A simple technique for mounting crustacean appendages for examination with the scanning electron microscope

Abstract

A simple, cheap, and yet effective technique to allow easy mounting and imaging of small crustacean (or arthropod) limbs or appendages under the scanning electron microscope using custom-made aluminum mounts, cut from commercial aluminum cans, is explained and illustrated.

Keywords: Appendage; Arthropod; Crustacea; Gonopod; Limb

The scanning electron microscope (SEM) has a long and rich history as a tool to produce high resolution, 3-dimensional images with considerable depth of focus, and has been aiding crustacean biologists to study and describe unique morphological features in a wide variety of taxonomic groups.

Improvements in specimen preparation obviously translate into better SEM imagery of crustaceans, and accordingly, there is copious literature on specimen preparation techniques to enhance image quality including removal of accumulated mucus, debris and epibionts which may limit or cover important morphological details (Felgenhauer, 1987; Fleischer et al., 1992; Szebeni and Hartnoll, 2005; Berke and Woodin, 2009; Keiler and Richter, 2011), examination of internal anatomy (Felgenhauer and Abele, 1985; Oshel, 1985; Lin, 2000; Brösing and Türkay, 2011), scanning of entire larvae (Scotto, 1980; Meyer et al., 2004), comparative cleaning and fixation procedures (Scotto, 1980; Osman et al., 2021), and drying methods, especially to avoid shrinkage (Meyer and Melzer, 2004).

While poor SEM mounting techniques can reduce the imaging field of view, thus limiting, or even compromising, the view of important morphological details and therefore imaging quality, the literature on mounting techniques seems far less extensive (Echlin, 2009).

The morphology of the first and second gonopods (G1 and G2, respectively) of brachyuran crabs is critical for species delimitation and description in many groups. The fine detail of gonopod microstructure is often imaged using SEM (Abele, 1971; Rodríguez and Williams, 1995; Tsuchida and Fujikura, 2000; Lianos et al., 2021 a ; 2021b; Mollemberg et al., 2021; Lasley et al., 2023). Brachyuran gonopods are paired appendages on the abdominal somites 1 and 2 modified into cylindrical tubes (either partially or fully closed along their length) involved in the transfer of spermatozoa in spermatophores into the female paired seminal receptacles (Guinot et al., 2013). The gonopods can be very complex morphologically and detailed 3-dimensional images of the distal ends are required to fully appreciate the gonopod morphological characteristics. Ideally, therefore, gonopods should be mounted vertically in relation to the plane of the SEM stub in order to be examined and photographed over 360 degrees in vertical, oblique and horizontal views. However, mounting gonopods (particularly small and thin ones) in a vertical position on a stub can be challenging. The fact that the gonopod basal area is very small relative to its length, means there is not enough support to hold the gonopod firmly in a vertical position on the stub, so it can easily collapse or flip on its side and be permanently damaged.

To circumvent this difficulty, we have developed a simple technique to firmly mount brachyuran gonopods on a standard aluminum SEM stub in a vertical position for easy examination through 360 degrees. This technique is explained and illustrated herein. It can actually be used to mount many kinds of thin arthropod appendages, or biological or non-biological structures, with special reference to ones with narrow bases relative to their length.

We refer the readers to the literature cited above for the standard methods for preparing gonopods for examination under SEM (cleaning, fixation and drying procedures) prior to permanent mounting.

Preparation for permanent mounting of thin crustacean/arthropod appendages with narrow bases relative to their length on a standard aluminum SEM stub using a custom-made, triangular, aluminum mount cut from a small piece of a commercially available aluminum can (Fig. 1 A -G):

1. 1) Have the gonopods (or other desired appendages/structures) appropriately cleaned, fixed and dehydrated prior to mounting;

2. 2) Cut a small rectangle out of a clean aluminum can (Fig. 1 A );

3. 3) Cover the rectangle with a similar sized piece of double-sided metallic adhesive tape (Fig. 1 B );

4. 4) Cut a small triangle out of the rectangle (Fig. 1 C );

5. 5) Fold the aluminum triangle into an L shape by bending the base at right angles (90^o), with the sticky side to the outside;

6. 6) Adhere the aluminum triangle onto a standard mounting aluminum stub by the bent base (Fig. 1 D );

7. 7) Under a stereomicroscope mount the gonopod in a vertical position by sticking the proximal lateral side of the gonopod onto the distal end of the aluminum triangle (Fig. 1 E );

8. 8) Coat the mounted specimen with gold using standard sputter-coating techniques (Fig. 1 E ).

Figure 1.
A, Small rectangle cut from an aluminum can. B, Rectangle covered with a similar sized piece of double-sided metallic adhesive tape. C, Small triangle cut out of the rectangle in B. D, Triangle folded into an L shape with the sticky side adhered onto a standard mounting aluminum stub by the bent base. E, Brachyuran first gonopod (Williamstimpsonia sp.) mounted in a vertical position onto the distal end of the aluminum triangle and then coated with gold. F, G, SEM photographs of the gonopod seen in (E) and of a feather from the head of a hummingbird (Archilochus colubris), respectively, using the technique described in the present work. Scale bars: A-E, 5 mm. F, G, 100 (m and 500 (m, respectively.

Generally, when mounting thin, tube-like structures in a vertical position, mount them singly rather than multiply to avoid two or more specimens blocking each other (overshadowing). Commercially available aluminum SEM stubs with mounting angles of 45° and 90° are available but can cost from US$8 to $35 per stub. This simple, cheap, and yet effective technique, should allow easy mounting and imaging of small crustacean (or arthropod) limbs (Fig. 1 E , F) or appendages under the SEM. Some further advantages of the custom-made aluminum mounts are that the number of holding supports per stub and angles can be adjusted to assist in viewing the specimen from the most advantageous perspective, the triangular tip allows for thin appendages to have a strong support without obstructing the possible view of structures located laterally on the specimen (Fig. 1 E , G), bending the mount can allow for larger/taller specimens to be mounted within the limited confines of the imaging chamber and that the aluminum triangular mount can be truncated to any shape or height to assist in mounting particular specimens. All of these advantages are better than acquiring a pre-defined commercial solution with pre-determined features. Since the custom mount is aluminum and the standard SEM stub is also aluminum there should be no issues of interference or charging of the mount or specimen while in the SEM compared to a standard mount (Fig. 1 F ).

ACKNOWLEDGEMENTS

We are grateful to Lara Maria Guimarães (MZUSP - Museum of Zoology, University of São Paulo) for sputter-coating the gonopod used in the illustration herein and Andrea Brothers for assistance with the SEM at AU. Jessica Colavite (MZUSP) for taking the light photographs and for preparing the illustrations for publication. The two anonymous reviewers are thanked for their valuable opinions that improved the manuscript.

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ADDITIONAL INFORMATION AND DECLARATIONS

• Consent for publication
  All authors declare that they have reviewed the content of the manuscript and gave their consent to submit the document.
• Funding and grant disclosures
  This research was supported by CNPq, grant Pq #309488/2020-6) to MT.
• Study association and permits
  Do not apply.
• Data availability
  All study data are included in the article.

Edited by

• Associate Editor
  Fernando L. Mantelatto

• Editor-in-chief
  Christopher Tudge

Data availability

All study data are included in the article.

Publication Dates

• Publication in this collection
  22 Nov 2024
• Date of issue
  2024

History

• Received
  09 Feb 2024
• Accepted
  21 June 2024