Dynamics of deletion genotypes in an experimental insect virus population

Defective viruses, that are deficient in certain essential genes, are maintained in the population by trans-complementation, exploiting the gene products of complete genotypes in co-infected cells. This process becomes prevalent only when cells are frequently infected by several virus particles, and only then will the fitness of defective viruses be subjected to frequency-dependent selection. Deletion variants that are not infectious per os are present in a multicapsid nucleopolyhedrovirus (SfMNPV, Baculoviridae) that infects the fall army worm, Spodoptera frugiperda. These variants enhance the pathogenicity and, therefore, the likelihood of transmission of the virus when co-infecting cells with complete genotypes, resulting in occlusion bodies (OBs) that may contain both genotypes co-occluded. Mixtures of complete (B) and defective (C) variants in ratios of 90% B+10% C, 50% B+50% C and 10% B+90% C were used to inoculate by injection S. frugiperda larvae. Viral OBs extracted from diseased insects were subjected to four or five successive rounds of per os infection. Following successive passages, genotype frequencies in all three experimental populations converged to a single equilibrium frequency comprising approximately 20% of deletion genotype C and approximately 80% of complete genotype B. This mirrors the relative proportions of deletion (22%) and complete (78%) genotypes observed in the wild-type SfMNPV population. The pathogenicity of experimental populations at the final passage was not significantly different from that of the wild-type isolate. In contrast, OBs of all genotype mixtures were significantly more pathogenic than OBs of genotype B alone. A population genetics model, in which virus populations were assigned linear frequency-dependent transmissibility values, was in remarkably close agreement to empirical data. Clearly, non-infectious deletion variants can profoundly affect the likelihood of transmission and the genetic structure and stability of virus populations.     

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-Tn5B1-4) 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.     

Variation in vein density and mesophyll cell architecture in a rice deletion mutant population

There is a need to develop rice plants with improved photosynthetic capacity and efficiency in order to enhance potential grain yield. Alterations in internal leaf morphology may be needed to underpin some of these improvements. One target is the production of a 'Kranz-like' anatomy, commonly considered to be required to achieve the desired levels of photosynthesis seen in C(4) crops. Kranz anatomy typically has two or three mesophyll cells interspersing adjacent veins. As a first step to determining the potential for such anatomical modifications in rice leaves, a population of rice deletion mutants was analysed for alterations in vein patterning and mesophyll cells in the interveinal regions. Significant variation is demonstrated in vein arrangement and the sequential distribution of major and minor veins across the leaf width, although there is a significant correlation between the total number of veins present and the width of the leaf. Thus the potential is demonstrated for modifying rice leaf structure. Six distinct rice mutant lines, termed altered leaf morphology (alm) mutants, were analysed for the architecture of their interveinal mesophyll cell arrangement. It is shown that in these mutant lines, the distance between adjacent minor veins and adjacent minor and major veins is essentially determined by the size of the interveinal mesophyll cells rather than changes in mesophyll cell number across this region, and hence interveinal distance changes as a result of cell expansion rather than cell division. This observation will be important when developing screens for traits relevant for the introduction of Kranz anatomy into rice. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details).     

Complementation of growth defect in an ampC deletion mutant of Escherichia coli

Abstract β-Lactamase genes of class-A ( Rtem ) and class-C ( ampC ) were placed under control of an inducible tac -promoter and expressed in Escherichia coli . Expression of RTEM had no observable effect on the growth properties of E. coli strains HB101 ( ampC ⁺) or MI1443 (Δ ampC ). E. coli MI1443 exhibited a decline in growth rate at mid-exponential phase which could be delayed by expression of AmpC at early-exponential phase. AmpC expression otherwise inhibited growth, particularly during the transition into exponential phase where growth was prevented altogether. We suggest that the AmpC β-lactamase, but not RTEM, may have an additional cellular function as a peptidoglycan hydrolase.     

