Insect Morphology in the Age of Phylogenomics: Innovative Techniques and Its Future Role in Systematics

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Entomological Science (2014) 17, 1–24 doi:10.1111/ens.12053

REVIEW ARTICLE

Insect morphology in the age of phylogenomics: innovative techniques and its future role in systematics

Frank FRIEDRICH1, Yoko MATSUMURA2, Hans POHL2, Ming BAI2,3, Thomas HÖRNSCHEMEYER4 and Rolf G. BEUTEL2 1Biozentrum Grindel und Zoologisches Museum, Universität Hamburg, Hamburg, Germany, 2Entomology Group, Institut für Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, Friedrich-Schiller-Universität Jena, Jena, Germany, 3Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; and 4Institut für Zoologie und Anthropologie der Universität Göttingen, Göttingen, Germany

Abstract A brief account of the history of insect morphology is given. Different techniques and analytical methods used in current projects on insect morphology and phylogeny and their optimized combined application are described. These include ﬁxation, dissection, maceration, histology (microtome sectioning), scanning electron microscopy (SEM), transmission electron microscopy (TEM), serial block-face scanning electron microscopy (SBFSEM), focused ion beam scanning electron microscopy (FIB/SEM), confocal laser scanning microscopy (CLSM), bleaching, micro-computed tomography (μCT), computer-based three-dimensional reconstruction, focus stacking of digital images, geometric morphometrics and the storage of morphological metadata. The role of insect morphology in the “age of phylogenomics” is discussed. Key words: confocal laser scanning microscopy, dissection, electron microscopy, focus stacking of digital images, geometric morphometrics, histology, micro computed tomography.

INTRODUCTION (1994), but without mentioning that “Morphologie” was coined by the German poet Johann Wolfgang von After a marked decline in the last decades of the last Goethe in 1779, and later independently by the German century, research in insect morphology and anatomy has anatomist Karl Friedrich Burdach in 1800. In contrast enjoyed a remarkable renaissance in the last ten years. to anatomy (Greek: aná = on, tomé = cut; investigation Innovative methods and new theoretical concepts have of the shape structure, and position of organs or body given new strong impulses to this discipline. An over- parts) morphology (Greek: morphé = form, shape, view of more traditional and modern techniques and lógos = word, teaching, ratio) always implies a com- their optimal combination is the main topic of this parative aspect and the concept of homology plays an contribution. important role. A detailed account of the history of insect mor- We owe the earliest scientific entomological infor- phology and anatomy was presented by Gupta (1994). mation to the famous Greek philosopher and natura- Therefore the historical development will be outlined list Aristotle (384–322 BCE). Among other groups of only briefly here. The different meanings of the terms animals, insects are also treated in his eminent work De morphology and anatomy were pointed out by Gupta Partibus Animalicum. However, the outstanding scholar pointed out that insects were so insignificant that they weren’t worth of the types of investigations dedicated Correspondence: Yoko Matsumura, Entomology Group, to vertebrates such as “fish”, “reptiles” and mammals. Institut für Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, Friedrich-Schiller-Universität Jena, In Western Europe the roots of scientific entomology Erbertstr.1, 07743 Jena, Germany. and insect morphology go back to the 17th century. Email: [email protected] In 1669 the Dutch naturalist, anatomist and micro- Received 20 June 2013; accepted 28 July 2013. scopist Jan Swammerdam (1637–1680) published his

