The industrial melanism mutation in British peppered moths is a transposable element.

Discovering the mutational events that fuel adaptation to environmental change remains an important challenge for evolutionary biology. The classroom example of a visible evolutionary response is industrial melanism in the peppered moth (Biston betularia): the replacement, during the Industrial Revolution, of the common pale typica form by a previously unknown black (carbonaria) form, driven by the interaction between bird predation and coal pollution. The carbonaria locus has been coarsely localized to a 200-kilobase region, but the specific identity and nature of the sequence difference controlling the carbonaria-typica polymorphism, and the gene it influences, are unknown. Here we show that the mutation event giving rise to industrial melanism in Britain was the insertion of a large, tandemly repeated, transposable element into the first intron of the gene cortex. Statistical inference based on the distribution of recombined carbonaria haplotypes indicates that this transposition event occurred around 1819, consistent with the historical record. We have begun to dissect the mode of action of the carbonaria transposable element by showing that it increases the abundance of a cortex transcript, the protein product of which plays an important role in cell-cycle regulation, during early wing disc development. Our findings fill a substantial knowledge gap in the iconic example of microevolutionary change, adding a further layer of insight into the mechanism of adaptation in response to natural selection. The discovery that the mutation itself is a transposable element will stimulate further debate about the importance of 'jumping genes' as a source of major phenotypic novelty.

Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

Assembly of tomato blistering mosaic virus-like particles using a baculovirus expression vector system.

The expression of several structural proteins from a wide variety of viruses in heterologous cell culture systems results in the formation of virus-like particles (VLPs). These VLPs structurally resemble the wild-type virus particles and have been used to study viral assembly process and as antigens for diagnosis and/or vaccine development. Tomato blistering mosaic virus (ToBMV) is a tymovirus that has a 6.3-kb positive-sense ssRNA genome. We have employed the baculovirus expression vector system (BEVS) for the production of tymovirus-like particles (tVLPs) in insect cells. Two recombinant baculoviruses containing the ToBMV wild-type coat protein (CP) gene or a modified short amino-terminal deletion (Δ2-24CP) variant were constructed and used to infect insect cells. Both recombinant viruses were able to express ToBMV CP and Δ2-24CP from infected insect cells that self-assembled into tVLPs. Therefore, the N-terminal residues (2-24) of the native ToBMV CP were shown not to be essential for self-assembly of tVLPs. We also constructed a third recombinant baculovirus containing a small sequence coding for the major epitope of the chikungunya virus (CHIKV) envelope protein 2 (E2) replacing the native CP N-terminal 2-24 amino acids. This recombinant virus also produced tVLPs. In summary, ToBMV VLPs can be produced in a baculovirus/insect cell heterologous expression system, and the N-terminal residues 2-24 of the CP are not essential for this assembly, allowing its potential use as a protein carrier that facilitates antigen purification and might be used for diagnosis.

Natural meroterpenoids isolated from the plant pathogenic fungus Verticillium albo-atrum with noteworthy modification action against voltage-gated sodium channels of central neurons of Helicoverpa armigera.

A new meroterpenoid, named acetoxydehydroaustin A (1) and the known meroterpenoid austin (2) were isolated from the plant pathogenic fungus Verticillium albo-atrum. Their structures were established based on general spectroscopic techniques and the relative configuration of compound 1 was determined by single-crystal X-ray diffraction analysis. We first investigated and identified their significant electrophysiological effects on the gating kinetics of voltage-gated sodium channels in central neurons acutely dissociated from Helicoverpa armigera using whole-cell patch clamp technique. Similar to the effects of pyrethroids on sodium late currents, both compounds produced concentration-dependent modification of sodium channels, prolonging the kinetics of channel inactivation to generate large persistent late currents during depolarization. However, different from the effects of tefluthrin and deltamethrin on sodium channels, two meroterpenoids did not induce tail currents during deactivation. Compounds 1 and 2 also caused depolarizing shifts in the voltage dependence of channel activation. The V0.5 shifted about 5.02mV and 6.32mV in the depolarizing direction by 50µM 1 and 50µM 2. The V0.5 of voltage-dependent inactivation shifted about 11.42mV and 11.62mV respectively in the hyperpolarizing direction by 50µM 1 and 100µM 2. In addition, they prolonged the time course of recovery from fast-inactivation for sodium channels. The effects of two compounds on the voltage-dependent gating substantially increased the size of sodium window currents. The overlapped area of window currents increased about 89.69% and 44.51% respectively by 10µM compound 1 and 10µM compound 2. These findings show that both compounds have effects on sodium channel activation, inactivation and window currents. The voltage-gated sodium channels in central neurons of H. armigera are the target sites of two meroterpenoid natural products.

Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae.

In the present work, we have investigate the cellular immune response of Galleria mellonella larvae against three strains of the gram-negative bacterium Actinobacillus pleuropneumoniae: low-virulence (780), high-virulence (1022) and the serotype 8 reference strain (R8). Prohemocytes, plasmatocytes, granulocytes, oenocytoids and spherulocytes were distinguished according to their size and morphology, their molecular markers and dye-staining properties and their role in the immune response. Total hemocyte count, differential hemocyte count, lysosome activity, autophagic response, cell viability and caspase-3 activation were determined in circulating hemocytes of naive and infected larvae. The presence of the autophagosome protein LC3 A/B within the circulating hemocytes of G. mellonella was dependent on and related to the infecting A. pleuropneumoniae strain and duration of infection. Hemocytes treated with the high-virulence strain expressed higher levels of LC3 A/B, whereas treatment with the low-virulence strain induced lower expression levels of this protein in the cells. Moreover, our results showed that apoptosis in circulating hemocytes of G. mellonella larvae after exposure to virulent bacterial strains occurred simultaneously with excessive cell death response induced by stress and subsequent caspase-3 activation.

The role of host cell physiology in the productivity of the baculovirus-insect cell system: Fluxome analysis of Trichoplusia ni and Spodoptera frugiperda cell lines.

The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is broadly used for the production of recombinant proteins and vaccine manufacture, yet the host physiological aspects that contribute to productivity are to be disclosed. This work provides the first quantitative analysis of the metabolic fluxes of High Five cells. This analysis was conducted in comparison with Sf9 cells, another major host for biologicals production via BEVS. Moreover, herein is presented, for the first time, quantitative data of the relative contribution of sugars and amino acids catabolism to the activity of the TCA cycle in Sf9 and High Five cells. High Five cells metabolic activity was markedly influenced by the amino acids concentration in culture medium, which determine the rates of amino acid catabolism, carbon overflow and by-product formation. This characteristic of High Five cells was reflected in the activities of anaplerotic metabolism and the TCA cycle, which may not work as a true cycle as a function of medium composition. This was not the case for Sf9 cells, in which the glucose carbon incorporation in the TCA cycle was significantly higher and lactate production minor. Following infection, the decrease in by-product accumulation rates was accompanied by an increase in net ATP synthesis in Sf9 and High Five cells, although through distinct mechanisms cell-line dependent. The impact of baculovirus infection on cellular metabolic status highlights the capacity of this virus to re-direct the cellular fluxome toward ATP production to support replication and progeny generation. These results pave the way to deepen our knowledge on the relationship between a host cell and the virus, contributing to disclosing the metabolic determinants that contribute to productivity. Biotechnol. Bioeng. 2017;114: 674-684. © 2016 Wiley Periodicals, Inc.

The structure of the atypical killer cell immunoglobulin-like receptor, KIR2DL4.

