pros and cons of scanning electron microscopy as a ... and cons of scanning electron microscopy as a...

Pros and cons of scanning electron microscopy as a research method in acarology

Zbigniew Adamski 1, 2 Eliza Rybska3 and Jerzy Błoszyk 4, 5

1Electron and Confocal Microscope Laboratory/2 Department of Animal Physiology and Developmental Biology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland,

3The Faculty Laboratory for Teaching Biology and Natural Science. Faculty of Biology, Adam Mickiewicz University, Poznan, Poland,

4Department of General Zoology/5 Natural History Collection, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland,

Microscopy is one of the most important techniques in taxonomical and morphological studies of mites and other arthropods. Morphological description is still a major way of description of new taxa. Leg structure, chaetotaxy, structure of setae, their number and location are among the most important features used in such studies. Therefore, light microscopy is extensively used in acarology. However, it has limitations, like low resolving power, relatively low magnification. What is more, transmitted light can pass through some weakly sclerotized structures. This may cause, that we will not notice some of those structures. The size of single setae may be as small as 15 microns or even less. Moreover, they often possess very short notches (1-2microns) which are often regarded as very important taxonomical features. Therefore, scanning electron microscopy becomes routine method in acarology. Scanning electron microscopes (SEM) enable us to see details under much higher magnification, with high resolution and without waves transmitting through the specimen.

Additionally, SEM has many other advantages. It gives possibility to measure details of structures precisely. Very tiny structures can be noticed and described. Next, some artefacts, like for example broken or lacking chaetae can be observed. In consequences, they cannot be mistakenly taken as shorter chaetae and the number of chaetae can be precisely counted. This tool gives also opportunity to document sculpture of exoskeleton. For example, SEM is necessary for proper documentation of sculpture within Labidostomidae. That speeds up the process of description.

However, the procedure of sample preparation can be time-consuming and lead to malformations. Therefore, sometimes light microscopy is a better method, than electron microscopy. Light microscopes are equipped with high resolution cameras nowadays. This opens new possibilities. The choice of method may be crucial for the research.

In this chapter we present some of our observations and remarks concerning both types of microscopes, limitations of their usage in acarology. We also give short review of SEM methods (direct observations, critical point drying, variable pressure) used in research on various groups of mites.

Keywords scanning electron microscopy; light microscopy; mites; taxonomy.

Introduction

Morphological analysis is one of the most important criteria in taxonomy. Such structures like size and shape of an animal, chaetotaxy, structure of mouthparts, scales, hairs or gential organs are used to describe new taxa. If the species does not reproduce sexually or it is parthenogenic, they became even more important in classification. Therefore, microscopy is a standard method in taxonomy of small organisms, like e.g. mites. Since it is estimated that there is approximately 1,000,000 mite species and c.a. 4-5% of them are described (Walter and Proctor, 1999), acarologists still have a plenty to do with morphological description of new species. Usually, light microscopy and drawings are used, too. However, scanning electron microscope (SEM) is more and more frequently used. Notes with use of SEM and TEM for acarological studies have been known for three-four decades at least (Sixl et al., 1971; Foelix and Axtell, 1972; Alberti et al., 1981). As we previously reported, electron microscopic methods have some virtues, which enable better examination and description of external structures of Acari (Adamski et al., 2008). High resolving power of SEM reveals details of structures, which are not well visible under light microscope, due to their very small size, transparency or location. Moreover, they are relatively less time-consuming than traditional drawings, especially when species with elaborated sculpture are described, e.g. Labidostomidae (Fig. 1). On the other hand, drawing can call special attention to the most important features and show them schematically. Next, light microscopy does not demand complicated methods of fixation, which can produce artifacts because of dehydration, vacuum etc. Therefore, all these three methods are used in acarology. In this paper we try to compare them, show their advantages and disadvantages for acarological studies.

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)

© 2012 FORMATEX 215

SEM methods in acarology.

