Pitarch Martí
email: africa.pitarch@ub.edu

João Zilhão
email: joao.zilhao@ub.edu

Francesco D'errico

Pedro Cantalejo-Duarte

Salvador Domínguez-Bella

Josep M Fullola

Gerd-Christian C Weniger

José Ramos-Muñoz

Africa Pitarch Martí

Pedro Cantalejo

JZ, FD, PCD Apm

Painting the place: insights from Cueva de Ardales into the symbolic role of caves in the Middle Paleolithic. Africa

Keywords: Cave art, symbolism, pigment, spectroscopic analyses, Iberian Peninsula

Cueva de Ardales (Málaga, Spain) is one of the richest and best-preserved Paleolithic painted caves of southwestern Europe, containing over a thousand graphic representations. Here, we study the red pigment in panel II.A.3 of "Sala de las Estrellas", dated by U-Th to the Middle Paleolithic, to determine its composition, verify its anthropogenic nature, infer the associated behaviors and discuss their implications. Using optical microscopy, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, micro-Raman spectroscopy, and X-ray diffraction we analyzed a set of samples from the panel and compared them to natural coloring materials collected from the floor and walls of the cave. The conspicuously different texture and composition of the geological samples indicatethat the pigments used in the paintings do not come from the outcrops sampled in the cave. We confirm that the paintings are not the result of natural processes and show that the composition of the paint is consistent with the artistic activity being recurrent. Our results strengthen the hypothesis that Neanderthals symbolically used these paintings and the large stalagmitic dome harboring them over an extended time span.

Significance Statement

The emergence of symbolic behavior in our genus is a controversial issue. The dating of paintings in three caves from the Iberian Peninsula supports the view that Neanderthals developed a form of cave art more than 20,000 years before the emergence of anatomical modernity in Europe. In this study, we confirm that the paintings on a large speleothem from one of these sites, Cueva de Ardales, were human made, and we show that the pigments were not collected at the outcrops sampled inside the cave. Variations in the composition of the paint correspond to differences in the age of the paintings, supporting the hypothesis that Neanderthals used the speleothems symbolically over an extended time span.

Main Text Introduction

The production of art is considered a big leap forward in the cultural evolution of humankind. It represents a means of recording and transmitting complex symbolic representations in a durable way [START_REF] Lorblanchet | Les Grottes ornées de la Préhistoire[END_REF][START_REF] Lorblanchet | The First Artists: In Search of the World's Oldest Art[END_REF]. However, despite the work of generations of researchers, many questions concerning the origin, chronology, technology, function and meaning of Paleolithic art remain open. Research conducted over the last two decades has focused on the earliest instances of graphic representation [START_REF] Joordens | Homo erectus at Trinil on Java used shells for tool production and engraving[END_REF][START_REF] Henshilwood | An abstract drawing from the 73,000-year-old levels at Blombos Cave, South Africa[END_REF], the interdisciplinary analyses of key cave sites [START_REF] Geneste | La grotte Chauvet à Vallon-Pont-d'Arc: un bilan des recherches pluridisciplinaires[END_REF][START_REF] Sanchidrián-Torti | La grotte de Nerja aujourd'hui. Synthèse des derniers travaux de recherche dans les zones ornées[END_REF][START_REF] Arias | Investigaciones en la Zona Arqueológica de La Garma (Omoño, Ribamontán al Mar), 2004-2011[END_REF][START_REF] Jaubert | The chronology of human and animal presence in the decorated and sepulchral cave of Cussac (France)[END_REF], the study of open air sites [START_REF] Zilhão | The rock art of the Côa valley (Portugal) and its archaeological context: first results of current research[END_REF][START_REF] Aubry | Palaeolithic engravings and sedimentary environments in the Côa River Valley (Portugal): implications for the detection, interpretation and dating of open-air rock art[END_REF][START_REF] Aubry | Nature vs. Culture: present-day spatial distribution and preservation of open-air rock art in the Côa and Douro River Valleys (Portugal)[END_REF], the presentation of new discoveries [START_REF] Garate | The cave of Atxurra: A new major Magdalenian rock art sanctuary in Northern Spain[END_REF][START_REF] Ruiz-Redondo | Expanding the horizons of Palaeolithic rock art: the site of Romualdova Pećina[END_REF][START_REF] Ruiz-Redondo | The Easternmost European Palaeolithic Artists: Iconography and Graphic Features at Kapova Cave (Southern Urals, Russia)[END_REF], and the dating of the earliest instances of cave painting [START_REF] Aubert | Pleistocene cave art from Sulawesi, Indonesia[END_REF][START_REF] Aubert | Palaeolithic cave art in Borneo[END_REF][START_REF] Aubert | Earliest hunting scene in prehistoric art[END_REF][START_REF] Pike | U-series dating of Paleolithic art in 11 caves in Spain[END_REF][START_REF] García-Diez | The chronology of hand stencils in European Palaeolithic rock art: implications of new U-series results from El Castillo Cave (Cantabria, Spain)[END_REF][START_REF] Hoffmann | U-Th dating of carbonate crusts reveals Neandertal origin of Iberian cave art[END_REF][START_REF] Brumm | Oldest cave art found in Sulawesi[END_REF].

Of particular relevance is that the application of U-series dating to stratigraphically associated calcite accretions has shown these artistic manifestations to be of much greater antiquity than hitherto thought. At El Castillo cave (Spain), a minimum age of 40.8 ka was obtained for a red disk [START_REF] Pike | U-series dating of Paleolithic art in 11 caves in Spain[END_REF], consistent with Neanderthal authorship of Europe's earliest cave art, as eventually corroborated by the non-figurative paintings and hand stencils from three Iberian sites dated to >64.8 ka [START_REF] Hoffmann | U-Th dating of carbonate crusts reveals Neandertal origin of Iberian cave art[END_REF]. Hand-stencil art from Borneo and a naturalistic painting from Sulawesi have yielded minimum ages of 39.9 ka and 43.9 ka [START_REF] Aubert | Pleistocene cave art from Sulawesi, Indonesia[END_REF][START_REF] Aubert | Palaeolithic cave art in Borneo[END_REF], convincingly demonstrating broad contemporaneity with the earliest European manifestations of this practice, as predicted [START_REF] Zilhão | Neandertal-modern human contact in Western Eurasia: issues of dating, taxonomy, and cultural associations[END_REF]. The Iberian evidence has been challenged [START_REF] Aubert | Early dates for 'Neanderthal cave art' may be wrong[END_REF][START_REF] Pearce | Trouble on the dating scene[END_REF][START_REF] Slimak | Comment on "U-Th dating of carbonate crusts reveals Neandertal origin of Iberian cave art[END_REF][START_REF] White | Still no archaeological evidence that Neanderthals created Iberian cave art[END_REF][START_REF] Pons-Branchu | U-series dating at Nerja cave reveal open system. Questioning the Neanderthal origin of Spanish rock art[END_REF], but all the criticisms have been exhaustively responded to [START_REF] Hoffmann | Dates for Neanderthal art and symbolic behaviour are reliable[END_REF][START_REF] Hoffmann | Response to Comment on "U-Th dating of carbonate crusts reveals Neandertal origin of Iberian cave art[END_REF][START_REF] Hoffmann | Response to Aubert et al.'s reply 'Early dates for 'Neanderthal cave art' may be wrong[END_REF][START_REF] Hoffmann | Response to White et al.'s reply: 'Still no archaeological evidence that Neanderthals created Iberian cave art[END_REF].

One of the early Iberian sites is Cueva de Ardales (Fig. 1, SI Appendix, Archaeological context), which has a long but intermittent history of research, beginning with Breuil more than a century ago [START_REF] Breuil | Nouvelles cavernes ornées paléolithiques dans la province de Málaga[END_REF] and continuing with the recent investigations, carried out by an international research team led by two of us (G-CW and JRM). Until now, however, the pigments composing the paintings in the cave, including those dated by U-series, remained unanalyzed. As part of a broader project to study the origin and evolution of southwestern Europe's Paleolithic painting technologies, this paper focuses on the microscopic and chemical analysis of panel II.A.3 (Fig. 2).

Based on the U-series dating of calcite samples, the age constraints for the red stains in panel II.A.3 are as follows: >45.9 ka in Curtain 5; >45.3 ka and <48.7 ka in Curtain 6; >65.5 ka in Curtain 8 [START_REF] Hoffmann | U-Th dating of carbonate crusts reveals Neandertal origin of Iberian cave art[END_REF]. These results place the artistic activity in the regional, Neanderthal-associated Middle Paleolithic, and there is nothing to suggest that the decoration of the panel's undated curtains might be of a different, later age.

Our aims here are twofold. Firstly, we intend to characterize the composition of the red pigment of panel II.A.3 (SI Appendix, Materials and Methods, Fig. S1). It has been suggested [START_REF] Aubert | Early dates for 'Neanderthal cave art' may be wrong[END_REF] suggested that the pigment could represent natural staining, which, macroscopic observation does not support [START_REF] Hoffmann | Response to Aubert et al.'s reply 'Early dates for 'Neanderthal cave art' may be wrong[END_REF] rejected as unsubstantiated. Secondly, we investigate whether patterns in pigment composition and technology can provide additional detail on the different phases of Middle Paleolithic artistic activity demonstrated by the dating. We also analyzed natural Fe-rich coloring materials collected from the floor and walls of the cave (SI Appendix, Materials and Methods, Fig. S2) to see if the chemical fingerprint of these geological materials was consistent with their being the source of the pigments used in the paintings.