The engagement of natural killer cell immunoglobulin-like receptors (KIRs) with their target ligands, human leukocyte antigen (HLA) molecules, is a critical component of innate immunity. Structurally, KIRs typically have either two (D1-D2) or three (D0-D1-D2) extracellular immunoglobulin domains, with the D1 and D2 domain recognizing the α1 and α2 helices of HLA, respectively, whereas the D0 domain of the KIR3DLs binds a loop region flanking the α1 helix of the HLA molecule. KIR2DL4 is distinct from other KIRs (except KIR2DL5) in that it does not contain a D1 domain and instead has a D0-D2 arrangement. Functionally, KIR2DL4 is also atypical in that, unlike all other KIRs, KIR2DL4 has both activating and inhibitory signaling domains. Here, we determined the 2.8 Å crystal structure of the extracellular domains of KIR2DL4. Structurally, KIR2DL4 is reminiscent of other KIR2DL receptors, with the D0 and D2 adopting the C2-type immunoglobulin fold arranged with an acute elbow angle. However, KIR2DL4 self-associated via the D0 domain in a concentration-dependent manner and was observed as a tetramer in the crystal lattice by size exclusion chromatography, dynamic light scattering, analytical ultracentrifugation, and small angle x-ray scattering experiments. The assignment of residues in the D0 domain to forming the KIR2DL4 tetramer precludes an interaction with HLA akin to that observed for KIR3DL1. Accordingly, no interaction was observed to HLA by direct binding studies. Our data suggest that the unique functional properties of KIR2DL4 may be mediated by self-association of the receptor.

Antennal-lobe tracts in the noctuid moth, Heliothis virescens: new anatomical findings.

As in other insects, three main tracts in the moth brain form parallel connections between the antennal lobe and the protocerebrum. These tracts, which consist of the antennal-lobe projection-neuron axons, target two main areas in the protocerebrum, the calyces of the mushroom bodies and the lateral horn. In spite of the solid neuroanatomical knowledge already established, there are still unresolved issues regarding the antennal-lobe tracts of the moth. One is the proportion of lateral-tract neurons targeting the calyces. In the study presented here, we have performed both retrograde and anterograde labeling of the antennal-lobe projection neurons in the brain of the moth, Heliothis virescens. The results from the retrograde staining, obtained by applying dye in the calyces, demonstrated that the direct connection between the antennal lobe and this neuropil is maintained primarily by the medial antennal-lobe tract; only a few axons confined to the lateral tract were found to innervate the calyces. In addition, these staining experiments, which allowed us to explore the arborization pattern of labeled neurons within the antennal lobe, resulted in new findings regarding anatomical arrangement of roots and cell body clusters linked to the medial tract. The results from the anterograde staining, obtained by applying dye into the antennal lobe, visualized the total assembly of axons passing along the antennal-lobe tracts. In addition to the three classical tracts, we found a transverse antennal-lobe tract not previously described in the moth. Also, these staining experiments revealed an organized neuropil in the lateral horn formed by terminals of the four antennal-lobe tracts.

Micropyle number is associated with elevated female promiscuity in Lepidoptera.

In the majority of insects, sperm fertilize the egg via a narrow canal through the outer chorion called the micropyle. Despite having this one primary function, there is considerable unexplained variation in the location, arrangement and number of micropyles within and between species. Here, we examined the relationship between micropyle number and female mating pattern through a comparative analysis across Lepidoptera. Three functional hypotheses could explain profound micropylar variation: (i) increasing micropyle number reduces the risk of infertility through sperm limitation in species that mate infrequently; (ii) decreasing micropyle number reduces the risk of pathological polyspermy in species that mate more frequently; and (iii) increasing micropyle number allows females to exert greater control over fertilization within the context of post-copulatory sexual selection, which will be more intense in promiscuous species. Micropyle number was positively related to the degree of female promiscuity as measured by spermatophore count, regardless of phylogenetic signal, supporting the hypothesis that micropyle number is shaped by post-copulatory sexual selection. We discuss this finding in the context of cryptic female choice, sperm limitation and physiological polyspermy.

VENOM FROM THE ECTOPARASITIC WASP Habrobracon hebetor ACTIVATES CALCIUM-DEPENDENT DEGRADATION OF Galleria mellonella LARVAL HEMOCYTES.

