Assessment of three microscopic techniques in observing morphology of pygidial glands of ground beetles

Abstract

Pygidial glands represent an exocrine glandular system situated in abdomen of ground beetles and other representatives of the suborder Adephaga within the order Coleoptera. This system plays a major role in the defense against predators by discharging its products (secretions) outwards. It includes two sets of secretory lobes, collecting canals, collecting reservoirs and efferent ducts. From a biological point of view, observing pygidial glands of ground beetles is important in taxonomy and promising due to potential medical significance of the secretions. In order to better understand glandular functional mechanisms, it is necessary to examine the morphological aspects in detail. Morphology of pygidial gland structures of certain ground beetle species (Insecta: Coleoptera: Carabidae) has been observed via three different microscopic techniques. Scanning electron microscopy (SEM), two-photon excitation fluorescence (TPEF) microscopy and conventional light microscopy (LM) were applied in order to identify complementarity of different methods of investigating the above-mentioned morphological structures. It is indisputable fact that the highest information content (level of details) on external morphology of biological structures is obtained by SEM. Examination of soft, fragile structures by SEM includes preparation of samples in a series of alcohol/acetone solutions of increasing concentrations up to 100% followed by a critical point drying (CPD), which is a well-established method for dehydrating biological tissues. In the case of pygidial glands of ground beetles, it has been noticed that tissue drying was excessive, resulting in the irreversible damage. Samples somewhat changed their shape and their volume shrunk, precluding detailed analysis. This is not the case when CPD is omitted, and dehydration is applied only through a series of ethanol solutions of increasing concentrations (30, 50, 70, 90 and 100%). Concerning simplicity of different techniques, undeniable advantage must be given to LM, although the flaw of this technique is its insufficient informativity regarding a detailed insight into micro- and nanostructures of pygidial gland system (e.g., arrangement of reservoir myofibrils; diameter of collecting and efferent ducts; diameter, thickness and a more detailed structure of radial canals of secretory lobes). Additional three-dimensional information is obtained by TPEF. On the basis of all advantages/disadvantages of the three microscopy techniques compared, it can be concluded that the combination of SEM and TPEF enables the most detailed morphological and anatomical surveys of analyzed biological structures .