remarkable book Historia Insectorum Generalis (“The (Weber 1933) and Grundriss der Insektenkunde (Weber Natural History of Insects”), focused on the develop- 1938) ). A highly productive but controversial North ment and metamorphosis of insects. Swammerdam’s American entomologist of the early 20th century was investigations were remarkably modern, already charac- Guy Chester Crampton (e.g. Crampton 1918, 1928). terized by careful dissections, a comparative approach Very important morphological contributions were made and the efficient use of microscopy. A prominent by another American insect morphologist, Gordon Italian researcher of this century was Marcello Malpighi Floyd Ferris. The understanding of thoracic structures (1628–1694) who discovered the excretory tubules was greatly improved by his profound studies (e.g. Ferris named after him, the insect heart and the anastomosing 1940). tracheal system (Gupta 1994). A distinguished natura- Outstanding morphological studies were published by list and entomologist of the 18th century was August Weber and his students at the University of Tübingen Johann Rösel von Rosenhof who published numerous (e.g. Wenk 1953; Weber 1955, 1960). The exceptional beautiful illustrations of insects and other arthropods beauty and detail-richness of the illustrations in Weber’s including developmental stages and, in some cases, also posthumously published monograph on the elephant anatomical details. His famous Insecten-Belustigung louse (Weber 1969) are still unsurpassed, and a similar (“Insect Amusement”) was published in 1740. He degree of perfection was reached by other members of already attempted to use a natural classification and can his group (e.g. Bierbrodt 1942). The tradition of the be considered as one of the founders of entomology in Weber school was continued by Gerhard Mickoleit (e.g. Germany. Many of Carl von Linné’s later descriptions of Mickoleit 1961, 1963) and students under his supervi- insects are based on Rösel’s work. Eminent entomolo- sion (e.g. Burmeister 1976; Rieger 1976). Another out- gists of the 18th century were Johann Christian Fabri- standing school of insect morphologists was the group cius and Pierre Latreille. However, their focus was more of Jean Chaudonneret at the Department d’Entmologie on classification than on morphology in a stricter sense. of the University of Dijon, France. Brilliant contri- An outstanding insect anatomist of the 18th century was butions were published by Chaudonneret himself Pierre Lyonnet (1708–1789) who described the incred- (e.g. Chaudonneret 1948, 1950–51) but also by some ible number of 1647 muscles in the goat moth (Cossus of his co-workers (e.g. Bitsch 1966). A fascinating and cossus) and discovered the peritrophic membrane, ima- extremely detailed anatomical study on the alder fly ginal discs and the prothoracic glands in the caterpillar Chauliodes formosanus was carried out by Takadi Maki (Tuxen 1973; Gupta 1994). at the former Imperial Taihoku University (Maki 1936). Carl Hermann Conrad Burmeister (1807–1892) was Another remarkable contribution by a Japanese insect an exceptional entomologist, zoologist and paleontolo- morphologist was the three-volume work on the evolu- gist of the 18th century. He published the first volume tion of the insect head, thorax and abdomen by Ryuichi of the Handbuch der Entomologie (Burmeister 1832), Matsuda (Matsuda 1965, 1970, 1976). The work was a remarkable contribution that was later translated mainly based on the results of earlier morphological into English (Gupta 1994). Charles Janet (1849–1932) studies but extremely useful as a reference work. was not only a dedicated entomologist and owner of The classical tradition of insect morphology was approximately 40 000 fossils, but was also an engineer, upheld at a very high level by scientists at the Zoologisk inventor and company director. Aside from his studies Museum in Copenhagen, notably by the eminent insect on plant biology and evolution he excelled as a pioneer systematist Niels Peder Kristensen. He published not of insect histology. His remarkable treatment of the only outstanding morphological treatments of lepi- anatomy of social insects was mainly based on serial dopteran key taxa (e.g. Kristensen 1968, 1984) and sections of stunning quality (Billen & Wilson 2008). an entire series of profound reviews of insect phylo- Two outstanding entomological works with simple geny (e.g. Kristensen 1975, 1991), but also landmark and virtually identical titles were published in the first volumes on systematics and morphology of Lepidoptera decade of the 20th century, Les Insectes by Louis Félix in the Handbook of Zoology series (Kristensen 1997, Henneguy (1904) and Gli Insetti by Antonio Berlese 2003). (1909). Other milestones in the study of insect structures Morphology-based insect systematics arguably were books published in the 1920s and 1930s by the reached a peak with the publication of the ground- American entomologists Augustus Daniel Imms (A breaking works of the German dipterist Willy Hennig General textbook of Entomology (Imms 1925) ) and (e.g. Hennig 1969), who also revolutionized phyloge- Robert Evans Snodgrass (The Principles of Insect Mor- netic reconstruction (Hennig 1950). In the last decades phology (Snodgrass 1935) ), and by the German mor- of the century, the detailed study of morphological phologist Herrmann Weber (Lehrbuch der Entomologie features of insects, especially internal structures,