Ectoparasitoids inject venom into hemolymph during oviposition. We determined the influence of envenomation by the parasitoid, Habrobracon hebetor, on the hemocytes of its larval host, Galleria mellonella. An increase in both intracellular Са(2+) content and phospholipase C activity of the host hemocytes was recorded during 2 days following envenomation by the parasitoid. The decreased hemocyte viability was detected 1, 2, and 24 h after the envenomation. Injecting of the crude venom (final protein concentration 3 µg/ml) into the G. mellonella larvae led to the reduced hemocyte adhesion. The larval envenomation caused a decrease in transmembrane potential of the hemocytes. These findings document the suppression of hemocytic immune effectors in the parasitized host larvae.

[Studies on macroscopic and microscopic characteristics of Ophiocordyceps xuefengensis].

The macroscopic characteristics, tissue, caterpillar body wall and powder of Ophiocordyceps xuefengensis in different batch numbers were observed and researched by the macroscopic and microscopic identification methods. The result shows that the morphology, size, abdominal annulations of caterpillar, etc. of 0. xuefengensis are the macroscopic identification characteristics, the caterpillar body surface mycelium, body wall sculpture and crochets on abdominal legs are the microscopic identification characteristics. These characters are stable and regular discriminant features, which are proved to be the identification basis of O. xuefengensis. In addition, The characters such as crochets on abdominal legs arrange in two parallel ellipse rings, the inner crochets are long strip, and the external toes are unciform, are specific.

The development of wing shape in Lepidoptera: mitotic density, not orientation, is the primary determinant of shape.

The wings of butterflies and moths develop from imaginal disks whose structure is always congruent with the final adult wing. It is therefore possible to map every point on the imaginal disk to a location on the adult wing throughout ontogeny. We studied the growth patterns of the wings of two distantly related species with very different adult wing shapes, Junonia coenia and Manduca sexta. The shape of the wing disks change throughout their growth phase in a species-specific pattern. We measured mitotic densities and mitotic orientation in successive stages of wing development approximately one cell division apart. Cell proliferation was spatially patterned, and the density of mitoses was highly correlated with local growth. Unlike other systems in which the direction of mitoses has been viewed as the primary determinant of directional growth, we found that in these two species the direction of growth was only weakly correlated with the orientation of mitoses. Directional growth appears to be imposed by a constantly changing spatial pattern of cell division coupled with a weak bias in the orientation of cell division. Because growth and cell division in imaginal disk require ecdysone and insulin signaling, the changing spatial pattern of cell division may due to a changing pattern of expression of receptors or downstream elements in the signaling pathways for one or both of these hormones. Evolution of wing shape comes about by changes in the progression of spatial patterns of cell division.

Identification and functional analysis of inter-subunit disulfide bonds of the F protein of Helicoverpa armigera nucleopolyhedrovirus.

The major envelope fusion protein F of the budded virus of baculoviruses consists of two disulfide-linked subunits: an N-terminal F2 subunit and a C-terminal, membrane-anchored F1 subunit. There is one cysteine in F2 and there are 15 cysteines in F1, but their role in disulfide linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein were analysed by site-directed mutagenesis. Results indicated that in a functional F protein, an inter-subunit disulfide bond exists between amino acids C108 (F2) and C241 (F1). When C241 was mutated, an alternative disulfide bond was formed between C108 and C232, rendering F non-functional. No inter-subunit bridge was observed in a double C232/C241 mutant of F1. C403 was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit disulfide bonds. The influence of reductant [tris(2-carboxyethyl) phosphine (TCEP)] and free-thiol inhibitors [4-acetamido-4'-maleimidylstilbene 2,2'-disulfonic acid (AMS) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] on the infectivity of HearNPV was tested. The results indicated that TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB had no inhibitory effect on viral infectivity. The data suggested that free thiol/disulfide isomerization was not likely to play a role in viral entry and infectivity.