Scale-dependent mechanisms in the population dynamics of an insect herbivore

A multiscale approach has lead to significant advances in the understanding of species population dynamics. The scale-dependent nature of population processes has been particularly clearly illustrated for insect herbivores. However, one of the most well-studied insect herbivores, the galling sawfly Euura lasiolepis, has to date been examined almost exclusively at fine spatial scales. The preference-performance, plant vigour and larval survival hypotheses are well supported by this species. Here, we test these hypotheses at a spatial scale larger than that previously considered, i.e. across a landscape in northern Arizona represented by an altitudinal gradient encompassing a series of drainages. We also develop a qualitative model for understanding the population dynamics of E. lasiolepis based on patterns of survival and mortality found in this study and previous ones. Gall density was highly variable across the altitudinal gradient, not explained by host plant variables, and thus a poor surrogate fot population abundance. These findings for the first time fail to support the plant vigour and preference hierarchy hypotheses for E. lasiolepis. Dispersal limitation most likely explains the lack of support for these hypotheses at this scale. By contrast, sawfly survival, gall abortion, parasitism and larval mortality were well explained by host plant quality variables and altitude. The larval survival hypothesis was well supported and is thus comparatively scale-invariant. A qualitative model developed here highlighted the importance of both willow water status and disturbance in determining host plant quality, as well as an apparent trade off between shoot length and plant moisture status in determining vital rates across the altitudinal gradient. This study thus demonstrated for the first time the scale-dependent nature of mechanisms underlying the population dynamics E. lasiolepis, and identified the interaction between parasitism and altitude as a novel mechanism underlying spatial patterns in the survival and mortality patterns of this species.     

Host ecology determines the relative fitness of virus genotypes in mixed-genotype nucleopolyhedrovirus infections

Mixed‐genotype infections are common in many natural host–parasite interactions. Classical kin‐selection models predict that single‐genotype infections can exploit host resources prudently to maximize fitness, but that selection favours rapid exploitation when co‐infecting genotypes share limited host resources. However, theory has outpaced evidence: we require empirical studies of pathogen genotypes that naturally co‐infect hosts. Do genotypes actually compete within hosts? Can host ecology affect the outcome of co‐infection? We posed both questions by comparing traits of infections in which two baculovirus genotypes were fed to hosts alongside inocula of the same or a different genotype. The host, Panolis flammea, is a herbivore of Pinus sylvestris and Pi. contorta. The pathogen, PfNPV (a nucleopolyhedrovirus), occurs naturally as mixtures of genotypes that differ, when isolated, in pathogenicity, speed of kill and yield. Single‐genotype infection traits failed to predict the ‘winning’ genotypes in co‐infections. Co‐infections infected and caused lethal disease in more hosts, and produced high yields, relative to single‐genotype infections. The need to share with nonkin did not cause fitness costs to either genotype. In fact, in hosts feeding on Pi. sylvestris, one genotype gained increased yields in mixed‐genotype infections. These results are discussed in relation to theory surrounding adaptive responses to competition with nonkin for limited resources.     

Spatiotemporal dynamics of an insect population in response to chemical substances

Insect pests represent a serious problem for the agriculture. The most popular control technic is the application of insecticide. This method, however, presents serious risks to the human healthy and to the environment so that new approaches, such as the applications of natural repellent, biological control and push–pull strategies, have been attempted. In this paper, we consider a spatially-discrete system described by Coupled Map Lattices to analyze the dynamics of an insect population when some chemical – insecticide or repellent – is spread over the population. The habitat is supposed to have plenty of resources so that insect individuals only move (flee) in response to the chemical concentration. The chemical substance, in turn, spreads by diffusion and by the wind. We show that the escape behavior of the insects and the wind can reduce the effectiveness of the chemical. Furthermore, we present the corresponding continuous equations that describe the system in the macroscopic scale.     

Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant

E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.     