To avoid artefacts, scanning microscopy uses various methods, which are to fix specimens properly. The large variety of methods is used. They mostly depend on the sclerotization of mites, thickness of their cuticle. The hard exoskeleton protects specimens from collapsing. We made a short review of methods used for scanning electron microscopy of mites, with reference to their sclerotization (Tab. I). For highly sclerotized taxa simple fixation or even only gold/silver/platinium sputtering is used. Mites are mounted on stubs, coated with one of the abovementioned elements and observed under SEM. We called this procedure “Sputtering Only - S”. It is often used when samples are collected in alcohol, for different purpose and then some individuals are used for microscopy. In this case they are already partially dehydrated, what may affect their structure. Therefore, this method is advisable for highly sclerotized specimens. Frequently, mites are pre-fixed, using glutaraldehyde, dehydrated, dried, mounted and coated with gold (”Conventional Fixation - CF”). For soft samples, critical point drying is used (“CPD”), together with CF and S. Both methods use high vacuum (“HV”). More and more frequently, cryogenic methods are used. They enable a researcher to dehydrate samples and preserve them from artefacts. (“LT-SEM”). Specimens are frozen, coating is thinner. Since that, this method may reveal some important features, fine details, which are not visible when S or CF methods are used. However, LT-SEM is not always a standard equipment, so its usage is often limited. LT-SEM uses microscopes with cold stage. This one can be also used together with variable pressure (VP). This method can be used for extremely soft specimens. Peltier stage is used and then specimens are observed in the atmosphere of 10-400Pa. This method does not demand coating. However, it is more time consuming and obtaining pictures of a good quality demands a lot of efforts. As we mentioned above, three techniques are used in taxonomy of mites: drawings, light microscopic pictures and scanning microscopy. Figure 2 compares pictures obtained with the use of these three methods. All of them show mites from different perspective, reveal different features. Obviously, drawing is very important during description of new taxa. However, it requires a lot of skills, patience and takes a lot of time. This can pay readers’ attention and to give weight to the most important taxonomic features. Moreover, SEM can reveal features, which are not visible with light microscopy, due to its lower resolution or transparency of samples. Scanning microscopy also gives some additional data, like precise measurement (Fig. 3). To sum up, all the three methods have their pros we suggest using all of them, if possible. However, one can notice, that scanning microscopy should be a standard method nowadays. Depending on the sclerotization, various methods may be necessary to preserve specimens.

Tab. I. Methods of scanning electron microscopy used in acarology.

Taxon Species SEM technique Reference SUPERORDER PARASITIFORMES

Order Ixodoidea

Ixodidae Amblyomma scalpturatum ? Onofrio et al. 2010

Amblyomma americanum S, CF Foelix and Axtell, 1972

Order Mesostigmata

Cohort Uropodina

Uropodoidea

Nenteria pandioni S Adamski et al. 2008 Janetiella pulchella S Błoszyk et al. 2004 Leiodinychus orbicularis S Błoszyk et al. 2011 Neoseius novus S Athias-Binche et al. 1993 Janetiella (Dynurella) stoechas S Athias-Binche 1988 Planodiscus capilliatus, Planodiscus kistneri

S Elzinga 1991

Oplitis paradoxa, Oplitis farrieri S Gorirossi-Bourdeau 1993 Uroobovella marginata, Uropoda orbicularis

S Błoszyk, Gwiazdowicz 2006



Oodinychus ovalis, Urotrachytes formicarius, Dinychura cordieri, Cilliba cassideasimilis, Neodiscopoma splendida, Urodiaspis tecta, Trematurella elegans, Olodiscus minima, Olodiscus misella, Dinychus perforatus, Janetiella pyriformis, Discourella baloghi

S Błoszyk 1999

Phaulodiaspis borealis S Błoszyk J., Bajaczyk R. 1999

Olodiscus minima, S Błoszyk, Stachowiak 1999 Uropoda orbicularis S Błoszyk et al. 2001 Uropoda orbicularis S Błoszyk et al. 2002 Uropoda orbicularis S Bejerlein, Błoszyk 2003 Janetiella pulchella* S Błoszyk et al. 2004 Uropoda orbicularis S Bajerlein, Błoszyk 2004 Uroobovella pulchella*, Oodinychus ovalis