Novel apoptosis suppressor Apsup from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus precludes apoptosis by preventing proteolytic processing of initiator caspase Dronc.

We previously identified a novel baculovirus-encoded apoptosis suppressor, Apsup, from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Apsup inhibits the apoptosis of L. dispar Ld652Y cells triggered by infection with p35-defective Autographa californica MNPV (vAcΔp35) and exposure to actinomycin D or UV light. Here, we examined the functional role of Apsup in apoptosis regulation in insect cells. Apsup prevented apoptosis and the proteolytic processing of L. dispar initiator caspase Dronc (Ld-Dronc) in Ld652Y cells triggered by overexpression of Ld-Dronc, LdMNPV inhibitor-of-apoptosis 3 (IAP3), or Hyphantria cunea MNPV IAP1. In vAcΔp35-infected apoptotic Ld652Y cells, Apsup restricted apoptosis induction and prevented processing of endogenous Ld-Dronc. Conversely, upon RNA interference (RNAi)-mediated silencing of apsup, LdMNPV-infected Ld652Y cells, which typically support high-titer virus replication, underwent apoptosis, accompanied by the processing of endogenous Ld-Dronc. Furthermore, endogenous Ld-Dronc coimmunoprecipitated with transiently expressed Apsup, indicating that Apsup physically interacts with Ld-Dronc. Apsup prevented the apoptosis of Sf9 cells triggered by vAcΔp35 infection but did not inhibit apoptosis or activation of caspase-3-like protease in vAcΔp35-infected Drosophila melanogaster S2 cells. Apsup also inhibited the proteolytic processing of L. dispar effector caspase Ld-caspase-1 in the transient expression assay but did not physically interact with Ld-caspase-1. These results demonstrate that Apsup inhibits apoptosis in Ld652Y cells by preventing the proteolytic processing of Ld-Dronc. Together with our previous findings showing that Apsup prevents the processing of both overexpressed Ld-Dronc and Bombyx mori Dronc, these results also demonstrate that Apsup functions as an effective apoptotic suppressor in various lepidopteran, but not dipteran, insect cells.

Detection of serum antibodies to hepatitis E virus in domestic pigs in Italy using a recombinant swine HEV capsid protein.

BACKGROUND: The hepatitis E virus (HEV) has been detected in both humans and animals, particularly pigs, worldwide. Several evidences, including human infection following consumption of raw contaminated meat, suggest a zoonotic transmission of HEV. In Italy, large circulation of genotype 3 HEV has been reported in swine, and recent studies have confirmed the involvement of this genotype in autochthonous human cases. RESULT: In this study 111 sera collected from healthy pigs in two Italian regions were tested for anti-HEV IgG antibodies. For specific HEV antibody detection in swine, we developed ELISA and Western blotting methods, using a truncated capsid (ORF2) protein lacking the first 111 amino acids of a swine HEV genotype 3 strain. The ORF2-based ELISA revealed anti-HEV antibodies in 104 out of 111 pigs compared with 102 detected with a commercial ELISA kit. A lower number of sera reacted with the recombinant ORF2 protein in a Western blotting format (81/111). Using a Latent class analysis (LCA), the estimated sensitivities for ELISA-ORF2 and ELISA-kit tests were 0.961 and 0.936, respectively, whereas specificities were 0.599 and 0.475. The estimated sensitivity of Western blotting was 0.775, and the specificity was 0.944. CONCLUSIONS: The overall results confirm the high prevalence of HEV seropositive healthy pigs in Italy. Through comparisons with a commercial ELISA test, the swine genotype 3 HEV antigen produced in this study was proven suitable to detect anti-HEV antibodies in pig sera by both ELISA and Western Blotting.

Galleria mellonella hemocytes destruction after infection with Pseudomonas aeruginosa.