Genealogy reconstruction from short tandem repeat genotypes in an Amazonian population

We have reconstructed partial genealogies in a sample of 44 SW Amazonian Rondonian Surui, in which 45 dinucleotide short tandem repeat polymorphisms had previously been typed. The genotypes of 488 pairs of individuals having an age difference of 13 or greater were compared, and parentage was excluded if a pair failed to share an allele at more than one locus. In order to test the power of this method, we computed the expected distribution of the number of exclusionary loci for such pairs of unrelated individuals, as well as that for individuals with different degrees of relatedness, and compared it to the observed distribution. We estimated that the pairs compared contained ∼20% of individual pairs with a first-cousin relation or closer. A total of 25 pairs were identified as possible parent-child. In three instances, we could identify two or more children having a common parent; we computed a relatedness coefficient in order to establish whether the children were full or half sibs. The genealogies inferred show that instances of polygyny and polyandry (or, alternatively, serial mating), in addition to apparent monogamy, can be found among the Surui. The Surui sample can be used as a model for paleoanthropological populations, in which the determination of relatedness can provide further insights into the social structure of past populations. We estimate that, depending on the history of the populations and the degree of inbreeding, 10–20 highly informative nuclear loci should be typed in order to infer genealogies with acceptable confidence. Am J Phys Anthropol 108:137–146, 1999. © 1998 Wiley-Liss, Inc.     

Degrees of heterogeneity and the survival of an insect population

Summary The results of simulation experiments with a stochastic model incorporating internal and external sources of heterogeneity suggest that the western tent caterpillar is a risk spreader of a type discussed byDen Boer (1968). By maintaining physiological plasticity in its population,M.c. pluviale can survive periods of bad weather and then expand rapidly in an improved climate. Intrinsic variability in the moths’ dispersal capacity also ensures that some will continue to contribute to populations in the climatic refuges where the species persists during unfavorable periods, while others can quickly colonize marginal areas as soon as favorable conditions return. This northern population of the western tent caterpillar seems distinctly less well adapted to a world in which the terrain and the weather are more homogeneous than on the Saanich Peninsula. As this series of papers is jointly produced by our whole group, the order of authorship on each is decided by chance, not protocol.     

Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line

p53 is a key modulator of a variety of cellular stresses. In human neuroblastomas, p53 is rarely mutated and aberrantly expressed in cytoplasm. In this study, we have identified a novel p53 mutant lacking its COOH-terminal region in neuroblastoma SK-N-AS cells. p53 accumulated in response to cisplatin (CDDP) and thereby promoting apoptosis in neuroblastoma SH-SY5Y cells bearing wild-type p53, whereas SK-N-AS cells did not undergo apoptosis. We found another p53 (p53DeltaC) lacking a part of oligomerization domain and nuclear localization signals in SK-N-AS cells. p53DeltaC was expressed largely in cytoplasm and lost the transactivation function. Furthermore, a 3'-part of the p53 locus was homozygously deleted in SK-N-AS cells. Thus, our present findings suggest that p53 plays an important role in the DNA-damage response in certain neuroblastoma cells and it seems to be important to search for p53 mutations outside DNA-binding domain.     

肠炎沙门氏菌鸡源株ompR 基因缺失株的构建及生物学特性与亲本株的比较

【目的】为了探讨ompR基因在肠炎沙门氏菌生物被膜形成及毒力中的作用。【方法】以肠炎沙门氏菌作为母本,运用自杀性载体pGMB151构建了ompR基因缺失株,结晶紫染色法和扫描电镜观察测定缺失株的生物被膜形成能力,细胞的吸附和侵入及小鼠攻毒试验测定缺失株的毒力。【结果】RT-PCR和蛋白表达证明了ompR基因缺失株构建成功;该缺失株不表达纤维素和菌毛,不形成生物被膜;上皮细胞吸附和侵入试验表明缺失株与野生株具有相同的吸附和侵入率;BALB/c鼠腹腔感染性试验表明,缺失株的半数致死量为106.67CFU,而野生株的半数致死量小于2 CFU。【结论】ompR基因既是肠炎沙门氏菌生物膜形成的调控基因,又是重要的毒力基因。     

Analysis of a naturally-occurring deletion mutant of Spodoptera frugiperda multiple nucleopolyhedrovirus reveals sf58 as a new per os infectivity factor of lepidopteran-infecting baculoviruses

The Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, is structured as a mixture of nine genotypes (A-I). Occlusion bodies (OBs) of SfMNPV-C, -D and -G pure genotypes are incapable of oral transmission; a phenotype which in SfMNPV-C and -D is due to the absence of pif1 and pif2 genes. The complete sequence of the SfMNPV-G genome was determined to identify possible factors involved in this phenotype. Deletions of 4860 bp (22,366-27,225) and 60 bp (119,759-119,818) were observed in SfMNPV-G genome compared with that of the predominant complete genotype SfMNPV-B (132,954 bp). However no genes homologous to previously described per os infectivity factors were located within the deleted sequences. Significant differences were detected in the nucleotide sequence in sf58 gene (unknown function) that produced changes in the amino acid sequence and the predicted secondary structure of the corresponding protein. This gene is conserved only in lepidopteran baculoviruses (alpha- and betabaculoviruses). To determine the role of sf58 in peroral infectivity a deletion mutant was constructed using bacmid technology. OBs of the deletion mutant (Sf58null) were not orally infectious for S. frugiperda larvae, whereas Sf58null rescue virus OBs recovered oral infectivity. Sf58null DNA and occlusion derived virions (ODVs) were as infective as SfMNPV bacmid DNA and ODVs in intrahemocelically infected larvae or cell culture, indicating that defects in ODV or OB morphogenesis were not involved in the loss of peroral infectivity. Addition of optical brightener or the presence of the orally infectious SfMNPV-B OBs in mixtures with SfMNPV-G OBs did not recover Sf58null OB infectivity. According to these results sf58 is a new per os infectivity factor present only in lepidopteran baculoviruses.     

Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp

The agp gene encoding ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis. By in vitro DNA recombination technology, agp deletion mutant (agp ^–) of cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutation led to a complete absence of glycogen biosynthesis. As compared with WT (wild type), a 60% decrease in ratio of the c-phycocyanine/chlorophyll a and no significant change in the carotenoid/chlorophyll a were observed in agp ^– cells. The agp ^– mutant had 38% less photosynthetic capacity when grown in light over 600 mol m^–2 s^–1. Under lower light intensity, the final biomass of the mutant strain was only 1.1 times of that of the WT strain under mixotrophic condition after 6 d culture. Under higher light intensity, however, the final biomass of the WT strain under mixotrophic conditions was 3 times that of the mutant strain after 6 d culture and 1.5 times under photoautotrophic conditions. The results indicate that there is a minimum requirement for glycogen synthesis for normal growth and development in cyanobacteria.     

Construction and characterization of an absolute deletion mutant of Escherichia coli ribonuclease II

Abstract The degradation of mRNA plays a central role in the control of protein synthesis. In Escherichia coli , the rnb gene encodes ribonuclease II (RNase II), one of the two main exonucleases involved in mRNA decay. We have constructed strain CMA201, in which the rnb promoter region and the gene were deleted from the chromosome and replaced by a tet^tr cassette. This is the first rnb absolute deletion mutant that shows the complete absence of rnb -specific mRNA. This strain has growth characteristics similar to the wild-type, even though it has no RNase II activity, and it should be useful in studies of mRNA metabolism.     

Functional amyloids in insect immune response

The innate immune system of insects consists of humoural and cellular responses that provide protection against invading pathogens and parasites. Defence reactions against these latter include encapsulation by immune cells and targeted melanin deposition, which is usually restricted to the surface of the foreign invader, to prevent systemic damage. Here we show that a protein produced by haemocytes of Heliothis virescens (Lepidoptera, Noctuidae) larvae, belonging to XendoU family, generates amyloid fibrils, which accumulate in large cisternae of the rough endoplasmic reticulum and are released upon immune challenge, to form a layer coating non-self objects entering the haemocoel. This amyloid layer acts as a molecular scaffold that promotes localised melanin synthesis and the adhesion of immune cells around the non-self intruder during encapsulation response. Our results demonstrate a new functional role for these protein aggregates that are commonly associated with severe human diseases. We predict that insects will offer new powerful experimental systems for studying inducible amyloidogenesis, which will likely provide fresh perspectives for its prevention.