2 Entomological Science (2014) 17, 1–24 © 2013 The Entomological Society of Japan Innovative techniques for morphology became less and less popular. This development was TRADITIONAL AND MODERN probably partly correlated with the rise of molecular TECHNIQUES IN INSECT systematics. Morphology was considered as second-rate MORPHOLOGY information by some systematists. In some primarily molecular studies, long lists with morphological charac- In the following sections, different technical approaches ters were included (e.g. Wheeler et al. 2001). However, are outlined to encourage and facilitate morphological in some cases the characters were uncritically extracted investigations of insects. Even though this is explicitly from the literature and inadequately coded. Today, most a practical introduction, covering all technical details systematists agree that the morphological characters would go far beyond the scope of this contribution. used in morphology-based or combined analyses should Therefore the study of original literature and detailed be well documented (e.g. Beetle Tree of Life project technical manuals is highly recommended. A similar (BToL, http://insects.oeb.harvard.edu/ATOL/description outline of technical approaches is also presented in the .htm); Lawrence et al. 2011). This attitude and new textbook “Insect Morphology and Phylogeny” (Beutel technological developments have given new strong et al. 2013). impulses to the structural investigation of insects. Inno- Features of techniques are summarized in Table 1. vative approaches such as micro-computer tomography (e.g. Hörnschemeyer et al. 2002) and computer-based Fixation reconstruction (e.g. Beutel & Haas 1998), an optimized Pinning and drying combined application of different techniques (e.g. Beutel A traditional and still widely used method of killing et al. 2011) and the concept of evolutionary morphol- insects is the use of ethyl acetate. Subsequently the speci- ogy (e.g. Wirkner & Richter 2010) have led to a remark- mens are dried, pinned and labeled. The long established able renaissance in insect morphology in the last decade. procedure is generally used by amateur collectors, but The purpose of this study, which is based on the also frequently by researchers, for instance in the frame- experience of several research projects, is to outline work of biodiversity projects. To store the pinned speci- recent developments in insect morphology and to give a mens in entomological boxes or insect cases has the brief overview over traditional and more recently intro- advantages of saving space and of easy accessibility. duced techniques and morphological analysis. This Dried material is useful for taxonomic investigations includes practical recommendations for structural inves- and for studying exo- and endoskeletal features includ- tigation and analysis, and for an optimized workflow ing the genitalia. Their use for detailed anatomical inves- leading to an increased efficiency in the acquisition tigations and extraction of DNA is limited but in of high-quality anatomical data. Although we mainly principle they can also be used for molecular phylogeny focus on practical aspects of structural investigations, (e.g. Ohshima & Yoshizawa 2006: see practical infor- we decided to include geometric morphometrics, which mation in Yoshizawa & Ohshima 2003; Ohshima & is an analytical procedure. High-quality visualized data Yoshizawa 2012). In rapidly dried insects soft tissues obtained with some of the techniques described here (e.g. muscles) and DNA are preserved to a certain are an excellent basis for this approach, which becomes degree. However, this is not the case if specimens are increasingly important in the context of functional and kept in vials with ethyl acetate for a longer time. Killing evolutionary studies. insects (especially larvae) in boiling water has the advan- The main aim of this review is to help students and tage that the external shape is well preserved without researchers interested in insect morphology to carry out shrinking artifacts. However, the material is useless for their projects. Since many techniques were developed in anatomical study. Germany and other European countries, this practical review is mainly based on this knowledge and research experience acquired at German institutions. However, Ethanol even though the situation in Japan is quite different and A simple and useful preservative widely used by insect the tradition of insect morphology distinctly younger, morphologists is 70–80% ethanol. Soft tissues are rea- some technical approaches developed by Japanese sonably well preserved and specimens remain relatively researchers are also considered here. Although most of flexible after fixation. Material preserved in ethanol can the techniques reviewed here are not widely used by be used for investigating skeletal features and can be Japanese scientists, the provided information on the stored for a long time provided that drying out is pre- specific situation in Japan may build bridges between vented (Fig. 2a). Usually it is also suitable for anatomi- research groups and accelerate morphological investiga- cal study (e.g. histological sectioning). However, the tions on insects and other groups of organisms. quality of the tissue preservation is lower compared to