The influence of infection with an entomopathogenic strain of Pseudomonas aeruginosa on Galleria mellonella hemocytes was investigated. Extensive bacteriaemia developed 18 h after infection. This was correlated with significant changes in morphology, viability and the spreading ability of immunocompetent hemocytes, namely granulocytes and plasmatocytes. Since bacteriaemia developed, membrane blebbing, cytoplasm vacuolization, cell and organelle swelling, and chromatin condensation were observed among others. These features are typical for apoptotic and autophagal cell death. A gradually increasing level of procaspase and its activation as well as lack of DNA degradation were also detected. Propidium iodide and acridine orange staining indicated that hemocytes become dead ultimately. Infection of G. mellonella larvae with P. aeruginosa also caused significant changes in the arrangement of the actin cytoskeleton in the hemocytes, which might be correlated with their restricted spreading ability.

Mitochondrial lineage sorting in action--historical biogeography of the Hyles euphorbiae complex (Sphingidae, Lepidoptera) in Italy.

BACKGROUND: Mitochondrial genes are among the most commonly used markers in studies of species' phylogeography and to draw conclusions about taxonomy. The Hyles euphorbiae complex (HEC) comprises six distinct mitochondrial lineages in the Mediterranean region, of which one exhibits a cryptic disjunct distribution. The predominant mitochondrial lineage in most of Europe, euphorbiae, is also present on Malta; however, it is nowadays strangely absent from Southern Italy and Sicily, where it is replaced by 'italica'. A separate biological entity in Italy is further corroborated by larval colour patterns with a congruent, confined suture zone along the Northern Apennines. By means of historic DNA extracted from museum specimens, we aimed to investigate the evolution of the mitochondrial demographic structure of the HEC in Italy and Malta throughout the Twentieth Century. RESULTS: At the beginning of the Twentieth Century, the European mainland lineages were also present at a moderate frequency in Southern Italy and Sicily. The proportion of 'italica' then steadily increased in this area from below 60 percent to near fixation in about 120 years. Thus, geographical sorting of mitochondrial lineages in the HEC was not as complete then as the current demography suggests. The pattern of an integral 'italica' core region and a disjunct euphorbiae distribution evolved very recently. To explain these strong demographic changes, we propose genetic drift due to anthropogenic habitat loss and fragmentation in combination with an impact from recent climate warming that favoured the spreading of the potentially better adapted 'italica' populations. CONCLUSIONS: The pattern of geographically separated mitochondrial lineages is commonly interpreted as representing long term separated entities. However, our results indicate that such a pattern can emerge surprisingly quickly, even in a widespread and rather common taxon. We thus caution against drawing hasty taxonomic conclusions from biogeographical patterns of mitochondrial markers derived from modern sampling alone.

Cloning and functional characterization of the Lymantria dispar initiator caspase dronc.

Ld652Y cells from the gypsy moth, Lymantria dispar, are extremely sensitive to various apoptotic stimuli, whereas BM-N cells from the silkworm, Bombyx mori, are relatively resistant to apoptotic stimuli. We previously cloned and characterized a B. mori homologue (bm-dronc) of Drosophila melanogaster dronc. In the present study, we cloned and characterized an L. dispar homologue of dronc (ld-dronc) comparatively with Bm-Dronc. The open reading frame of ld-dronc consisted of 1329bp that was predicted to encode a 443 amino-acid polypeptide with a molecular mass of 50,706Da and 54-57% amino acid sequence identity with Dronc homologues from other lepidopteran insects identified to date. Ld-Dronc had a long prodomain, large p20 domain, and small p10 domain, and a catalytic site composed of (308)QTCRG(312), which was distinct from the sites QACRG in Bm-Dronc and QMCRG in Dronc homologues of several other lepidopteran insects. Transiently expressed Ld-Dronc underwent proteolytic processing in the lepidopteran cell lines L. dispar Ld652Y, Spodoptera frugiperda Sf9, and B. mori BM-N, and dipteran D. melanogaster S2, but only triggered apoptosis in the lepidopteran cell lines. Endogenous Ld-Dronc underwent processing in Ld652Y cells upon infection with vAcΔp35, but not in mock-infected Ld652Y cells, supporting the involvement of Ld-Dronc in apoptosis induction. In vAcΔp35-infected apoptotic cells, Ld-Dronc underwent proteolytic processing more rapidly and extensively than Bm-Dronc. Similar results were obtained for Ld-Dronc and Bm-Dronc expressed transiently in S2, Ld652Y, Sf9, and BM-N cells. Taken together, these findings suggest that the intrinsic properties of Dronc proteinsare responsible, at least in part, for the differing sensitivity of Ld652Y and BM-N to apoptosis induction upon NPV infection.

