The CLSM observations of spider web threads revealed a homogenous autofluorescence easily used for the 3D reconstructions. The fluorescence is limited to the range of blue/green wavelength, generally between 450 and 630 nm. It is important to note that the same signal is given by the threads included in amber and by the silk of the extant spider webs.

Spectral analysis of the threads reveals a similar spectral signature with an emission peak for amber-included spider webs at 469 nm, and an emission peak of 505 nm for garden spider webs (Fig. 2).

Amber pieces from La Garnache contain in abundance filamentous structures presented in two more or less complementary forms: web-like structures and isolated or sets of threads. As distinguishable from optical observations, the web structures can occupy space without a privileged direction, with a loose network extending over several centimetres in length (Fig. 1). The 3D reconstructions performed by the CLSM provide similar features for the web organization of La Garnache (Fig. 3A) and present-day samples (Fig. 3B). Clusters of threads are fairly common, but despite the high density of some of these clusters, they do not really affect the transparency of the amber (Fig. 3C). In some cases, the alignment of threads can be observed in the direction of stretching of the casting resin (Fig. 3D,E).

The web is generally composed of threads of different thickness (1 μm to 20 μm in diameter) delimiting principal and secondary elements of the web construction (Fig. 1 and Fig. 4 A and B). The architecture of the web is characterized by junction points of threads, often marked by thickening. The junction point may involve more than two threads and as many as four. From these branching points, threads fall in different directions and thus in several optical planes (Fig. 1 and Fig. 3). Triangular and trapezoidal figures resulting of branching and junctions are often visible delimiting an irregular frame (Fig. 3A). The threads can form loops that close on themselves (Fig. 4A,B), as also visible on present-day webs (Fig. 4C). Threads may be more intertwined, forming twisted braids (Fig. 4 and Fig. 5), similar to present-day features (Fig. 4E). Very often, the threads are associated as strongly intertwined pairs with parallel orientations and similar twists and kinks (Fig. 4F), a setting again common in current webs (Fig. 4G).

A number of the threads show more or less loose kinks and spirals (Fig. 5B–E), frequent in current spider webs (Fig. 5F). As Brasier et al. (2009) emphasized concerning the spider web preserved in the Cretaceous amber of Hastings, twisting of the threads may reflect tension, and kinks result from the relaxation of initial tension. In some cases, the winding of the threads is accomplished by the formation of a short circular spiral circular (Fig. 5G–I). Such geometry was reported by Benjamin et al. (2002) in the construction of some current spider webs.

Infrequent droplets may fall irregularly along the threads (Fig. 6A–B). Although some spherical droplets are visible, they are more or less elongated in the direction of the thread.

Under classical microscopy, the threads included in amber, like extant spider web threads, appear as “full” homogeneous filaments that may be irregularly twisted, sometimes imparting a rosary appearance which might suggest cells of filamentous microorganisms such as bacteria or microfungi. The CLSM observations highlight this character more precisely and allow us to see that the thickness of the threads may vary; irregularly distributed thickenings are often observed (Fig. 6 C).

When we deal with filamentous networks in amber, we can question their real nature: filamentous microorganisms or spider web?

Brasier et al. (2009) pointed out that the spider threads identified in amber invariably lack any evidence for cellular organization, such as regular cell wall divisions or reproductive bodies. On the contrary, Ascaso et al. (2003) remarked that mycelia present in amber show a clear differentiation evidenced by CLSM, including the mode of preservation. Our observations under optical microscopy and CLSM confirm that the filamentous network of the supposed spider web from La Garnache amber exhibits no concentric differentiation, no walls, no central lumen, no partitioning, and no constrictions, unlike many filamentous organisms observed in the same samples (Fig. 6D), and, more generally, in other Cretaceous amber. These observations allow us to provide supplementary criteria for the discrimination of the spider web in amber using CLSM. The spider webs do not alter the transparency of the amber, whereas dense networks of filamentous mycelia give a milky or opaque appearance to the amber (Saint Martin et al., 2012 and Saint Martin et al., 2013). Moreover, the autofluorescent ability of spider silk and their spectral characteristics could also be a criterion of discrimination. Provocatively, we can hypothesize that some misidentified filaments observed in amber and considered mycelia of microorganisms could be in fact fragments or threads of spider webs. The examination of the material under CLSM then proves a good means of investigation and discrimination.

The characteristics of the webs evidenced in the La Garnache amber are highlighted by the autofluorescence exhibited by the threads. In extant organic silks, the fibre is mainly composed of proteins called fibroins and more especially spidroin for the spider silks. Recent studies (Hayashi and Lewis, 2001, Lefèvre et al., 2011 and Motriuk-Smith et al., 2005) provide important insights into fibroin-spidroin structure, showing that these proteins are high molecular-weight biopolymers composed of an alternating amino acid rich blocks between non-repetitive N- and C-terminal domains. The fibroin contains three aromatic amino acid residues (tryptophan, tyrosine, and phenylalanine), which may contribute to their intrinsic fluorescence (Georgakoudi et al., 2007). Tryptophan is the most used to assess protein structure, since it has the highest quantum yield and extinction coefficient of the three aromatic amino acids (Georgakoudi et al., 2007).