Background to the site

Cueva de Ardales is situated close to the eponymous village, in Málaga, southern Spain (Fig. 1). The cave is 1577 m long and features two superimposed levels: the Lower and the Upper Galleries. The site was discovered in 1821 after an earthquake re-opened a cave entrance previously sealed by colluvial sediments, but it was not until 1918 that the Paleolithic rock art was found by Henri Breuil [START_REF] Breuil | Nouvelles cavernes ornées paléolithiques dans la province de Málaga[END_REF]. Over a thousand graphic representations, mostly attributed to the Upper Paleolithic, have been described. They include both figurative and non-figurative engravings and paintings grouped into two hundred and fifty-two panels [START_REF] Cantalejo | La cueva de Ardales: arte prehistórico y ocupación en el paleolítico superior: estudios, 1985-2005[END_REF]. Most abstract red paintings are on speleothems and located near the entrance rather than at the back, i.e., in those parts of the cave in which archaeological excavation has corroborated broadly coeval use of the space by Middle Paleolithic Neanderthals [START_REF] Ramos | The archaeological context of early rock art in Cueva Ardales (Spain)[END_REF]. Panel II.A.3, which is the focus of the present study, is located in an impressive stalagmitic dome of Sala de las Estrellas (Fig. 2), in the Lower Gallery. More detailed information on the cave setting, research history, art and human use is provided in (SI Appendix, Archaeological context)

Results

Micro-samples from panel II.A.3. The samples are composed of hematite, aluminosilicates (clay minerals and micas), calcite and, in some cases, quartz and amorphous carbon (Table 1, SI Appendix, Results). Analyses also detected traces of P and S that may come from small amounts of sulphates and phosphates. SEM observation of these micro-samples indicates a mineral origin, since none of them features the particle morphologies typically found in biomineralizations (e.g., filaments, coccoid forms, beads-on-a-string, rods arranged in rows, biofilms; [START_REF] Northup | Lava Cave Microbial Communities Within Mats and Secondary Mineral Deposits: Implications for Life Detection on Other Planets[END_REF][START_REF] Daza | Allophanic and ferric root-associated stalactites: biomineralization induced by microbial activity (Galeria da Queimada lava tube, Terceira, Azores)[END_REF][START_REF] Macdonald | Hunter-gatherers harvested and heated microbial biogenic iron oxides to produce rock art pigment[END_REF]. The shape and size of crystals in the micro-samples are also consistent with a mineral habit. Closer analysis reveals interesting textural and compositional differences (SI Appendix, Results, Fig. 3, Fig. S3 to S7). Samples from Curtains 5 (P-ARD-06) and 8 (P-ARD-03 and P-ARD-04) are composed of tightly bound agglomerates of sub-micrometric to micrometric platy Fe-rich minerals and clays while in Curtain 6 (P-ARD-05) the hematite and aluminosilicate particles do not appear as agglomerates but are recorded in the form of individual particles. The red stains of Curtain 9 (P-ARD-02) differ from those of Curtains 5 and 8 for the presence of coarse isolated mica platelets (15-30 µm) and the lack of hydrated clay minerals. Unlike Curtain 6, and similarly to Curtain 5 and 8, hematite and clay particles occur in Curtain 9 in the form of agglomerates. In addition, Curtain 6 (P-ARD-05) reveals the presence of amorphous carbon (SI Appendix, Results, Fig. S8), not detected in the other samples. Geological samples. Six types of Fe-rich deposits that could have potentially been used as pigments were identified in the cave. They are composed of heterogeneous materials ranging from loose ochraceous sediment to compact violaceous rocks (SI Appendix, Materials and Methods, Fig. S2, Table S2, Results, Fig. S11). SEM analysis makes it clear that these materials share no similarities with the samples from panel II.A.3 (SI Appendix, Results, Fig. S12, Table S3). The XRD analysis (SI Appendix, Results, Table S4) shows that only two of the geological samples (G-ARD-01 and G-ARD-11) include hematite, the mineral responsible for the color of the red staining on the stalagmites, but none of them is comparable to those from the panel: sample G-ARD-01 is composed of micrometric to submicrometric granular, massive and acicular Fe/Mn-rich crystals (Mn reaching concentrations ranging from 0.5 to 2 %), Fe-rich sulphate spherules, and K-rich micas (Si, K, Al, Ca, Ti, Mg), while sample G-ARD-11 consists of clusters of 2 µm disc-shaped Fe-rich crystals in a foliated matrix of clays (Si, K, Al, Mg, Ti, Ca, Mn).

Speleothem sample.

Microscopic and Elemental nalyses performed on the speleothem layer covering one stains (SI Appendix, Results, Fig. S13) show that it is mainly composed of lowmagnesium calcite. Small amounts of aluminum, probably from of Al-rich hydroxide, were also detected. No iron or clay minerals were detected.

Discussion

The analysis of the micro-samples collected on the panel indicates, as suggested on the basis of close observation of the panel by naked eye [START_REF] Hoffmann | Response to Aubert et al.'s reply 'Early dates for 'Neanderthal cave art' may be wrong[END_REF], that the staining is mineral in origin and cannot be interpreted as the result of microbial activity. The staining cannot be interpreted either as the result of natural geological processes typically occurring in caves such as fluvial flows, infiltration from soils, percolating waters, or weathering of the walls [START_REF] Ford | Karst Hydrogeology and Geomorphology[END_REF]. Although flooding may coat the walls and even the roof of a cave, most accumulation occurs on the floors and is, in general, widespread. In Cueva de Ardales, traces of a deposit formed by flooding are visible neither on the floor nor on the walls of the chamber in which panel II.A.3 is located. In addition, clay platelets transported by flooding generally show, under SEM, broken or rounded edges [START_REF] Osborne | Carboniferous clay deposits from Jenolan Caves, New South Wales: implications for timing of speleogenesis and regional geology[END_REF], which is not the case with our samples. Deposition of iron oxides by dripping water would produce a diffuse red staining of the calcite while the deposit interpreted as paint occurs in the form of a distinct layer on top of and/or covered by calcite Inclusion within the calcite of iron-rich particles present in drip water would not lead to the formation of loose hematite and clay platelets, as observed in panel II.A.3. Weathering is the only process that could produce thin layers of well-preserved iron oxides and clay platelets but is inconsistent with the exclusive affection of a small area in the middle of a stalagmite located in a large room on the walls of which no similar deposits are observed. Microscopic and chemical analysis of the calcite layer overlying the pigment do not detect these compounds, which discard the hypothesis that iron and clay rich mineral could originate from the speleothem. Furthermore, in terms of morphology, the paintings are markings characterized by a central area with high color density surrounded by an aureole that features a gradual reduction in the concentration of red matter [START_REF] Hoffmann | Response to Aubert et al.'s reply 'Early dates for 'Neanderthal cave art' may be wrong[END_REF]. This pattern suggests an application of the paint by splattering as experimentally reproduced [START_REF] Errico | The technology of the earliest European cave paintings: El Castillo Cave, Spain[END_REF]. Our results reject speculations that the panel might be the result of natural processes [START_REF] Aubert | Early dates for 'Neanderthal cave art' may be wrong[END_REF].

It has been proposed that some red stains found in Paleolithic decorated caves may be the consequence of accidental contact rather than due to a deliberate will of marking the caves' walls [START_REF] Medina-Alcaide | Painted in red: In search of alternative explanations for European Palaeolithic cave art[END_REF]. In a narrow passage it is indeed possible that visitors wearing ochered clothes or body painting might inadvertently touch the walls, but, in the case of panel II.A.3, accidental staining can be excluded because the painted dome is in the middle of a very large chamber. In addition, the traces of color are found in both salient and recessed areas of the stalagmitic drapery. Indeed, some of the folds of this drapery where color can be seen are very deep and even beyond arm's reach; the only way pigment accretions could have reached some of the places where they can be observed is as drops and droplets blown via experimentally reproduced techniques [START_REF] Errico | The technology of the earliest European cave paintings: El Castillo Cave, Spain[END_REF].

The striking differences between the geological and the archaeological samples indicate that none of the cave deposits sampled in this study were used as sources for the pigments used to paint panel II.A.3. Furthermore, we observed neither substantial changes in the intensity and width of the hematite bands in the archaeological samples' Raman spectra nor features suggesting that goethite-rich raw materials could have been heat-treated. No support therefore exists for a hypothesis whereby goethite-rich material naturally present in the cave was heated to produce the paint on panel II.A.3. Our results strongly support that the Paleolithic artist(s) used Fe-rich lumps collected in geological formations from an as yet unknown source likely to be found outside the cave. Future research will need to survey local Fe-rich formations to establish whether the ochre used to paint the stalagmites is found nearby or comes from more distant sources.

The differences observed in the composition of the micro-samples from panel II.A.3 may be attributed to different causes. Assuming a single episode of artistic activity, conducted by single or multiple artists, slight differences in composition might be due to incomplete homogenization of the mixture applied to the draperies, or to different persons using pigment powders of diverse geological origin and produced with distinct techniques (e.g., because those persons belonged to different cultural traditions or came from different regions). Alternatively, such variations might be due to the fact that different draperies were painted at different times with a slightly different paint recipe or with a different source being used each time. These alternatives can be assessed against available dating evidence. The markings in Curtains 5 and 8 date to, respectively, >45.9 and >65.5 ka and, therefore, we cannot exclude that they represent a single painting episode taking place at some point in time before 65.5 ka ago. Such a hypothesis would be consistent with our finding that the markings were made in both curtains with a quite similar paint, composed of agglomerates of fine-grained platy Fe-rich minerals and clays. Curtain 6 was painted between 45.3 and 48.7 ka ago and therefore must represent a different incursion. Our finding that the Curtain 6 pigment differs from that applied to Curtain 8 -it is also composed of clay-sized platy particles of hematite and aluminosilicates, but such particles are in the case of Curtain 6 scattered rather than forming agglomerates-suggests variation through time in the nature of the colorants used. The pigment in Curtain 9 -composed of agglomerates of fine-grained Fe-rich minerals and clays associated with coarser K-rich micas-differs from the paint used in Curtains 5, 6 and 8 for the presence of isolated mica platelets and the lack of hydrated clay minerals.