Table 1 Summary of features of morphological techniques tal et Dissection Histology SEM TEM SBFSEM FIB CLSM μ-CT . Preferable e.g. 70% e.g. FAE – Primary and Same as TEM Same as TEM e.g. 70% ethanol, e.g. FAE (also fixation ethanol, FAE secondary FAE; bleaching immobilized fixation is useful living (see text) organisms) Data Usually fast, Time-consuming Fast Very time Slow but less Slower than SEM, Fast Fast (depending acquisition depending consuming than TEM faster than on equipment) speed on object and SBFSEM structures Alignment Perfect Problematic 3D images can Problematic, loss Perfect Perfect Perfect Perfect be obtained by or deformation focal stacking of sections of micrographs (see text) Sample size Minimum <1mmto Less than 10 cm, Approximately <1 mm Up to a few mm; Usually <3 mm, Diameter about about 2 mm >100 mm limited by cell organelle depends on also very flat 0.5–50 mm (depending (depends on chamber size level (nm) setup (e.g. larger samples (depends on on aim) embedding and specimen anatomy of equipment) medium and stage of SEM sensilla) microtome) Color Present, usually Artificial Grey scale or Grey scale or Grey scale or Grey scale or Artificial, Grey scale or information modified by staining false color false color false color false color depending false color fixation representation representation representation representation on laser representation

03TeEtmlgclSceyo Japan of Society Entomological The 2013 © wavelength Voucher Samples Slides Samples Usually digital Digital images Digital images Samples Samples noooia Science Entomological images and section series Data size – – Small to medium Medium (some Large (tens of Large (up to tens Small to large Large (several (MB) MB) GB/data set) of GB/data set) (MB to GB) tens of GB) Maximum Low High Medium to high Very high Very high Very high Low to medium Medium to high resolution† †Comparison of maximum resolution: [low] Dissection (stereo microscope) < CLSM <μ-CT < Histology (compound light microscope) < SBFSEM (5 × 5 × 25 nm/pixel) < FIB (5 nm/pixel) ≤ TEM

(2014) [very high]. SEM is mainly used to investigate surface structures and the magnification (and resolution) varies widely depending on the aim, the object of study and the equipment. A direct comparison with the other methods (mainly focused on anatomy) is therefore difficult. It is important to keep in mind that a higher resolution obtained using a specific technique does not mean that it is superior to others. –, not applicable. 17, 1–24 Innovative techniques for morphology