Virulence-related traits of epidemic Acinetobacter baumannii strains belonging to the international clonal lineages I-III and to the emerging genotypes ST25 and ST78.

BACKGROUND: Acinetobacter baumannii is responsible for large epidemics in hospitals, where it can persist for long time on abiotic surfaces. This study investigated some virulence-related traits of epidemic A. baumannii strains assigned to distinct MLST genotypes, including those corresponding to the international clones I-III as well as emerging genotypes responsible for recent epidemics. METHODS: Genotyping of bacteria was performed by PFGE analysis and MLST according to the Pasteur's scheme. Biofilm formation on polystyrene plates was assessed by crystal violet staining; resistance to desiccation was evaluated on glass cover-slips when kept at room-temperature and 31% relative humidity; adherence to and invasion of A549 human alveolar epithelial cells were determined by the analysis of viable bacteria associated with or internalized by A549 human alveolar epithelial cells; Galleria mellonella killing assays were used to analyze the virulence of A. baumannii in vivo. RESULTS: The ability to form biofilm was significantly higher for A. baumannnii strains assigned to ST2 (international clone II), ST25 and ST78 compared to other STs. All A. baumannii strains survived on dry surfaces for over 16 days, and strains assigned to ST1 (international clone I) and ST78 survived for up to 89 and 96 days, respectively. Adherence to A549 pneumocytes was higher for strains assigned to ST2, ST25 and ST78 than other genotypes; a positive correlation exists between adherence and biofilm formation. Strains assigned to ST78 also showed significantly higher ability to invade A549 cells. No significant differences in the killing of G. mellonella worms were found among strains. CONCLUSIONS: Elevated resistance to desiccation, high biofilm-forming capacity on abiotic surfaces and adherence to A549 cells might have favoured the spread and persistence in the hospital environment of A. baumannii strains assigned to the international clones I and II and to the emerging genotypes ST25 and ST78.

Characterization of Atg8 in lepidopteran insect cells.

Yeast Atg8 and mammalian microtubule-associated protein light chain 3 (LC3) are landmark proteins essential for autophagy. Here the lepidopteran Atg8, a homolog of LC3, is characterized. Sequence analysis reveals that Atg8 proteins are highly conserved in lepidopteran species. The abundance of endogeous Atg8 and the ratios of Atg8 conjugation to phosphatidylethanolamine (Atg8-PE)/Atg8 are different among several lepidopteran cell lines and different tissues of Helicoverpa armigera larvae. Both the density of fluorescent pre-autophagosomal structures with GFP-Ha Atg8 and the abundance of Atg6 are positively correlated with levels of Atg8-PE in different cell lines. The mutant GFP-Atg8(G116A) has lost the function in punctual formation, suggesting that G116 is important for autophagy. Exogenous factors have significant influences on the conversion of Atg8 in lepidopteran cells. Bacillus thuringiensis enhances the degradation of Atg8 in Spodoptera litura Sl-HP cells. Atg8-PE degrades gradually with extension of amino acid starvation, and bafilomycin A1 can block the decrease through the inhibition of autophagosome fusion with lysosome. Interestingly, high pH is more effective than amino acid starvation in Bombyx mori Bme cells to induce the conversion of BmAtg8 to BmAgt8-PE. Change of the quality of fetal bovine serum in the culture medium results in alteration of the ratio of Atg8-PE/Atg8 in some lepidopteran cell lines.