To summarize, the dating evidence implies a minimum of two incursions. Based on Occam's Razor, similarity in composition is more likely to reflect appurtenance to the same painting event, and it is the opposite for dissimilarity. This is the more so since, in addition to their different compositions, the Ardales samples also feature different grain sizes, which is consistent, although in itself not demonstrative, of the use of a different paint. Thus, combining both lines of evidence (dating and composition), we can be certain that our samples represent a minimum of two painting events, and we can additionally suggest that the real number probably is of at least three, maybe even four. Additional precision on the number of painting episodes is currently not possible; it depends on the acquisition of additional dating evidence.

As the paintings are the result of recurrent addition, questions arise about it being a piece of art, subject to a sort of rejuvenation or restoration. Rejuvenation of motifs has been shown to occur in rock art, and ethnographic research has demonstrated that repainting is a common practice amongst traditional communities [START_REF] Yates | Society Shamanism and Rock Paintings: Aspects of the Use of Rock Art in the South-Western Cape, South Africa[END_REF][START_REF] Love | Rock paintings of the Worora and their mythological interpretation[END_REF][START_REF] Elkin | Rock paintings in north west Australia[END_REF][START_REF] Lommel | The Art of the Fifth Continent -Australia[END_REF][START_REF] Crawford | The Art of the Wandjina[END_REF][START_REF] Mowaljarlai | Repainting of images on rock in Australia and the maintenance of Aboriginal Culture[END_REF][START_REF] O'connor | Painting and Repainting in the West Kimberley[END_REF][START_REF] May | New Insights into the Rock Art of Anbangbang Gallery[END_REF]. Deliberate modification/restoration has also been proposed for Paleolithic cave art (e.g., the Pech Merle horses; (1)), and would seem to be ubiquitous in sites of the Spanish Levantine rock art tradition, which features panels that are thought to have been restored, altered, or expanded, either for ritual purposes or for the restoration of degraded figures [START_REF] Domingo | Técnica y ejecución de la figura en el arte rupestre levantino. Hacia una definición actualizada del concepto de estilo: Validez y limitaciones[END_REF][START_REF] Vallverdú | Las superposiciones en el arte rupestre levantino: antiguas propuestas y nuevas evidencias para un período de reflexión[END_REF]. Examples include Cova Remigia, in the Valltorta-Gassulla area of Castellón, where total or partial repainting, addition of new elements or of another colour have been described, suggesting graphical and narrative re-appropriation [START_REF] López-Montalvo | An approximation to the study of black pigments in Cova Remigia (Castellon, Spain). Technical and cultural assessments of the use of carbon-based black pigments in Spanish Levantine Rock Art[END_REF]. In Coves de la Saltadora (Castellón), the detection of a chemically distinct pigment led to interpret one figure as repainted [START_REF] Roldán | Análisis in-situ de pigmentos de las pinturas rupestres de los abrigos VII, VIII y IX de La Saltadora mediante fluorescencia de rayos X[END_REF]. A clear example of two-color combination has been recently documented in Barranco de Segovia (Letur), where the use of red over black was interpreted as enhancement of the original's value [START_REF] Mateo | Un ejemplo de bicromía en el arte levantino del Barranco Segovia (Letur, Albacete)[END_REF]. Other examples of intentional overpainting that modified the nature and identity of the imagery are found at Ceja de Piezarrodilla (Albarracín), Cueva del Chopo (Obón), Canto Blanco (Jumilla), Prado de las Olivanas (Tormón), or Cueva de la Vieja (Albacete), amongst others [START_REF] Martínez | Rock Art of the Spanish Levant[END_REF][START_REF] Piñón | Las pinturas rupestres de Albarracín (Teruel)[END_REF][START_REF] Utrilla | La figura humana en el arte levantino aragonés[END_REF].

In the case of panel II.A.3 of Cueva de Ardales, assessing whether image renovation occurred could be evaluated via the identification of distinct pigment layers separated by Ca/Mg-rich accretions. However, such a micro-stratigraphic study would entail significant damage to both painting and canvas, which is precluded by one of the key premises of our studynot to damage the paintings during our sampling work. It is unlikely, however, that such is the behavior reflected by our results. Ethnographic examples of rock art restoration show that this practice is often applied to abstract or figurative representations presenting characteristics (shape, details, color association) that fade out over time and that need to be rejuvenated in order to guarantee, on the one hand, the visual recognition of the diagnostic features identifying them as discrete, recognizable symbols by the concerned members of the group and, in some cases, to renew the symbolic link between the place and the representations. By restoring the painting, the group cultivates its ancestral link with a place charged with meaning and renews the art that binds the group to the place. Commitment to the land, however, is generally not the only reason for making rock art and one cannot directly use ethnographic examples to infer ancient cultural behavior. The techniques used to apply the paint in panel II.A.3 of Cueva de Ardales and the resulting markings do not allow the recognition of discrete features. This suggests that cultivating the link with the place, rather than associating it with a particular representation, must have been the main reason for marking the stalagmites. Restoration of an image makes sense when the image itself carries symbolic information and is the focus of the artistic activity but what we see at Cueva de Ardales is distinct: the carrier of the symbolic information is, in this case, the large stalagmitic dome harboring the panel, not the panel itself. Put another way, treating the dome as the canvas is useful shorthand but should not be taken to imply that this large formation is no more than a convenient surface used to appose markings and that these markings are in and of themselves the repositories of symbolic information irrespective of where they were made. Instead, the dome is the symbol and the paintings are there to mark it as such, not the other way around. In this context, recurrent marking is not akin to the restoration or modification of a pre-existing motif to maintain, enhance or modify its meaning. Rather it must stand instead for the renewed assertion of the symbolic value of the dome itself.

Based on the results of this study, we hypothesize that panel II.A.3 is not "art" in the narrow sense of the word --"the making of objects, images, music, etc. that are beautiful or that express feelings, or "the activity of painting, drawing, and making sculpture" (https://dictionary.cambridge.org/dictionary/english/art)-but rather the result of graphic behaviors intent on perpetuating the symbolic significance of a space. The evidence from Bruniquel cave, in France, shows that Middle Paleolithic Neanderthals were involved in symbolic activities taking place deep inside the karst that involved the intentional modification of speleothems and their use in the construction of complex arrangements [START_REF] Jaubert | The chronology of human and animal presence in the decorated and sepulchral cave of Cussac (France)[END_REF]. Evidence from Cueva de Ardales confirms the view that speleothems played a fundamental role in the symbolic systems of some Neanderthal communities. Paint-marking using splattered red pigment on such large, imposing domes as that which panel II.A.3 decorates can thus be seen as a development deeply rooted in a long-standing tradition, of which other examples exist at Ardales. Rock art may therefore have begun in Europe as a form of place marking, with the Middle Paleolithic hand stencils and geometric signs seen at other Iberian cave sites (Maltravieso, La Pasiega, El Castillo and Gorham's Cave; [START_REF] Pike | U-series dating of Paleolithic art in 11 caves in Spain[END_REF][START_REF] Hoffmann | U-Th dating of carbonate crusts reveals Neandertal origin of Iberian cave art[END_REF][START_REF] Errico | The technology of the earliest European cave paintings: El Castillo Cave, Spain[END_REF][START_REF] Rodríguez-Vidal | A rock engraving made by Neanderthals in Gibraltar[END_REF]) representing much the same type of symbolic behavior. We predict that more markings bearing similarities with those from Cueva de Ardales will be identified in the future in the Iberian Peninsula and dated to the Middle Paleolithic. Although Upper Paleolithic cave art is technically and thematically more complex, signs and hand stencils play a prominent role in it [START_REF] Vialou | L'Art des grottes en Ariège magdalénienne[END_REF]. It is possible that markings such as those identified at Cueva de Ardales and other Iberian sites represent the prolegomena of a long process during which new needs linked to social complexification have triggered the development of novel symbolic traditions supported by the development of more varied and innovative technical practices.

Materials and Methods

Permission to sample the paintings was granted by the Department of Culture of the Regional Government of Andalusia. Subsequent to visual assessment of the pictorial representations, five samples were collected. Micro-agglomerates of pigments were recovered by scratching the outer surface at a unique spot. We paid special attention not to leave visible traces of the sampling. Photographs were taken before and after the extraction procedure to precisely document the location of the sampled area. To assess whether Fe-rich deposits present in the cave could have provided the pigmentatious material sampled in panel II.A.3, eight geological samples of natural coloring materials were collected from different Fe-rich deposits. The pigment and geological samples were submitted to a variety of microscopic, elemental, and mineralogical analyses. More information on methods is given in (SI Appendix, Materials and Methods, Fig. S1-S2; Table S1-S2).

Figure captions Figure 1 .Figure 2 .

 captions12 Figure captions Figure 1. Cueva de Ardales, Spain. Geographic location and plan of the cave. The position of recently excavated areas (zones 2, 3, 5), panel II.A.3 where samples P-ARD-02 to 06 were

Figure 3 .

 3 Figure 3. Close-up view of red stains, microphotographs and selected SEM images in BSE mode of red micro-samples extracted from panel II.A.3. A: curtain 9; B, C: curtain 8; D: curtain 6; E: curtain 5; F, K: P-ARD-02; G, L: P-ARD-03; H, M: P-ARD-04; I, N: P-ARD-05; J, O: P-ARD-06. Arrows in A to E indicate the extraction spot. Scale bars in A to E = 1 cm.

Acknowledgments

Authors are grateful for the support and assistance of the team who worked in the cave, including D. S. Fernández Sánchez, T. Kellberg, A. Moreno-Márquez, T. Otto and M. Rotgänger as well as the Centre for the Interpretation of Prehistoric remains from the Guadalteba -Ardales Museum. Authors also thank D. Hoffmann for sending us the speleothem sample overlying the pigment layer. Authors would also express their gratitude to X. Alcobé, D. Artiaga and J. Garcia from the Scientific and Technological Centers -University of Barcelona for their valuable help during the analyses. Permission to conduct research was granted by the Regional Department of Culture of the Regional Government of Andalusia (Consejería de Cultura -Junta de Andalucía, permit number: SIDPH/DI.201564100003000).