material treated with fixatives (e.g. Bouin’s fluid, see times of 1–5 hours are appropriate, and after this speci- below). In specimens kept for several years, muscles mens have to be extensively washed in absolute ethanol. often become detached from their sites of origin and insertion. Usually specimens lose or change their color- Dissection ation and become more fragile. Simple dissection often yields results rapidly, especially For the extraction of DNA specimens are usually when larger insects are investigated. Razor blades, hand- fixed in 98–100% ethanol (preferably not denatured). sharpened pins and fine forceps are simple but useful This material is suitable for histological section series. tools. In well-preserved specimens (e.g. FAE) the attach- However, artifacts are often caused by dehydration and ment sites of muscles can be investigated and move- the specimens become very inflexible and brittle. ments can be simulated by pulling muscles with forceps. Dissections can be carried out in glycerine or alcohol. Bouin’s fluid Preparation in water is useful for visualizing tracheae, This excellent fixative is a saturated aqueous solution which are air-filled and thus appear silvery-white. The of picric acid (15 parts), concentrated formaldehyde contrast between them and muscles can be enhanced by (5 parts) and glacial acetic acid (1 part). It should be applying methylene blue solution. Specimens cut sagit- prepared before use and stored in a refrigerator. Fixation tally and stabilized by a small lump of Plasticine with a between two hours to several days is required depending fitting concavity can provide a good impression of the on the size of the specimen. Subsequently, specimens spatial arrangement of structures. In critical-point-dried should be washed several times in 70% ethanol until or freeze-dried specimens muscles can be removed sub- the yellow coloration (picric acid) vanishes from dipp- sequently without damaging the skeletal attachment ing ethanol. Contact with the skin should be strictly sites. Steps of dissection can be documented by drawings avoided. or microscopic pictures. Duboscq Brazil is a slightly modified alcoholic Dissections can be efficient but have limitations. version. Nine parts of saturated alcoholic solution of Minor structures can be easily overlooked, especially in picric acid (10 g in 100 mL absolute ethanol) are mixed small insects. Useful results can not be obtained for with four parts concentrated formaldehyde and one part insects below 2 mm. The precise histological properties glacial acetic acid. Duboscq Brazil is applied and stored of tissues can not be assessed. Moreover, the specimens like Bouin’s fluid. are more or less destroyed after the dissection. K.-D. Klass (Museum of Zoology, Dresden) has devel- Formaldehyde – acetic acid – ethanol oped a specific dissecting and drawing technique. In Formaldehyde – acetic acid – ethanol (FAE (FAA)) solu- anatomical investigations at least two samples for each tion, which is composed of 6 parts concentrated form- species are used: one is macerated in KOH to assess aldehyde, 10 parts absolute ethanol and 1 part glacial the three-dimensional configuration of sclerites and acetic acid, is relatively easy to prepare and very useful membranes. The second alcohol-preserved specimen is for insect morphology. It should be stored in a refrig- dissected on a silicon-embedded laboratory dish. The erator. Landowsky’s fluid is similar but contains a higher cuticle is extended and fixed with pins to expose the portion of ethanol (17 parts). Specimens should be fixed muscles. The cuticle is considered as a continuous plane for 1–24 h. Both solutions are less toxic than fixatives that is extensively folded and includes areas that are containing picric acid and therefore are more convenient more or less strongly invaginated or evaginated. The in their application. They are good fixatives for ana- details can not be treated here but examples are found tomical work based on dissection, histological section in Klass and Matushkina (2012) and Schneider and series and micro computed tomography (μCT). Klass (2013). Kahle’s fluid is a modified version composed of Dissection is the most popular method in Japan, 15 parts ethanol, 5 parts concentrated formaldehyde, but Japanese researchers tend to focus on sclerotized 1 part glacial acetic acid and 30 parts distilled water. It parts (for detailed explanation of methods see, e.g. is mainly used by North American insect morphologists. Takagi 1970; Ôhara 2006; Ohshima 2013), especially for assessing diagnostic characters for species identifica- Carnoy’s fluid tion in a taxonomic framework. In this context the male Due to the high alcohol content this mixture of six parts intromittent organ plays a dominant role, recently also absolute ethanol, three parts chloroform and one part including the endophallus (=internal sac). Not only scle- glacial acetic acid penetrates specimens very fast, but rites are used but also reconstructions of membranous causes strong dehydration. This can result in shrinking swellings occurring during copulation (e.g. Dang 1993; and hardening of tissues if applied too long. Fixation Owada 1995; Imura 2007a,b). In Germany Hünefeld

