunin and free alpha 1-microglobulin.

Alpha-Globulins↗
PubMed1996

Immediate-early baculovirus vectors for foreign gene expression in transformed or infected insect cells.

Baculovirus expression vectors are used routinely for foreign gene expression and are under intense development as improved biological pesticides. Conventional baculovirus expression vectors are recombinant viruses that can express a foreign gene in insect cells under the control of the polyhedrin promoter, which provides high-level transcription during the very late phase of infection. For some applications, including foreign glycoprotein production and insect pest control, it might be advantageous to have baculovirus vectors that could express foreign gene products in uninfected cells or earlier after infection. To fulfill this need, we have constructed a new set of plasmids that can be used to clone and express foreign genes under the control of a baculovirus ie1 promoter, which is active in uninfected insect cells and throughout infection. We used a subset of these new plasmids to isolate recombinant baculoviruses containing various foreign genes and compared expression of these genes by the resulting immediate-early baculovirus vectors and by conventional baculovirus vectors. As expected, the immediate-early vectors began to express each foreign gene earlier in infection but, by 36-48 h postinfection, the conventional vectors had produced more of each foreign protein. Conventional baculovirus vectors also produced more enzymatic activity from two different procaryotic genes than the immediate-early baculovirus vectors. However, immediate-early vectors produced as much or more enzymatic activity from two different eucaryotic genes encoding secretory pathway proteins than the conventional vectors, even at 48 h postinfection. Hence, this report describes a new set of plasmids that can be used to clone and express foreign genes under the control of the baculovirus ie1 promoter and suggests that immediate-early baculovirus vectors might be as useful as conventional baculovirus expression vectors for producing biologically active eucaryotic secretory pathway proteins.

Animals↗
PubMed1997

Rat dihydroorotate dehydrogenase: isolation of the recombinant enzyme from mitochondria of insect cells.

Mammalian dihydroorotate dehydrogenase (EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis is located in the mitochondrial inner membrane with functional connection to the respiratory chain. From the cDNA of rat liver dihydroorotate dehydrogenase cloned in our laboratory the first complete sequence of a mammalian enzyme was deduced. Two hydrophobic stretches centered around residues 20 and 357, respectively, and a short N-terminal mitochondrial targeting sequence of 10 amino acids was proposed. A recombinant baculovirus containing the rat liver cDNA for dihydroorotate dehydrogenase was constructed and used for virus infection and protein expression in Trichoplusia ni cells. The targeting of the recombinant protein to mitochondria of the insect cells was monitored by activity determination of dihydroorotate dehydrogenase in subcellular compartments in comparison to succinate dehydrogenase activity (EC 1.3.5.1), which is a specific marker enzyme of the inner mitochondrial membrane. The results of subcellular distribution were verified by Western blotting with anti-dihydroorotate dehydrogenase immunoglobulins. The activity of the recombinant enzyme in the mitochondria of infected insect cells was found to be about 570-fold above the level of dihydroorotate dehydrogenase in rat liver mitochondria. By cation exchange chromatography of the Triton X-114 solubilisate of mitochondria, dihydroorotate dehydrogenase was purified to give a specific activity of 15 U/mg at pH 8.0. This was a marked progress over the six-step purification procedure of the enzyme from rat liver which resulted in a specific activity of 0.7 U/mg at pH 8.0. The characteristic flavin absorption spectrum obtained with the recombinant enzyme gave strong evidence that the rodent enzyme is a flavoprotein. By enzyme kinetic studies K(m) values for dihydroorotate and ubiquinone were 6.4 and 9.9 microM with the recombinant enzyme, and were 5.0 and 19.7 microM, respectively, with the rat liver enzyme. After expression of only truncated forms of human dihydroorotate dehydrogenase, the present successful generation of the complete rodent enzyme using insect cells and the efficient procedure will promote structure and function studies of the eukaryotic dihydroorotate dehydrogenases in comparison to the microbial enzyme.