Guillaume Delalieux

Anne-Catherine Moquet

come

Guest editorial

Do CSR management control systems walk the talk? From Bhopal to Rana Plaza, 30 years of CSR practices Rana Plaza disaster: what lessons can be learned over the past 30 years of corporate social responsibility management practices?

The collapse of the Rana Plaza building of 2013 was the last disaster that caught the media's and academics' attention out of a long list of disasters that happened in the past decades. As the explosion of the Union Carbide factory at Bhopal in India in December 1984, to limit ourselves to industrial globalization in developing countries, many catastrophes happened in developing countries as a consequence of the increase of the wave of delocalization of industrial factories from well-regulated developed countries into weakly regulated developing countries. These catastrophes are reminiscent of older industrial catastrophes that paved the way of the history of industrial capitalist development, in developed countries in the 19th and 20th centuries [START_REF] Aggeri | Les entreprises et le développement durable[END_REF]. The Triangle Shirtwaist factory fire in 1911 in New York, caused the death of 146 people and dozens of injured, generating important media coverage and popular protest when the macabre circumstances were described by the press, with the doors to the stairwells and exits locked to prevent workers from taking unauthorized breaks. The fire led to stricter legislation and changes in safety prevention standards and participated to develop labor unionism [START_REF] Drehle | Triangle: The Fire That Changed America[END_REF].

Similar catastrophes in developing countries' factories most of the time [1] did not result in stricter legislation or strengthening of labor unionism but in the development of private mechanisms of corporate social responsibility (CSR).

The different coalitions of NGOs, industrials, governments and labor unions formed after each catastrophe or scandal in the 1990s, resulting in the form of multi-stakeholder initiatives (MSIs) or private mechanisms of CSR. As explained by [START_REF] Bartley | Certifying forests and factories: states, social movements, and the rise of private regulation in the apparel and forest products fields[END_REF], these practices were favored by the neoliberal institutional context, favoring private mechanisms considered as more efficient and cheaper for public finance than public intervention. These CSR practices, such as private mechanisms of certification, extra-financial reporting or management capacity building, were developed only after the coalition failed two previous attempts. They first failed to implement stricter importation policies in developed countries, banning goods involved in social and environmental dumping as in the case of Austria in 1992 because of the GATT considering social and environmental norms as barriers to trade. They then failed to convince producing countries such as Indonesia to strengthen their own legislation like protecting their forest, a request that Europeans did not apply to their own forest during their industrial development as duly noted by the Indonesian Government.

Thus, CSR can be viewed as a disputed concept [START_REF] Brabet | Le champ conteste de la responsabilité sociale des entreprises[END_REF][START_REF] Brabet | Corporate social responsibility and its models in a contested field[END_REF] for which multiple stakeholders struggle and collaborate. As such, its definition depends on how each of them ideologically analyzes controversies and the outcome is related to power influences between their different CSR ideas. This history of the path non-taken [START_REF] Veyne | Do workers benefit from ethical trade? Assessing codes of labour practice in global production systems[END_REF] is then important to understand the explosion of CSR mechanisms, the market for virtue (Vogel, 2010), during the past 30 years, as an alternative to governmental regulation. In this context, MNCs implement CSR management tools to prevent state intervention and carry on maximizing shareholder value [START_REF] Chamayou | La Société Ingouvernable: une Généalogie du Libéralisme Autoritaire[END_REF].

When stakeholders' willingness [START_REF] Brabet | Le champ conteste de la responsabilité sociale des entreprises[END_REF][START_REF] Brabet | Corporate social responsibility and its models in a contested field[END_REF] is admitted by MNCs, management literature has demonstrated how biased and selective this MSI coalition process is, after the Nike scandal in Pakistan for instance [START_REF] Khan | A dark side of institutional entrepreneurship: soccer balls, child labour and postcolonial impoverishment[END_REF], showing how invisible mechanisms of power operates: privileging northern consumers' preferences (no child work) while ignoring the demands of local workers in developing countries, e.g. pay rises or freedom of association and bargaining. In a similar way, De Neve (2013,2014) showed the ethnocentrism of CSR mechanisms developed in Indian factories, importing the "iron cage" bureaucratic power model, controlling and imposing workers' productivity and imposing strict discipline and obedience to management.

Corporate social responsibility vs shareholder value: two institutionally paralyzing managerial logics Can CSR regulations govern the logic of capital [START_REF] O'laughlin | Governing capital? Corporate social responsibility and the limits of regulation: governing capital? CSR and the limits of regulation[END_REF]? Questioning the ability of CSR devices initiated by MNCs to improve social and environmental conditions in developing countries implies explaining the paradoxical strengths inside this type of private organization.

Even if top executive management supports internal CSR programs and initiatives, the first back managerial actions that maximize shareholder value [START_REF] Boyer | L'impossible Éthique Des Entreprises[END_REF][START_REF] Moquet | Le Contrôle Sociétal[END_REF]. The top management has constantly to deal with this double bind -CSR concerns vs financial value as schizophrenic constraintsbut in the end, because of existing neoliberal governance systems and despite it goes against their personal convictions, they will choose to put resources where financial value might be optimized on a short-term basis.

These MNCs' systems of governance, therefore, explain that highest priority programs address the shareholder value, thus belonging to the neoliberal logic and that secondary managerial programs deal with CSR concerns as part of the CSR logic, both being internal competing institutional logics (Thornton and Ocasio, 1999, p. 804). That is why MNCs cannot be considered as organizations with a simple logic as it is deemed when CSR managerial tools are observed with an institutional external view. At an operational level, managers take part in one of these two managerial logics by implementing CSR or neoliberal action plans. These paradoxical logics are sometimes surprisingly visible at the corporate level when, for instance, CSR senior director's office is next to the tax directors who are in charge of the controversial international fiscal optimization of the accumulated free cash flows.

In some way, corporate purchasers are a good example of these paradoxical logics where the neoliberal one takes over the CSR one [START_REF] Quairel | La diffusion de la RSE par la relation fournisseurs: injonctions paradoxales ou partenariat de progrès?[END_REF]. Again, purchasers implement CSR and shareholder value devices. However, at this step, managerial practices become more complex as both institutional CSR and neoliberal logics are different in home and host countries [START_REF] Tan | MNC strategic responses to ethical pressure: an institutional logics perspective[END_REF]. If you consider purchase practices in home countries, you can observe that CSR tools have expanded in parallel with regulation, in particular in European countries, the last example being the law on Duty of Vigilance enforced in 2017 in France. Through these institutionalist lenses, CSR devices embody legitimacy flows [START_REF] Moquet | Les technologies de la responsabilité sociétale ou l'invention du manager responsable: le cas lafarge[END_REF] initiated by management as (only) symbolic solutions to comply with new CSR rules or answer to disputed working conditions-related issues. CSR initiatives are then made of voluntary actions and binding regulation that stems from hard or soft laws. That is why CSR managerial practices are ambivalent: they are at the same time very ineffective in tackling working-related issues and very effective in marginalizing workers' demands and promoting anti-unionism practices. When corporate purchasers implement CSR tools, they are well aware of the organizational hypocrisy [START_REF] Brunsson | The Organization of Hypocrisy: Talk, Decisions and Actions in Organizations[END_REF][START_REF] Dumez | L'hypocrisie organisationnelle[END_REF] that urges them to develop new CSR practices, knowing that it will postpone more constraining rules and focus on managerial actions that will maximize financial results. CSR mechanisms can be understood as a response from managers to resolve this double bind. Until now, this situation explains why CSR tools are counterproductive and lead at best to a no change in the working conditions in developing countries.

Do current corporate social responsibility management control systems improve poor workers' conditions in developing countries?

In what conditions is it then possible to give more internal power to CSR tools and managers? As part of the CSR institutional logic, some internal CSR managers act as tempered radicals [START_REF] Meyerson | Tempered radicalism and the politics of ambivalence and change[END_REF], who can be defined as:

[. . .] individuals who identify with and are committed to their own organizations, and are also committed to a cause, community or ideology that is fundamentally different from, and possibly at odds with, the dominant culture (Meyerson and Scully, 1995, p. 589).

Convinced by their own cause and conscious of the limitation of the dominant neoliberal institutional logic of their organization, they work out tactics to fight against neoliberal logic [START_REF] Moquet | Le Contrôle Sociétal[END_REF]. Hence, they advocate for social improvement using neoliberal logical arguments. Their actions enable only small incremental steps that help to give more institutional power to the CSR institutional logic and, in the best cases, give birth to effective CSR progress [START_REF] Capron | La Responsabilité Sociale D'entreprise[END_REF][START_REF] Moquet | Le Contrôle Sociétal[END_REF], even if these initiatives are limited to small pockets of CSR-driven values. The enforcement of the law on Duty of Vigilance is then a good example that could potentially bring those tempered radicals new opportunities for improving working conditions in developing countries. Hence, they could argue the requested diligence plan to give momentum to improve social conditions in developing countries. Progress is nevertheless limited to individual initiatives that do not change the dominant neoliberal institutional logic. It nevertheless may lead to neoliberal logic to take into account some CSR considerations. In this conceptual framework, MNCs' neoliberal institutional logic has no other choice but to change to take into account CSRdriven concerns for future corporate actions. However, when it comes to the operational level, this change requires cooperation and struggles between multiple private and public actors on the local and global level, and then give the opportunity to those tempered radical managers to use these external forces to convince internal operational managers and senior officers that now the institutional neoliberal logic needs to move on to new needs that can no longer be ignored. In that case, MNCsas part of the dominant institutional neoliberal logichave no choice but to recognize stakeholders' willingness [START_REF] Brabet | Le champ conteste de la responsabilité sociale des entreprises[END_REF][START_REF] Brabet | Corporate social responsibility and its models in a contested field[END_REF].