et al. (2013) recently developed a new appliance to loss of the original coloration and sclerites are rendered reverse the endophallus for easy observation. This mini- more or less transparent. The latter effect can be com- mizes damage of the membranous region and reflects the pensated by applying chlorazol black or pyrogallol natural shape during copulation. staining, which renders sclerotized structures blackish. A morphological concept, which is still unusual in The staining is reversible and can be removed by Japan, was developed by T. Saigusa (Fukuoka, Kyushu) washing with ethanol. The cleared specimens can be and co-workers. Similar to K.-D. Klass’s approach, the stored in glycerin or 70% ethanol or critical-point-dried cuticle including membranous and semi membranous and used for detailed SEM study. areas is considered as a continuous sheet, and the body Alternative substances are sodium hydroxide organization results from modeling this structural entity. (NaOH), lactic acid (C3H6O3), lactophenol (lactic An illustration method was developed to reflect com- acid + distilled water + glycerin + phenol; 1:1:2:1) or plicated morphological configurations exactly and to diaphanol (chlorine dioxide – acetic acid). An alterna- define highly sclerotized parts and membranous areas tive method is to let insects rot slowly in distilled water following R. E. Snodgrass’s drawing procedures (see at room temperature. After some days or few weeks examples in Shirôzu & Saigusa 1971; Ueda & Saigusa (depending on size) the decaying soft tissue can be 1982; Sugimoto & Saigusa 2001; Yoshizawa & Saigusa removed using fine forceps. An advantage is that the 2001). Other examples of precise depictions of complex cuticle retains its original coloration. structures were provided by K. Yoshizawa (Hokkaido University) and K. Suzuki (Toyama) who intensively Histology investigated the wing and wing base morphology in Semi-thin sections are still widely used for investigating a phylogenetic context. Especially, Yoshizawa and internal structures and histological features. Tradition- co-workers (Yoshizawa & Ninomiya 2007; Ninomiya ally, specimens are embedded in paraffin or celloidin & Yoshizawa 2009; Yoshizawa 2011; Yoshizawa & (see, e.g. Heddergott 1939). These substances are soft Wagatsuma 2012) have investigated the homology and compared to exo- and endoskeletal structures of most phylogenetic utility of the wing articulation. Aside from insects and do not rigidly interconnect with the cuticle. providing phylogenetic information on a higher system- The use of teneral (freshly-molted) specimens or of spe- atic level, establishing the homology, shape and connec- cific chemicals can reduce the effects, but the resulting tion of each sclerite is crucial in a functional context sections are comparatively thick (5–50 μm) and show (e.g. Wootton 1979). Another rarely used but valuable a high rate of artifacts, for instance deformation, loss technique adopted by K. Suzuki is manual dissection of of structures, or interfolding of parts of the sections. insects anesthetized with ether to observe anatomical An advantage is that after the removal of the paraffin details of internal reproductive organs in vivo. This contrast-rich staining, protocols can be applied (e.g. apparently also reveals informative characters (Suzuki methylene blue + basic fuchsine, Masson’s trichrome 1988). Using this approach, male and female reproduc- stain). Recently traditional embedding media were tive organs of chrysomelid beetles were investigated more and more replaced by hard epoxy resins as already intensively (Suzuki 1974, 1988; Suzuki & Hara 1975; previously used in transmission electron microscopy. Suzuki & Windsor 1999; Matsumura & Suzuki 2008). These new media yielded distinctly thinner sections The observation of internal reproductive organs using (0.5–1.5 μm). Furthermore, deformations and the loss dried specimens is possible in principle but has its limi- of sections were greatly reduced. tations (Suzuki 1994). In any case, the simple classical Resin encompasses a broad variety of substances technique of using binocular microscopes combined of similar mechanical properties, such as methacrylate, with a pair of forceps is still highly efficient for mentally epoxy or styrene resin. A specific epoxy resin, araldite, reconstructing three-dimensional images of structures turned out to be highly suitable for sectioning even and movements. strongly sclerotized insects (and other arthropods). Presently it is the most popular embedding medium in Maceration entomology. However, other resins used for TEM are Maceration is often used to dissolve internal soft parts also suitable. The preparation is almost identical to the for a detailed study of skeletal elements. The most procedure described for TEM (see below). Specimens frequently used agent is potassium hydroxide (KOH, should be well-fixed using FAE or buffered glutaralde- approximately 5% aqueous solution), which macerates hyde, but samples directly stored in 70% ethanol are soft tissue efficiently, especially at higher temperatures. also suitable. For the latter, a post-fixation with FAE Specimens should be kept at 60°C for some hours prior to embedding is advisable. In order to increase depending on size. Prolonged maceration results in the the permeability for fixatives and the resin, removing