Amino Acid Sequence↗
PubMed1998

Expression and purification of human stromelysin 1 and 3 from baculovirus-infected insect cells.

Stromelysin 1 (ST1) is a member of the matrix metalloproteinase (MMP) family probably involved in extracellular matrix degradation. Stromelysin 3 (ST3), considered by sequence homology to be a member of the MMP family of proteases, is specifically expressed in the stroma adjacent to the invasive tumoral cells, but its role in cancer progression remains to be elucidated. Genes encoding ST1 and ST3 were expressed in lepidopteran insect cells using the baculovirus expression vector system. Recombinant baculoviruses were obtained after cloning the full-length cDNA of ST1 and ST3 in plasmids pBacPAK1 and pBacPAK9, respectively. Sf9 insect cells infected with the recombinant baculovirus overexpressed the zymogen proST1 (60 kDa) in an insoluble form, a peak of expression being reached from 24 h postinfection. After solubilization in 8 M urea, and further refolding, activation, and purification, 0.3 mg of mature ST1 (30 kDa), purified to 90% homogeneity, was obtained per 5 x 10(8) infected cells. Recombinant ST1 exhibited proteolytic activity on alpha2-macroglobulin, casein, fibronectin, alpha1-antitrypsin, and laminin. The recombinant zymogen proST3 (55 kDa) was expressed as a soluble form in insect cells, maximal expression occurring at 72 h postinfection. After purification to 95% homogeneity, 2.5 mg of proST3 was obtained per 5 x 10(8) infected cells. A number of proteases including plasmin, urokinase, and ST1 were shown to be able to cleave proST3 giving rise to defined bands of 50-30 kDa. The ST3 mature form of 45 kDa (mST3) was also expressed in the baculovirus system and the obtained protein, 2. 5 mg per 5 x 10(8) infected cells purified to 80% homogeneity, was shown to be active on both casein degradation and alpha2-macroglobulin entrapment assays. Our results suggest that the baculovirus system offers a convenient and efficient means to produce ST1 and ST3 in order to carry out further biochemical studies.

Animals↗
PubMed2000

Purification and characteristics of recombinant human folylpoly-gamma-glutamate synthetase expressed at high levels in insect cells.

Folylpoly-gamma-glutamate synthetase activity is central to the operation of folate metabolism and is essential for the survival of mammalian stem cell populations but the very low levels of endogenous expression of this enzyme have greatly limited its study. We now report the expression of cytosolic folylpoly-gamma-glutamate synthetase (FPGS) cloned from human leukemic cells in baculovirus-infected insect cells at levels of 4-5% of the total soluble protein of the cells. As was the case with endogenously expressed mammalian FPGS, recombinant enzyme was quantitatively blocked at the amino terminus in spite of the large-scale production in insect cells. A three-step purification procedure resulted in an overall yield of 7-35 mg per liter of culture with a recovery of about 50% and purity approximately 95%; pure enzyme was stable to storage for extended periods. Pure protein had a specific activity of 25 micromol h(-1)mg(-1) with aminopterin as a substrate and used a broad spectrum of folates as substrates. The pure enzyme also carried out ATP hydrolysis in the absence of a folate substrate or glutamic acid; this partial reaction occurred at a k(cat) about 0.4% that of the full reaction. In vitro, this single protein added several (1-8) moles of glutamic acid per mole of folate analog, the same spectrum of folate polyglutamates as seen in vivo. The quantities of pure enzyme achievable in insect cells should allow functional and structural studies on this enzyme.

Adenosine Triphosphatases↗
PubMed2000

Endogenous glutathione-binding proteins of insect cell lines: characterization and removal from glutathione S-transferase (GST) fusion proteins.