As their emergence at the beginning of the 1990s [START_REF] Bartley | Certifying forests and factories: states, social movements, and the rise of private regulation in the apparel and forest products fields[END_REF], MSIs control mechanisms of working conditions in supplier factories located in developing countries, literally exploded to enlarge the market for virtue (Vogel, 2010). A quasi-market composed of private mechanisms of certification, consulting services (management capacity building) and extra-financial reporting such as SRI (Levy et al., 2010) run by (not-) for-profit organizations. As their origin, numerous academic articles and reports [2] from experts have insisted on their limited efficiency to detect or correct working condition issues across the global supply chain, this literature being a thickening contribution for understanding MSI internal organizational weaknesses (O'Rourke, 2006;Wells, 2007;Locke, 2012;Anner, 2012;Chung, 2015;Lund-Thomsen, 2012). While most of the literature in management continues to favor a reformist perspective on CSR mechanisms, calling for their improvement, amelioration and strengthening, one of the main criticisms of CSR practices and MSI impact is directed beyond these practices, at the institutional level. Different studies have clearly emphasized the difficulty for such MSIexcluding their internal weaknessesto efficiently control and improve the global supply chain in weak institutional context (corruption, social and environmental dumping, failing resources, etc.).

On the contrary, this audit regime governing control mechanisms of working conditions seems rather effective in preventing workers' right to improve, wages to increase or freedom of association to emerge [START_REF] Gall | Global anti-Unionism: Nature, Dynamics, Trajectories and Outcomes[END_REF]Le Baron et al., 2017;Scheper, 2017). To a certain extent, CSR control mechanisms help to maintain industrial hegemony on global value chains (GCVs), thus performing a passive revolution [START_REF] Kourula | The micro-level foundations and dynamics of political corporate social responsibility: hegemony and passive revolution through civil society[END_REF] granting menial ethnocentric improvements (De Neve, 2014; [START_REF] Khan | A dark side of institutional entrepreneurship: soccer balls, child labour and postcolonial impoverishment[END_REF] favored by northern consumers while preventing at the same time strategic changes to occur for local workers such as freedom of bargaining. For some researchers (Guénin-Paracini and Gendron, 2010), these mechanisms have another important function, besides bringing profits to their shareholders: they provide useful scapegoats that can be blamed when CSR disasters happen, focusing attention on them, their weaknesses and lack of reliability while sweeping under the carpet deeper and structural inequalities at the basis of capitalism (Newell, 2008). As such, CSR mechanisms provide another good example of neoliberal technologies of government (Murphy, 2010) that, behind a rhetoric veil of transparency, participation and dialogue, operates a depoliticization process, a "CSRization" [START_REF] Sum | Wal-Martization and CSR-ization in developing countries[END_REF] of social and environmental issues, securing social elite domination (Lipschutz, 2005).

What are the clues for more effective corporate social responsibility practices? If we go back to the origins of CSR practices as told by [START_REF] Bartley | Certifying forests and factories: states, social movements, and the rise of private regulation in the apparel and forest products fields[END_REF] mentioned at the beginning of this introduction, one has to remind that MSIs were developed as a default solution, after failing attempts to develop institutional answers, e.g. include social and environmental clauses in commercial treaties or empower local administration to control working conditions. The historical path taken with the explosion of MSIs was opened by neoliberal institutions, favoring private mechanisms of certification largely subsidized at the beginning by governments (Clinton administration) over direct governmental interventions. Maybe the time has come to reflect on the paths not taken by history as follows: social and environmental norms to regulate importations of goods for importing countries and stronger local legislation, an administrative system to control and judicial system to sanction for producing countries as practical illustrations of an embedded coregulated RSE [START_REF] Brabet | Le champ conteste de la responsabilité sociale des entreprises[END_REF][START_REF] Brabet | Corporate social responsibility and its models in a contested field[END_REF]?

Alternatives to classical CSR mechanisms already exist. The International Labor Organization (ILO) better factory Cambodia program [START_REF] Polaski | Combining global and local forces: the case of labor rights in Cambodia[END_REF][START_REF] Oka | Brands as labour rights advocates? Potential and limits of brand advocacy in global supply chains[END_REF][START_REF] Bastianutti | Genèse et développement d'un dispositif de RSE dans les chaînes de valeur globales: le programme better factories Cambodia, dispositif alternatif à l'audit social[END_REF] implemented a reliable control program of working conditions in factories as a substitute for failing local authorities, even if this program is not free from criticism [START_REF] Arnold | Governmentalizing Gramsci: topologies of power and passive revolution in Cambodia's garment production network[END_REF]. INTERPOL developed different programs aiming on the contrary to strengthen and empower local administrative and judicial forces through capacity building programs. However, these programs clearly lack public financing to secure their independence. In the wake of the Rana Plaza disaster, the Accord agreement signed between International organizations, the government of Bangladesh, factories and buyers was an attempt to propose an alternative to MSI: Alamgir and Banerjee (2019) showed how this agreement while providing the legitimacy of MNCs, did little to improve workers' conditions and counter aggressive pricing and procurement policies. In this case, government involvement, including repression of opposition such as labor unions, offers a good example of a neoliberal reconfiguration of state power to refuse industrial democracy [START_REF] Donaghey | When industrial democracy meets corporate social responsibilitya comparison of the Bangladesh accord and alliance as responses to the Rana Plaza disaster[END_REF] while meeting global production network pressures.

Collective commercial negotiations on importation regulations are at a dead-end; on the contrary, bilateral agreements signed between countries tend to rely on audit mechanisms to avoid more social and environmental dumping. This institutionalization of this audit regime in governmental (Lebaron et al., 2017) or international regulations is taking a step further the neo-liberalization of the state initially observed by [START_REF] Bartley | Certifying forests and factories: states, social movements, and the rise of private regulation in the apparel and forest products fields[END_REF].

In this special issue of SBR

The first paper written by Clément Sehier shows that codes of conduct and social audits are instruments that benefit MNCs, and therefore, illustrate their domination in factories. The context of this study takes place in Chinese factories as part of the Chinese export industry. When positioning themselves in a "business case" tactic for a cost-benefit optimization, MNCs adopt a compliance approach that is imposed on their suppliers. In the worst cases, the implementation of this compliance approach has the opposite effects and leads to the destruction of existing social conditions. In the Chinese specific context, the civil society of developed countries put pressure on MNCs to change for more restrictive social purchasing criteria and Chinese authorities' attempts to improve labor laws even if they are still against collective rights. The results of this study demonstrate that the compliance approach is ineffective as it is disconnected from the daily factories' practices. The dominant shareholder-value management logic is proven by the MNCs' opposition to the 2007 labor law initiated by Chinese authorities. The inequitable redistribution of value-added within the GVCs tends to push the pressure on factories to respect conduct codes without transferring the financial means that would enable them to improve social working conditions. Then, factories face paradoxical situations with no other way than hypocritical organizational behavior where CSR instruments become symbolic.

In the second paper, Mohammad Islam and Christina Stringer address the issue of the transfer of economic upgrading to social upgrading based on a study conducted in Bangladesh's apparel industry. The authors point out the power asymmetry between the buyer firms and their direct and indirect subcontractors. Because of their dominant position, the buyer firms benefit from the major part of the value-added within the GVCs. Social upgrading is then considered as the result of co-constructed governance that mixes private, public and social structures. The authors analyze the working conditions in six Bangladesh industrial firms through the completion of 90 semi-structured interviews. The first result of this paper highlights the small steps achieved by the enforcement of the local social law upgrading regarding minimum wage and unions just after the Rana Plaza disaster. The second result of this study shows that governance within tiered Bangladesh firms leads to the limitation of social upgrading. Working conditions are better in the first-tier firms, are downgraded in the second-tier firms and deteriorate even more in the third-tier firms. Faced with the pressure of the buyer firms to comply with the conduct codes, first-tier firms subcontract to second-and third-tier firms for which noncompliance will be less visible. Therefore, MSIs made of private, public and social governance is necessary to improve social working conditions in tier firms within a GVC.

The third paper reflects on the role of development aid in private governance and its contradictions through the GVC concept lens of social upgrading. To do this, Laurence Beierlein proposes to analyze the Accord on fire and building safety in Bangladesh (hereinafter the Accord) founded in 2013, which was chaired by the ILO just after the Rana Plaza disaster. The exploration of this initiative is particularly relevant now that, despite wide international support, the Accord is confronted with a withdrawal of its license to operate in the country. Building on the numerous existing literature on the case, this recent event, thus, gives the opportunity to examine the contribution of development aid that supports governance instruments to effectively uphold social standards in developing countries. GVC literature underpins both development aid approaches to the private sector and social contestation on the uneven balance of power to the disadvantage of manufacturers from producing countries and their workers at the far end of the chain. It also reveals the complexity of the upgrading concept and shows the problematic disconnection between economic and social upgrading and the difficulties of reconciling both on the long term through private initiatives, even if these are supported and operationally carried out with the help of the ILO. An in-depth exploration of the Accord has been performed thanks to multiple secondary sources coming from academic literature and public documentary sources. While the Accord as an operational program has proven to be effective despite some limitations, it has endured early termination due to the contestation by local actors of its low inclusiveness, this struggle confirming the criticism made to private regulatory initiatives. The binding feature of the Accord that conferred legitimacy to the initiative has revealed itself a weak clause. The lack of transparency of its enforcement demonstrates a shortfall in MNCs' accountability. The Achilles' heel of the Accord lies in its incapacity to change the rules of the game and leads to even strengthening the balance of power in the advantage of the MNCs and the disadvantage of manufacturers from producing countries. The implementation of the Accord tends also to constrain the local producers' business model through increased compliance costs while MNCs' one is only affected by a limited financial contribution to the Accord. To conclude, the Accord's end shows that it can clearly be viewed as a "hegemonic" governance instrument. Further research on responses to governance deficits is necessary to investigate the consumer-end of the value chain and how to re-embed markets in society.

