Detailed three‐dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy

Abstract

SummaryResilin is a rubber-like protein found in the exoskeleton of arthropods. It often contributes large proportions to the material of certain structures in movement systems. Accordingly, the knowledge of the presence and distribution of resilin is essential for the understanding of the functional morphology of these systems. Because of its specific autofluorescence, resilin can be effectively visualized using fluorescence microscopy. However, the respective excitation maximum is in the UV range, which is not covered by the lasers available in most of the modern commercial confocal laser scanning microscopes. The goal of this study was to test the potential of confocal laser scanning microscopy (CLSM) in combination with a 405 nm laser to visualize and analyse the presence and distribution of resilin in arthropod exoskeletons. The results clearly show that all resilin-dominated structures, which were visualized successfully using wide-field fluorescence microscopy (WFM) and a 'classical' UV excitation, could also be visualized efficiently with the proposed CLSM method. Furthermore, with the application of additional laser lines CLSM turned out to be very appropriate for studying differences in the material composition within arthropod exoskeletons in great detail. As CLSM has several advantages over WFM with respect to detailed morphological imaging, the application of the proposed CLSM method may reveal new information about the micromorphology and material composition of resilindominated exoskeleton structures leading to new insights into the functional morphology and biomechanics of arthropods.

Evidence for a material gradient in the adhesive tarsal setae of the ladybird beetle Coccinella septempunctata

“…The CLSM analyses revealed the presence of considerable proportions of resilin in the adhesive tarsal setae, indicated by the blue autofluorescence of resilin 24 . At the tips of the setae, the material composition is strongly dominated by resilin, whereas the central and proximal parts of the setae mainly consist of other materials (other proteins and very likely mainly chitin), indicated by the dominance of green and red autofluorescences in these structures (Fig.…”

“…For all analyses and measurements, fresh forelegs of C. septempunctata females, collected in the botanical garden of the university in Kiel, Germany, were used. For the CLSM analyses, the beetles were frozen at À 70°C, and later the legs were removed as described earlier 24 . The beetles used for all other analyses and measurements were kept in plastic boxes on clean moist paper towels to prevent leg contamination and animal desiccation.…”

“…Fresh beetle forelegs were transferred to glycerine (Z99.5%, free of water, two times distilled, Carl Roth GmbH & Co. KG) and mounted using high-precision cover slips (thickness ¼ 0.170 mm ± 0.005 mm, refractive index ¼ 1.52550±0.00015, Carl Zeiss Microscopy GmbH, Jena, Germany) as described earlier 24 . The presence and the distribution of four different autofluorescences within the second adhesive pad of the legs were analysed using the confocal laser scanning microscope Zeiss LSM 700 (Carl Zeiss Microscopy GmbH) equipped with an upright microscope (Zeiss Axio Imager.M1m) and four solid-state lasers (wavelengths: 405, 488, 555, 639 nm) and applying a previously described method 24 . In contrast to this method, instead of a longpass emission filter transmitting light with wavelengths Z560 nm, a longpass emission filter transmitting light with wavelengths Z640 nm was used to detect the autofluorescence excited by 639 nm.…”

“…Previous studies on other biological materials [21][22][23] have indicated that AFM nanoindentation is a well-suited method to analyse local mechanical properties of small volumes of the material of the adhesive tarsal setae. CLSM has been shown to have a great potential for visualizing the morphology and the material composition of arthropod exoskeleton structures with sub-micrometre resolution 24 making this technique ideal to evaluate a supposed material gradient within the adhesive tarsal setae.…”

Evolutionary phenomics and the emerging enlightenment of arthropod systematics

“…1E). Specimens also do not require exotic media: Canada balsam, euparal, glycerine jelly (Michels 2007;Michels and Gorb 2012) or agarose are the usual embedding media. CLSM can also be used for visualisation of internal structures through the cuticle in transparent specimens, which rapidly yields information about the site of origin of muscles corresponding with external cuticle modifications (Fig.…”