After affinity purification on immobilized glutathione, insect-cell-derived glutathione S-transferase (GST) fusion proteins contain variable amounts of protein contaminants of about 23-24 kDa. We have isolated these glutathione-binding proteins from the widely used Sf9 and Hi5 insect cell lines and characterized them by LC-MS and N-terminal sequencing. Based on the observation that these proteins have higher affinity for glutathione than GST fusions, we have found that by using differential elution conditions the amount of such contaminants in GST fusion preparations can be strongly reduced directly during the affinity purification step. The main interest of these results is that they are not restricted to a specific construct, but rather they seem to apply to various insect-cell-derived GST fusions.

Animals↗
PubMed2000

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells.

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.

Animals↗
PubMed2000

Bioreactor-scale production and one-step purification of Epstein-Barr nuclear antigen 1 expressed in baculovirus-infected insect cells.

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated malignancies and is essential for EBV-genome maintenance. Antibodies to EBNA1 are abundantly detected in serum of most EBV carriers but EBNA1 escapes recognition by effector T-lymphocytes. To further study the functional and immunological characteristics of EBNA1 it is important to have sufficient quantities of purified EBNA1 available. This paper describes a simple, reproducible method for the production and purification of EBV-encoded EBNA1 expressed in insect cells (bEBNA1). For quantification of EBNA1 expression levels in cell lines and for monitoring bEBNA1 purification and overall yields we developed a quantitative and EBNA1-specific capture ELISA. We observed that EBV-positive cell lines express EBNA1 at different levels, with the B cell lymphoblastoid cell line X50/7 having the highest production. However, much larger quantities (380-fold) were obtained by expressing bEBNA1 in recombinant-baculovirus-infected Sf9 insect cells. Scaling-up experiments revealed that bEBNA1 expression kinetics and protein stability are identical in 1-liter stirred bioreactors when compared to expression in stationary culture flasks. Optimal expression was reached after 72 h following inoculation at 1 pfu/cell, when insect cell viability was about 50%. For purification the nuclear fraction containing most of the bEBNA1 (>95%) was isolated. Solubilized bEBNA1 was purified by a one-step oriP DNA-Sepharose affinity purification procedure, using biotinylated PCR-amplified family of repeats (FR)-domain products immobilized onto streptavidin agarose. A >200-fold specific enrichment was reached and yields of bEBNA1 with an estimated purity of >95%.

Animals↗
PubMed1999

Modelling density-dependent resistance in insect-pathogen interactions.

We consider a mathematical model for a host-pathogen interaction where the host population is split into two categories: those susceptible to disease and those resistant to disease. Since the model was motivated by studies on insect populations, we consider a discrete-time model to reflect the discrete generations which are common among insect species. Whether an individual is born susceptible or resistant to disease depends on the local population levels at the start of each generation. In particular, we are interested in the case where the fraction of resistant individuals in the population increases as the total population increases. This may be seen as a positive feedback mechanism since disease is the only population control imposed upon the system. Moreover, it reflects recent experimental observations from noctuid moth-baculovirus interactions that pathogen resistance may increase with larval density. We find that the inclusion of a resistant class can stabilise unstable host-pathogen interactions but there is greatest regulation when the fraction born resistant is density independent. Nonetheless, inclusion of density dependence can still allow intrinsically unstable host-pathogen dynamics to be stabilised provided that this effect is sufficiently small. Moreover, inclusion of density-dependent resistance to disease allows the system to give rise to bistable dynamics in which the final outcome is dictated by the initial conditions for the model system. This has implications for the management of agricultural pests using biocontrol agents-in particular, it is suggested that the propensity for density-dependent resistance be determined prior to such a biocontrol attempt in order to be sure that this will result in the prevention of pest outbreaks, rather than their facilitation. Finally we consider how the cost of resistance to disease affects model outcomes and discover that when there is no cost to resistance, the model predicts stable periodic outbreaks of the insect population. The results are interpreted ecologically and future avenues for research to address the shortfalls in the present model system are discussed.

Animals↗