In the next paper, Léna Masson reflects on MNCs' regulation in developing countries based on a review of Locke's book The Promise and Limits of Private Power. Locke recognizes that private and public regulation used alone are both ineffective to ensure acceptable working conditions in developing countries. He is nevertheless convinced that a combination of private and state regulation would succeed in enhancing working conditions and workers' rights. In reaction to this argument, Léna Masson shifts the questioning from regulation to dysfunction of our neoliberal economy and the way brands are managed. These are the real causes of workplace issues in relation to site relocations and the emergence of poor workers in developing countries. Moreover, Léna tackles the problem of public regulation. She argues that Locke is ignoring state regulation in developed countries. Thus, the French Duty of Vigilance law illustrates that states of developed countries can potentially influence MNCs' behaviors and become part of a co-constructed regulation. However, the major flaw of this new regulation is that it could be considered as a new form of imperialism.

Guillaume Delalieux and Anne-Catherine Moquet, the authors of the last paper, a question to what extent the French Duty of Vigilance law could prevent more efficiently social and environmental concerns in GVCs. Along the way, they point out a due diligence paradox in the French law on CSR: while the spirit of this law was to harden soft law mechanism, might this apparent hardening cause a softening of hard law mechanism? Even if representatives of civil society pushed MNCs to implement CSR management practices more than 20 years ago, those initiatives suffer important limitations that lead to inaction or even reversal of progress to address CSR issues. In worst cases, beyond decoupled practices, organizational hypocrisy seems to be the last chance to respond to double binds. NGOs get round the usefulness of CSR certification and audit by organizing their own anonymous investigations. The French Duty of Vigilance law requires MNCs to implement reasonable due diligence actions related to social and environmental concerns. The assessment of its reasonableness by the judge is in itself a challenge regarding the gravity of violations and the organizational and financial resources of the organization to face it. This new law specifies that the diligence plan has to be built with the company's stakeholders, but past MSIs have proven that they do not represent a breakthrough compared to private CSR unilateral mechanisms. With no direct sanctions, the effectiveness of this law depends on the perceived non-compliance risks by managers. By promoting an implicit stakeholder ideology, this law underestimates the complexity of multilevel actors for whom social and environmental upgrading is not necessarily the choice. Based on the business case argument, defenders of this law expect responsible consumers to back virtuous firms that will comply with it and implement CSR devices. Thus, the weaknesses and failures of CSR auditing show that the business case argument is a modern myth. Moreover, the interpretation of the legal text by managers and the effectiveness of the enforcement of the law is not questioned. The risk in relation to the enforcement of this new law is to strengthen CSR mechanisms and doing so to institutionalize a neoliberal bureaucracy that hinders the emergence of more emancipatory alternative mechanisms for workers. Alternatives to the law on Duty of Vigilance lie in the crafting of less heterogeneous international regulations at the local and global levels. To illustrate concretely this idea, better factories Cambodia ILO initiative or INTERPOL programs rely on international organizations acting for operational assistance, these initiatives being based on the capacity to empower local governments on a long-term basis, so that they can preserve their environmental resources and protect human rights. The inclusion of social and environmental clauses in international commercial treaties is another illustration of these possible alternatives providing that the monitoring of compliance is not dependent on private mechanisms and linked to binding legislation. 2. 2019 Clean Clothes Campaign report on social auditing: https://cleanclothes.org/news/2019/wego-as-far-as-brands-want-us-to-go retrieved on September 24, 2019.

Guillaume

  Delalieux Department of Management, Sciences Po Lille, Lille, France and CERAPS, Lille, France, and Anne-Catherine Moquet Universite Paris-Est Creteil Val de Marne Institut d'Administration des Entreprises Gustave Eiffel, Creteil, France Notes 1. The aftermath of the Rana Plaza is a good example, the binding agreement signed between MSI lasted a few years, until the different actors realized that the amount of investment needed to secure factories would threaten the competitivity of the textile sector in Bangladesh (ref. Jamali).


Sassan Asgari
email: s.asgari@uq.edu.au

Dennis K Bideshi
email: dbideshi@calbaptist.edu

Yves Bigot
email: yves.bigot@inra.fr

Brian A Federici
email: brian.federici@ucr.edu

Ascoviruses (Ascoviridae, Ascovirus, and Toursvirus)

Keywords: Apoptosis, Ascoviridae, Ascovirus, caspase, Iridoviridae, Iridovirus, Hymenoptera, Lepidoptera, Megavirales, Noctuidae, parasitism, parasitoid, Toursvirus, Yponomeutidae. Glossary

Members of the family Ascoviridae infect larvae and pupae of insects that belong to the order Lepidoptera, and cause a chronic, but fatal disease in the host. They are horizontally transmitted by endoparasitoid wasps during subsequent parasitization events via contamination of the wasp's ovipositor. The virus typically induces a controlled apoptosis in the host cells leading to the cleavage of the host cells into numerous vesicles in which most virions are produced. This leads to a milky-white colouration of the hemolymph, that is a unique diagnostic feature of ascovirus infection. Ascoviruses have a circular double stranded DNA genome and are closely related to iridoviruses, from which they apparently evolved.

The family Ascoviridae is one of the newest families of viruses, established in 2000 to accommodate several species of a newly recognized type of DNA virus that attacks larvae of insects of the order Lepidoptera. It consists of two genera, Ascovirus and Toursvirus; for practicality, here we refer to the collective members of the family Ascoviridae as ascoviruses. Viruses of this family produce large, enveloped virions, measuring 130 nm in diameter by 300-400 nm in length, and when viewed by electron microscopy have a reticulated surface appearance. They are typically bacilliform or reniform in shape, and contain a circular double-stranded DNA genome that, depending on the species, ranges from 119-200 kbp. Whereas the virions of ascoviruses are structurally complex like those of other large DNA viruses that attack insects, such as those of iridoviruses (family Iridoviridae) and entomopoxviruses (family Poxviridae), they differ from these in two significant aspects. First, ascoviruses are transmitted from diseased to healthy lepidopteran larvae or pupae by female endoparastic wasps when these lay eggs in their hosts. Second, ascoviruses have a unique cell biology and cytopathology in which shortly after infecting a cell, they induce apoptosis and then rescue the developing apoptotic bodies and convert these into virion-containing vesicles. This aspect of viral reproduction apparently evolved to disseminate virions to the larval hemolymph (blood) where they could contaminate the ovipositors of female wasps so that the virus could be transmitted to new hosts. Ascoviruses appear to occur worldwide, wherever there are endoparasitic wasps and larvae of species belonging to the lepidopteran family Noctuidae. However, as these viruses have been discovered relatively recently and their signs of disease are not commonly known in the scientific community, relatively few ascovirus species have been described.

History

The first ascoviruses were discovered during the late 1970s in southern California where they were found causing disease in larvae of moths belonging to the lepidopteran family Noctuidae. Diseased larvae were recognized by the presence of hemolymph that was very white and opaque, in marked contrast to the hemolymph of healthy larvae which is translucent and slightly green (Figure 1). The color and opacity of the hemolymph in diseased larvae was shown to be due to the presence of high concentrations of vesicles that contained virions (Figure 2). The white hemolymph and virion-containing vesicles are diagnostic for the disease, and the name for this group, ascoviruses (derived from the Greek asco meaning 'sac'), was chosen on the basis of the latter characteristic. Since the discovery of the first ascovirus, ascoviruses have been isolated as the cause of disease in many species of noctuid larvae. In addition, an ascovirus that attacks the pupal stage of a species belonging to the family Yponomeutidae was discovered in the 1990s in France.

Distribution and Taxonomy

With respect to distribution, ascoviruses have been reported from the United States, Europe, Australia, and Indonesia, and it is highly probable that they occur worldwide. This is because their most common hosts, larvae of lepidopteran species belonging to the family Noctuidae, the largest family of the order Lepidoptera, as well as their most common vectors, endoparasitic wasps of the families Braconidae and Ichnuemonidae, are distributed throughout the world. Although only a few ascovirus species have been described to date, there are probably many, including variants, that occur worldwide. Thus, given the common occurrence of their hosts and vectors, it is possible that ascoviruses are very common insect viruses. That they have not been discovered in large numbers, for example like baculoviruses (family Baculoviridae), is probably because they cause a chronic disease with few easily detectable signs, making it difficult for individuals not familiar with the disease to recognize diseased larvae in field populations. At present, five species of ascoviruses are officially recognized based on a combination of properties including the relatedness of 28 key genes coding among others for the DNA polymerase and major capsid protein, the percent identity between their sequenced genomes, their lepidopteran host range, and their tissue tropism (Table 1). They are split into two genera, Ascovirus and Toursvirus. The type species of the Ascovirus genus is Spodoptera frugiperda ascovirus 1a (SfAV-1a), with the other species being Trichoplusia ni ascovirus 2a (TnAV-2a) and Heliothis virescens ascovirus 3a (HvAV-3a). To date, the Toursvirus genus contains only one virus species originally called Diadromus pulchellus ascovirus 4a (DpAV-4a), and now named Diadromus pulchellus toursvirus (DpTV-4a). The Arabic numeral reflects the order in which each species was formally recognized, whereas the lower case letter indicates variants of the type species. Variants from the type species are recognized by different consecutive lower case numbers; for example, TnAV-2b and 2c would represent two different variants of TnAV-2a recognized subsequently. Herein, these viruses are referred to by their acronyms without the numerical and lower case suffix. Members of the Ascovirus genus have been isolated from many more insect species than those listed above, but these isolates have turned out to be variants of known ascoviruses, and therefore they have not been named after the host from which they have been isolated. For example, ascoviruses related to TnAV and HvAV have been isolated from noctuid species such as Autographa precationis, Helicoverpa zea, Helicoverpa armigera, and Helicoverpa punctigera; however, they do not bear the name of their host of isolation. What this implies is that ascoviruses belonging to the TnAV and HvAV species have a broad and overlapping host range among different noctuid species, although this has only been tested experimentally to a limited extent.

