22.04.2022

A new technique for lightening chitinized structures

For some time now, the taxonomic publications and descriptions of new tarantula species by some Chinese biologists have been remarkable for the excellent quality of the images in the publications. Taxonomic distinguishing features such as the bulbs of the males or stridulation organs are shown very clearly and remarkably brightly, so that the shape and number of keels on the bulbs can be seen and understood much better than in comparable publications by other researchers in which the Structures are shown in their often dark original colors, because usually the bulbs and the chelicerae consist of quite dark chitin. Dark keels or dark stridulation spines on the above mentioned body segments are therefore often difficult to distinguish from the dark background and form little contrast to it. This is different within the tarantula-related publications by the Chinese researchers. There, the mentioned structures are often shown relatively brightly, so that the number and shape of even the smallest keels, as well as the stridulating bristles, can be seen very well (see Lin et al. 2022a; Lin et al. 2022b). Pic 1 shows the bulb of a Haplocosmia sp. that could be examined in a museum collection in its original colouration and Pic 2 shows the bulb with its colouration of Haplocosmia sherwoodae from the original Chinese description of this species from Lin et al. (2022b). Since both males are about the same size and generally colored identically, it can be assumed that the bulbs should also be similarly dark colored, as can be seen in Pic 1. We therefore asked ourselves how the Chinese researchers could photograph the structures, especially the bulbs of the males and the stridulation organs so clearly, brightly and distinctly visible, when these structures are actually and usually much darker. One guess was that the structures might be lightened by some chemicals. So we searched the Internet for possible substances that would make it possible to lighten the chitinized structures. We found an article by a biologist from the University of Wuerzburg (Germany) who made ants more or less transparent using 35% hydrogen peroxide (H2O2), trypsin enzyme solution and sodium tetraborate. (Stueben & Linsemair 2008). However, our goal was not to make the structures transparent, but only to lighten them up. We therefore organized the basic substance, namely hydrogen peroxide in a 30% solution (Pic 3). Hydrogen peroxide is known for its bleaching properties. It is often used in a highly diluted version as a cleaning agent, as it has a disinfecting, antibacterial and antiseptic effect. It is freely available in Germany up to a concentration of 12%. Care should be taken when handling hydrogen peroxide, especially when its content is 30% or more in a dilute solution, as it is corrosive and highly combustible. Therefore, you should only work with appropriate protective clothing, i.e. overalls (A), protective goggles (B) and rubber gloves (C) (Pic 4).

The first attempt to lighten a bulb with hydrogen peroxide brought a satisfactory result. We separated a bulb of a large male Theraphosinae from the Subtegulum, photographed it embedded in diluted ultrasonic gel (Pic 5) and then placed this bulb in the 30% H2O2 solution for 12 hours. We then transferred the bulb to an alcohol bath made of 99.8% ethanol for a further 12 hours in order to remove the remaining hydrogen peroxide from the bulb, because when embedding the bulb directly in diluted ultrasonic gel for photographic documentation, severe bubble formation occurs in the gel if the H2O2 has not yet washed out of the object. After this procedure, the bulb could now be photographed bubble-free. He was clearly brighter (Pic 6). We repeated the same process with a palptibiae of another Theraphosinae and the male genital apparatus attached to it (Pic 7). Here, the whole structure was also clearly brighter (Pic 8 ). Keels on the embolus now appeared clearer and with higher contrast than in the original state. It was noticeable that the structures in the alcohol bath initially brightened further and thus sometimes became too bright. Another Haplocosmia sp. bulb was therefore only stored in H2O2 for 6 hours (Pic 9) and then cleaned in ethanol for 12 hours. As you can see, the bulb is brighter now, but not as bright as if it had been in H2O2 for 12 hours (Pic 10). So the degree of brightness of a structure can be affected by the time it is in the H2O2.

In the meantime, we had a lively scientific email exchange with the authors of the above mentioned description of Haplocosmia sherwoodae about the photographic methods they used when photographing the spider structures. Dr. Li confirmed to us that he does not use any brightening chemicals, but only several illumination systems. Therefore, anyone who does not have the appropriate lighting equipment available can use the method of brightening structures that we have developed (see von Wirth & Hildebrandt 2022).

Reference:

LIN, Y. J., YAN, X. Y. & LI, S. Q. (2022a). Two new species of the genus Chilobrachys (Araneae, Theraphosidae) from China. ZooKeys 1081: 99-109

LIN, Y. J., YAN, X. Y. & LI, S. Q. (2022b). A new species of the tarantula genus Haplocosmia (Araneae, Theraphosidae) from Tibet, China. Biodiversity Data Journal 10(e82682): 1-6.

STÜBEN, M & K. E. LINSENMAIR (2008): Advances in insect preparation: bleaching, clearing and relaxing ants (Hymenoptera: Formicidae). Myrmecological News 12, pp. 15-21

von Wirth, V. & K. Hildebrandt (2022). Bleaching of taxonomically important body structures in spiders using the example of tarantulas (Araneae: Theraphosidae). BTS Journal (37) 1, pp. 34-41