The spectral analysis performed on the La Garnache amber revealed very similar emission spectra with the spectra reported by Rice et al. (2008) using two-photon microscopy of silkworm silk fibroin-based biomaterials. These authors observed peaks around 465 nm for dehydrated biomaterial and around 500 nm for hydrated biomaterial. The blue shift of the emission spectra of dry silk fibroin has been explained by Rice et al. (2008) as being a response to a more polar environment for the dry fibroin. Furthermore, fluorescence of amino acids, such as tryptophan has long been known to be sensitive to the polarity of their local environment (Gaines et al., 2010, Vivian and Callis, 2001, Weber, 1960). This is consistent with our data where amber spider webs, representing the dehydrated form show a blue-shifted spectrum in comparison to garden spider webs, which were mounted in Eukitt^® mounting medium prior to observation, thus, representing the hydrated form.

The autofluorescence signal observed and its spectral characteristics therefore suggest that amber preserved, at least in part, the original character of the organic material that constitutes the spider web.

Spiders produce several types of webs generally associated with different families, including spiral orb webs, tangle webs, cobwebs, sheet webs, and funnel webs. Orb webs represent the best known type of spider web and are woven by numerous families of spiders, especially the Araneidae.

In the fossil record, it is mostly the droplets associated with spider web threads that have attracted attention (Alimohammadian et al., 2005, Brasier et al., 2009 and Knight et al., 2010; Peňalver et al., 2006; Zschokke, 2003). However, Peñalver et al. (2006) clearly described in Cretaceous amber from Spain an evident radial architecture organised in a single plane characteristic of orb webs. More generally, the webs are effectively rather recognized as orb webs and the araneid spiders often invoked as the weavers (Peňalver et al., 2006; Poinar and Buckley, 2012, Poinar and Buckley, 2014 and Zschokke, 2003).

According to Blackledge et al. (2011), the orb webs are distinguished from other types of spider webs in their suspension in the air column upon discrete networks of frame threads and their two-dimensional (2D) capture surfaces. One of the main characteristics is the radial aspect of the construction. The webs observed in amber from La Garnache do not present specific characteristics typifying the orb webs as follows: the network of threads is three-dimensional and their organization appears quite confused, the threads have loops and multiple connection points are possible, no radial element is distinguishable, and the droplets are infrequent. Thus, the webs of La Garnache may be more likely cobweb or sheet web types.

It was considered that a cobweb is a highly complex, disordered, three-dimensional meshwork (Moore and Tran, 1999). More recently, Benjamin and Zschokke, 2002 and Benjamin and Zschokke, 2003 stated that cobweb spiders, as the Theridiidae, spin three-dimensional webs apparently organized chaotically but actually constructed using stereotyped behaviours that result in some structural elements. Features of the webs of La Garnache resemble this type of three-dimensional construction that seems to show no specific organization (see Fig. 3). Other aspects, such as the connections at short spirals could also evoke the characters of sheet webs described by Benjamin et al. (2002). Nevertheless, it seems very difficult to give a more precise assignment in the absence of identifiable anchorage dispositive and of the overall web geometry defining the weaving strategies (Benjamin and Zschokke, 2003 and Zevenbergen et al., 2008). However, some features of observed webs can reveal construction processes and behaviour of the responsible weaver spider. For instance, the thickness variations along the same thread (as seen in Fig. 6C) may be explained by the fact that usually a cobweb reinforces original guy lines (Moore and Tran, 1999). The general architecture of the web trapped in amber exhibits two types of areas regarding the density of threads as observed in Fig. 1 and Fig. 3A: areas with spaced lines and areas with more interconnected lines. This kind of organisation was observed in modern cobweb and explained by two different specific functions: widely spaced lines are devoted to prevent collapse, while interconnected lines serve as a meshwork for catching prey (Moore and Tran, 1999). The general architecture of webs may also reflect the physiological state of the spider. The studies of modern spider web by Zevenbergen et al. (2008) showed that “spiders in poor body condition spin webs that are more effective at capturing prey than the webs spun by spiders in good body condition, providing a functional benefit to behavioural plasticity in these spiders”. Effectively the authors observed that spiders in poor body condition spin cobwebs presenting many threads coated with glue (gum-footed threads) at their base, while spiders in good condition spin webs that lack gum-footed threads. From this point of view, concerning the architecture of the webs found in La Garnache amber, we may hypothesize that the web was woven by an almost starved spider because there are only a few glue droplets observed.

According to Breton (2012), spider webs encompassed in modern resin exuded along pine trunks would be cob-webs, which confirms our own observations (Figs 6E–G).

The abundance of spider web remains, and sometimes their density, in the amber of La Garnache, allows us to assume that weaver spider activity was important in the production environment of the original resin. Current observations of coniferous trees show the pervasiveness of more or less complete spider webs, or at least the threads of disorganized webs, near or in contact with resin in production (Fig. 6E–G). Some spider webs have been encompassed in resin flows near their location. Deriving thread clusters have also been included within resin and aligned along the flow of the resin.

The silk of spider webs is reputed to be fragile and degraded very quickly. The inclusion in resin is probably the only way to preserve them in geologic time. However, even preserved in amber, silk has certainly undergone physicochemical changes that altered its original nature. The differences in the spectral analysis of the silk from the amber of La Garnache and an extant web could result from these changes. More comprehensive analysis of the spider webs preserved in amber and various types of current spider webs are needed to discriminate what amounts to the effects of preservation or the specificity of silks depending on kinds of spider.