Virion Structure and Composition

Depending on the species, the virions of ascoviruses are either bacilliform or reniform in shape, with complex symmetry, and very large, measuring about 130 nm in diameter by 300-400 nm in length. Although variations occur in the virion structure among various ascovirus species, their structural components appear to be essentially the same. Ascovirus virions consists of an inner particle surrounded by an outer envelope (Figure 2). The inner particle is complex containing a DNA/protein core as well as an apparent internal lipid bilayer surrounded by a distinctive layer of protein subunits. Thus, the virion appears to contain two lipid membranes: one associated with the inner particle and the other forming the lipid component of the envelope. In negatively stained preparations, virions have a distinctive reticulate appearance, which is thought to be due to superimposition of subunits on the surface of the internal particle with those in the envelope. As indicated by the size and complexity of the virions, the genome of ascoviruses is large, and consists of a single molecule of double-stranded circular DNA. The genomes of SfAV, TnAV, HvAV, or variants of these, in the genus Ascovirus, and TvAV-4a the sole virus in the genus Toursvirus have been sequenced (Table 1). The SfAV-1a genome is 157 kbp and codes for at least 120 proteins, whereas the TnAV genomes are slightly larger, 174-186 kbp, and codes for at least 165-178 proteins. The genomes of HvAV variants range from 186-200 kbp and code for approximately 180-190 potential proteins. The DpTV-4a genome is 119 kb in size and encodes about 119 proteins. Based on gel analyses, ascovirus virions contain more than 12 structural polypeptides ranging in size from 12 to 200 kDa. Furthermore, proteomic analysis revealed that that SfAV-1a virion is composed of at least 21 proteins, whereas similar analyses identified at least 67 proteins in the virion of HvAV-3i. The marked difference in the virion protein profiles of SfAV-1a and HvAV-3i could be due to inadvertent contamination with non-structral proteins or a higher level of structural complexity in the HvAV-3i virion. Regardless, the two major virion proteins in ascovirus virions are, invariably, the major capsid protein and members of an unusual large cationic protein family (ascovirus P64 homologs) that functions in condensing and encapsidating ascovirus genomic DNA. In addition to proteins and the DNA genome, the presence of an envelope as detected by electron microscopy, as well as experiments with detergents and organic solvents, indicate that virions contain a substantial lipid component. And, as in other enveloped viruses of eukaryotes, it is likely that the virion also contains carbohydrate in the form of glycoproteins, though at present none have been identified.

Transmission and Ecology

One of the most interesting features of ascoviruses is that their transmission from host to host appears to be dependent on their being vectored by female endoparasitic wasps (order Hymenoptera). Ascoviruses are extremely difficult to transmit per os, with typical infection rates averaging less than 15% even when larvae are fed as many as 10 5 virion-containing vesicles in a single dose. In contrast to this, infection rates for caterpillars injected with as few as 10 virion-containing vesicles are typically greater than 90%. Moreover, experiments with parasitic wasps show that they can effectively transmit ascoviruses to their noctuid hosts. For example, when females are allowed to lay eggs in ascovirus-infected noctuid caterpillars, thereby contaminating their ovipositor, and then allowed to lay eggs in healthy larvae, the majority of the latter contract ascovirus disease. Interestingly, though the parasitoid eggs hatch in their infected noctuid hosts, the parasitoid larvae die as the ascovirus disease develops in the caterpillar. Under field conditions, the prevalence of ascovirus disease in caterpillars is correlated with rates of parasitization by endoparasitic wasps. When wasps from these populations are collected in the field and allowed to oviposit in healthy caterpillars reared in the laboratory, the latter often exhibit ascovirus disease within a few days. Thus, laboratory and field studies provide sound evidence that the primary mechanism for the transmission of ascoviruses attacking noctuid larvae is through being vectored mechanically by parasitic wasps. No evidence has been found in the lepidopteran hosts for transovum or transovarial transmission. In the case of DpTV (genus Toursvirus), the association of the virus with its wasp and caterpillar hosts is much more intimate. DpTV DNA is carried in wasp nuclei as a circular molecule, and small numbers of virions are produced in the oviducts of females. However, the virus does not cause noticeable pathology in the wasp host. The females lay eggs in the pupal stage of the lepidopteran host, Acrolepiopsis assectella, introducing small numbers of toursvirus virions along with the wasp eggs. These virions invade lepidopteran host cells, replicate, and initiate destruction of major host tissues. The wasp larva then emerges from the egg and feeds on the host tissues and toursvirus virions. The DpTV genome is carried extrachromosomally by both male and female wasps, where it is apparently transmitted from generation to generation transovarially. These observations make ascoviruses the only known group of viruses pathogenic to insects that are primarily dependent on vectors for their transmission. Now that the characteristics of the disease are known, field studies in the southeastern United States and California are beginning to show that ascoviruses are probably the most common type of virus to occur during most of the year in populations of several important noctuid pests, including the cabbage looper, Trichoplusia ni, fall armyworm, Spodoptera frugiperda, and the corn earworm, Helicoverpa zea. Prevalence rates range from 10% to 25%, depending on the species and time of the year, with the highest rates of infection, as noted above, being correlated with high levels of parasitization. In South Carolina, ascovirus infection rates as high as 60% have been reported in populations of noctuid larvae at the end of summer.

Host Range

The experimental host range of ascoviruses varies with the viral species. TnAV, HvAV, and SeAV have a broad host range and are capable of replication in a variety of noctuid species, as well as in selected species belonging to other families of the order Lepidoptera. Alternatively, the experimental host range of SfAV is limited to other species of the genus Spodoptera. DpTV can replicate in hymenopteran and lepidopteran hosts closely related to its natural host species, A. assectella. To propagate virus in the laboratory, all ascoviruses can be grown in their larval or pupal hosts. To infect caterpillars, they are injected with virus in the third, fourth, or early fifth instar, and virion-containing vesicles are harvested from the hemolymph 5-7 days later. While ascoviruses can be propaged in lepidopteran host cell lines, virus pathology usually is not as distinct as that of in vivo. In addition, virus replication in vitro does not appear to be as high as that in vivo.

Pathology and Pathogenesis

Signs of Disease

In both natural and laboratory environments, the initial signs of ascovirus disease are very subtle, and this probably accounts for why ascoviruses were discovered only recently. The most obvious sign of disease within 24 h of infection is a decrease in the normal rate of feeding. The feeding rate continues to slow as the disease progresses, and as a result larvae fail to gain weight or advance in development. Healthy larvae, particularly in the early stages of development, will easily quadruple their weight and size in a period of 3 or 4 days, whereas ascovirus-infected larvae cease to grow and may actually lose weight. This feature of ascovirus disease is almost impossible to detect in infected larvae in the field. However, it is easily noticed under laboratory conditions when infected and healthy larvae are reared side by side over a period of a few days. A second feature easily noted in the laboratory is that ascovirus diseases are chronic, though usually fatal. When infected during early stages of development, ascovirus-diseased larvae often survive for 2 or 3 weeks beyond the time at which most healthy larvae have completed their development and pupated. Signs of disease other than these are minor, but include the inability to completely cast the molted cuticle, a bloated thoracic region, and a white or creamy discoloration and hypertrophied appearance of the larval body at advanced stages of disease development.

Cytopathology and Cell Biology

In comparison to all other known viruses, the most unique property of ascoviruses is the unusual cytopathology that leads to the formation of the virion-containing vesicles. This process resembles apoptosis, and previous studies of the SfAV-1a genome have shown that it encodes a functional executioner caspase, synthesized 9 h after infection, which by itself is capable of inducing apoptosis (Figure 3); at present, no other virus is known to encode a functional caspase. Although in vitro studies have demonstrated that knock-down of SfAV-1a caspase by RNA interference significantly reduced the formation of apoptotic vesicles, it is unclear whether caspase homologs in other ascoviruses participate in this process. For example, in similar studies a caspase-like gene in HvAV-3e was shown not to be implicated in apoptosis but was essential for virus replication. At the cellular level in vivo, the disease begins with extraordinary hypertrophy of the nucleus accompanied by invagination of sections of the nuclear envelope, followed by a corresponding enlargement of the cell. Cells typically grow from 5 to 10 times the diameter of uninfected cells. As the nucleus enlarges, the nuclear envelope ruptures and disintegrates into fragments. At about this stage, the cell plasmalemma begins to invaginate along 'planes' toward the now anucleate cell center. Concomitantly, sheets of membrane form closely adjacent to mitochondria that accumulate along the planes. As this process continues, the membrane sheets coalesce and join the invaginating plasmalemma, thereby cleaving the cell into a cluster of 20 to more than 30 vesicles, ranging in size from 5 to 10 µm in diameter. This aspect of ascovirus cellular pathology resembles the formation of apoptotic bodies during apoptosis. However, rather than dissipate as the cell dies, the developing apoptotic bodies are rescued by the virus and progress to form vesicles in which virions continue to assemble. These virion-containing vesicles, also referred to as viral vesicles, typically remain in the tissue until the basement membrane ruptures, though on occasion cell hypertrophy can be so great that the enlarging cell erupts out through the basement membrane of the infected tissue, releasing large fragments of the infected cell directly into the hemolymph. Analysis of ascovirus genomes shows that, unlike many other large DNA viruses, ascoviruses encode several lipid-metabolizing enzymes that are likely involved in the process of converting developing apoptotic bodies into virion-containing vesicles. Recent transcriptome analysis have shown that genes coding for a patatin-like phospholipase, a PlsC phosphate acyltransferase, a fatty acid elongase, and an esterase/lipase in SfAV-1a are highly expressed during later stages of virus infection in vivo. These enzymes are known to modify fatty acid chain length and the degree of lipid saturation, presumably generating novel lipid components in the virion and/or the membranes of viral vesicles. Regarding the latter, it is possible that these lipids, in part, function in providing stability to viral vesicles as they circulate in hemolymph for an extended period encompassing several days or weeks, and when these vesicles are disseminated through oviposition by female parasitic wasps. Another interesting aspect of this cytopathology is that the mitochondria, apparently under ascovirus control, are reprogrammed to assist synthesis of membranes that cleave the cell into the viral vesicles in which most replication occurs. Although the process by which viral vesicles are cleaved from cells varies among different ascoviruses, the histopathology is similar among virtually all ascoviruses. Vesicles accumulate in the tissues where they are formed, but as these tissues degenerate during disease progression, the basement membrane of infected tissues deteriorates and ruptures, allowing the vesicles to spill out into the hemolymph. There they accumulate reaching concentrations as high as 10 7 -10 8 vesicles per ml within 3-4 of infection. There is some evidence that viral replication proceeds within the vesicles as they circulate in the hemolymph, and thus this tissue must also be considered one of the tissues attacked by ascoviruses. In fact, because such high concentrations of viral vesicles are found in the hemolymph, this tissue could be considered a major site of infection, particularly if it is eventually shown that these viruses continue to replicate in the vesicles as they circulate in the hemolymph. Despite the chronic nature of the disease caused by ascoviruses, virion-containing vesicles are present in the hemolymph within 2 or 3 days of infection. When the virus replicates in cells in vitro, the vesicles are formed within 12-16 h of infection. The rapid development and circulation of the viral vesicles in the hemolymph probably evolved to enhance transmission of the virus by parasitic wasps.