S Błoszyk et al. 2006a

Cilliba cassideasimilis, Cilliba rafalskii

S Błoszyk et al. 2006b

Platysetosus occultus S Dylewska et al. 2006 Olodiscus minima, Olodiscus misella, Urodiaspis tecta, Urodiaspis pannonica, Dinychus perforatus

S Błoszyk, Rozwałka 2008.

Polyaspidoidea Trachytes aegrota, Trachytes minima, Polyaspinus cylindricus,

S Błoszyk 1999

Trachytes kaliszewskii S Błoszyk, Szymkowiak 1996 Acroseius womerslei, Acroseius tuberculatus

S Błoszyk et al. 2005

Trachytes aegrota, Trachytes pauperior, Trachytes lamda

S Błoszyk, Rozwałka 2008.

Cohort Gamasina Dermanyssidae Dermanyssus gallinae S, CPD Locher et al. 2010 Macrochelidae Macrocheles glaber S Błoszyk J. et al. 2002 Varroidae

Varroa destructor CF, CPD, HP Kanbar and Engels 2005

SUPERORDER ACARIFORMES

Tenuipalpidae

Raoiella spp. LT-SEM Ethan et al., Brevipalpus phoenicis LT-SEM Wergin et al., 2000

Raoiella indica LT-SEM Ochoa et al., 2007

Tarsonemidae Polyphagotarsonemus latus CPD, LT-SEM Martin, 1991 Histiostomatidae

Histiostoma spp LT-SEM Wergin et al., 2000

Loxanoetus sp. CF, HP Al-Arfaj et al., 2007 Tydeidae Lorryia formosa LT-SEM Wergin et al., 2000 Eriophyidae

Aculops pelekassi LT-SEM Wergin et al., 2000 Aculops pelekassi CF, S Huang and Wang, 1997 Phyllocoptruta oleivora LT-SEM Wergin et al., 2000 Abacarus hystrix LT-SEM Wergin et al., 2000 Phyllocoptes bougainvilleae LT-SEM Wergin et al., 2000; Ochoa

et al., 2007 Aceria anthocoptes LT-SEM Wergin et al., 2000



Bdellidae Trachymolgus purpureus LT-SEM Fisher et al., 2011 Labidostommidae Labidostomma denticulata S Błoszyk J., Rozwałka R.

2008. Scutacaridae Scutacarus baculitarsus Freeze-dried Bins 1982 Order Sarcoptiformes Suborder Oribatida Trhypochthoniidae Archegozetes longisetosus CPD Heethoff and Koerner, 2007 Cohort Astigmata Acaridae Acarus siro LT-SEM Melnyk et al., 2010

Tyrolichus casei LT-SEM Melnyk et al., 2010 Echimyopodidae Blomia tropicalis ? Mariana et al., 2007

CF – conventional fixation, S – sputtering only, CPD – critical point drying, VP – variable pressure, FESEM - field emission scanning electron microscope, LT-SEM – cryo-SEM, Low-Temperature SEM, ? – method is not mentioned in the text. * Janetiella pulchella (= Urobovella pulchella)

A B

C D

Fig. 1. Sculpture of Labidostoma sp. Whole mite (A), details of sculpture (B, C), trichobothrium and its surrounding (D).



Fig. 2. Examples of mites presented with the use of SEM (1, 3), light microscope (5) and illustrated by drawings (2, 4, 6). 1, 2 – Janetiella pulchella, 3, 4 – Oodinychus ovalis, 5, 6 – Apionoseius sp.



Fig. 3. Nenteria pandioni. SEM reveals fine differences between setae, like variability of their length (A), shape (B and C) or can enables the researchers to distinguish between broken (D) setae and the complete, needle-like shaped ones (C).

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