Tissue Tropism

The cytopathology of ascoviruses is consistent among different viral species; however, considerable variation occurs with respect to the tissues attacked, that is, in which replication occurs. TnAV, HvAV, and SeAV exhibit a relatively broad tissue tropism infecting the tracheal matrix, epidermis, fat body, and connective tissue. Differences exist between these species in that some HvAV variants infect the epidermis much more extensively than TnAV variants, whereas some of the latter can also replicate more extensively in fat body cells, but appear only to do this when larvae are infected early in their development. Alternatively, the type species, SfAV, and its variants have a very narrow tissue tropism, with the fat body being the primary site of infection. DpTV occurs in the nuclei of all tissues of its wasp host, but appears to only produce progeny in ovarial tissues. In its lepidopteran pupal host, it attacks and replicates in a wide variety of tissues.

Replication and Virion Assembly

Although there have been few biochemical studies of viral DNA replication or protein synthesis, studies carried out with ascoviruses in vivo and in vitro show that progeny virions first appear about 12 h after infection. Virion assembly is initiated after the nucleus ruptures, and occurs prior to and during the cleavage of the cell into viral vesicles. The first recognizable structural component of the virion to form is the multilaminar layer of the inner particle. Based on its ultrastructure, this layer consists of a unit membrane and an exterior layer of protein subunits. As the multilaminar layer assembles, a dense nucleoprotein core aggregates on the interior surface. This process continues until the inner particle is complete. After formation, the inner particle is enveloped by membranes within the cell or vesicle. These membranes are apparently synthesized de novo. Thus, the assembly of the virions is reminiscent of that in other viruses with complex virions, such as the iridoviruses, herpesviruses, and poxviruses, where the virions differentiate after association of the precursors of virion structural components. After formation, the virions of the TnAV ascovirus accumulate toward the periphery of the vesicle where they often form inclusion bodies, that is, aggregations of virions (Figure 2). In SfAV, occlusion bodies are formed in which the virions are actually occluded in a 'foamy' vesicular matrix that consists of a mixture of protein and minute spherical vesicles. When viewed with phase microscopy, these viral inclusion and occlusion bodies are phase bright, and are largely responsible for the highly refractile appearance of the vesicles.

Ascoviruses do not typically form the types of occlusion bodies characteristic of other types of DNA insect viruses, such as baculoviruses and entomopoxviruses.

Origin and Evolution

The subject of viral evolution over millions of years has received relatively little study due to the lack of a fossil record. Moreover, viruses are considered polyphyletic, and thus most of the more than 70 families of viruses are thought to have originated independently. In this regard, ascoviruses may provide a unique opportunity to obtain insights into virus evolution over long periods. Phylogenetic comparisons of ascovirus genes sequenced to date including those coding DNA polymerase and major capsid protein as well as 20 other proteins indicate that these viruses evolved from a lepidopteran iridovirus (family Iridoviridae). Iridoviruses, in turn, appear to share a common ancestor with two families of Megavirales, Marseilleviridae and Pithoviridae which attack certain aquatic unicellular organisms.

Future Perspectives

At present, too little is known about ascoviruses to assess whether they are or will turn out to be of economic importance. Their poor infectivity per os makes it highly unlikely they will ever be developed as viral insecticides, especially given the successful advent of insect-resistant transgenic crops. However, as more entomologists become familiar with the disease caused by ascoviruses, it may be shown that in habitats rarely treated with chemical insecticides, such as transgenic crops, these viruses are responsible for significant levels of natural pest suppression, particularly where parasitic wasps are abundant. Such findings would encourage even greater emphasis on the development of biological control and other more environmentally sound methods of pest control. With respect to the cell biology of viral vesicle formation, ascoviruses provide an interesting model for how apoptosis can be manipulated at the molecular level. Additionally, study of the unusual process by which ascoviruses rescue the developing apoptotic bodies to form viral vesicles could lead to insights into how cells manipulate the cytoskeleton and mitochondria. Finally, it is possible that viral vesicles will provide a unique anucleate cellular system for studying the replication of a complex type of enveloped DNA virus in vitro. Figure 3 Major stages of cellular pathogenesis caused by a typical ascovirus, a process that resembles apoptosis. After infection, the nucleus enlarges and the nuclear membrane invaginates, and then lyses.

Figures

Figure 1

 1 Figure 1 Major characteristics of the disease typically caused by ascoviruses in lepidopteran larvae. (A) and (B) Healthy and ascovirus-infected larvae, respectively, of the cabbage looper, Trichoplusia ni, infected with TnAV. Note the opaque white hemolymph in the infected larva. (C) Spot plate containing hemolymph from healthy (left) and infected larvae (right). (D, E) Sections through lobes of fat body from a healthy and infected larva, respectively, of the fall armyworm, Spodoptera frugiperda, infected with SfAV ascovirus. Note the greatly hypertrophied cells in the fat body of the infected larva. The cells in most of this tissue have already cleaved into viral vesicles. N, nuclei.Figure 2 Structural and morphological characteristics of ascovirus virions and virion-containing vesicles. (A) Wet mount preparation viewed with phase microscopy of hemolymph from a Spodoptera frugiperda larva infected with Spodoptera frugiperda ascovirus (SfAV). The spherical refractile bodies are virion-containing vesicles. (B, C) Electron micrographs of ultrathin sections through viral vesicles produced by Trichoplusia ni ascovirus (TnAV) and SfAV, respectively. (D) Matrix of the occlusion body produced by SfAV. The occlusion consists of virions, protein, and small spherical vesicles. (E, F) Negatively stained virions of SfAV and TnAV, respectively. Note the reticulate appearance of the virions. (G, H) Electron micrographs of ultrathin cross sections through inner particles of SfAV after formation (G) and during envelopment (H). (I) Ultrathin cross section through a fully developed virion of TnAV.Figure3Major stages of cellular pathogenesis caused by a typical ascovirus, a process that resembles apoptosis. After infection, the nucleus enlarges and the nuclear membrane invaginates, and then lyses.

Figure 2

 2 Figure 1 Major characteristics of the disease typically caused by ascoviruses in lepidopteran larvae. (A) and (B) Healthy and ascovirus-infected larvae, respectively, of the cabbage looper, Trichoplusia ni, infected with TnAV. Note the opaque white hemolymph in the infected larva. (C) Spot plate containing hemolymph from healthy (left) and infected larvae (right). (D, E) Sections through lobes of fat body from a healthy and infected larva, respectively, of the fall armyworm, Spodoptera frugiperda, infected with SfAV ascovirus. Note the greatly hypertrophied cells in the fat body of the infected larva. The cells in most of this tissue have already cleaved into viral vesicles. N, nuclei.Figure 2 Structural and morphological characteristics of ascovirus virions and virion-containing vesicles. (A) Wet mount preparation viewed with phase microscopy of hemolymph from a Spodoptera frugiperda larva infected with Spodoptera frugiperda ascovirus (SfAV). The spherical refractile bodies are virion-containing vesicles. (B, C) Electron micrographs of ultrathin sections through viral vesicles produced by Trichoplusia ni ascovirus (TnAV) and SfAV, respectively. (D) Matrix of the occlusion body produced by SfAV. The occlusion consists of virions, protein, and small spherical vesicles. (E, F) Negatively stained virions of SfAV and TnAV, respectively. Note the reticulate appearance of the virions. (G, H) Electron micrographs of ultrathin cross sections through inner particles of SfAV after formation (G) and during envelopment (H). (I) Ultrathin cross section through a fully developed virion of TnAV.Figure3Major stages of cellular pathogenesis caused by a typical ascovirus, a process that resembles apoptosis. After infection, the nucleus enlarges and the nuclear membrane invaginates, and then lyses.

Tentative species Spodoptera exigua ascovirus 5a SeAV-5a Spodoptera exigua ascovirus 6a

SeAV-6a Helicoverpa armigera ascovirus 7a

HaAV-7a Helicoverpa punctigera ascovirus 8a

HpAV-8a Subsequently, the plasmalemma of the cell invaginates and coalesces with cytoplasmic membranes, apparently formed de novo, thereby dividing the cell into a cluster of virion-containing vesicles. These vesicles dissociate and are liberated into the hemolymph as the basement membrane of infected tissues degenerates. Virion assembly becomes apparent as the nuclear membrane lyses, and continues throughout all subsequent stages of vesicle formation.