appendages (e.g. legs) is recommended. Specimens are gradually dehydrated in an ethanol series (up to pure ethanol) and transferred over several steps of ascending acetone–Araldite mixtures into pure resin. The infiltration can be facilitated by applying vacuum. Finally the samples are separately placed in silicon molds filled with resin. After a minimum of two days hardening at 60°C the resin blocks can be removed from the molds and trimmed as described for TEM. Ultra-microtomes used for TEM sectioning are usually suitable for producing semi-thin sections (up to 1 μm thickness), but specific microtomes are used in most cases. Sections are prepared using glass- or diamond-knifes. The former are cheap, but the latter are more durable and produce fewer artifacts. After cutting, every section is transferred to a drop of water on a microscope slide with a thin needle or eyelash glued on Figure 1 Microtome and histological sectioning by R Machida a glass pipette. As the sections are often compressed by (Sugadaira Montane Research Center, University of Tsukuba). sectioning, they are placed on a heating plate (60°C) for Image by K Sekiya. stretching. The dried sections can be stained using basic solutions. In contrast to paraffin embedding, the resin is usually not removed, which impedes several staining essential for investigating anatomical details. The com- protocols. However, a combination of toluidine blue, bination of fast, artifact-free computed tomography borax (each 4 parts of 1% watery solution) and (CT-scans) for broader sets of taxa combined with pyronin G (1 part of 1% watery solution) applied for histological sections of selected specimens is the ideal about one minute at 40–60°C is a good staining method. solution. After drying, the sections are sealed using cover glasses In Japan, some researchers in ecology and evolution- mounted with agents as Pertex or Eukit. After this they ary developmental biology (evo-devo) prefer paraffin can be stored for a long time and investigated also using (e.g. Ishikawa et al. 2008; Ishikawa & Miura 2009; oil immersion microscopy at high magnification (100×). Yao & Katagiri 2011). The aim is mainly to compare A practical description for using other epoxy resins is volumes of body elements and to verify the presence or available in Pernstich et al. (2003). absence of structures among species. In some contexts Until quite recently the microscopic study of hundreds deformation artifacts typical for paraffin sectioning are of sections was time-consuming and the reconstruc- more or less irrelevant and results can be obtained with tion of structures required strong imaginational skills. lower costs and less time. Today, motorized microscopes (e.g. slide scanner) semi- Machida (Sugadaira Montane Research Center, automatically digitize sections at high-resolution and University of Tsukuba) and coworkers have greatly in very short times. Computer-based three-dimensional contributed to insect embryology and phylogeny alignment and visualization functions (see below) (Fig. 1). To investigate the morphology and develop- greatly facilitate fast analysis and documentation of ana- ment of delicate embryos they used methacrylate resin tomical data. (Technovit 7100; Kulzer, Frankfurt AM, Germany) and Despite considerable progress in histology the sections a tungsten carbide steel knife (Superhard Knife; Meiwa are never completely free of artifacts. The loss of termi- Co., Mie, Japan) (Fig. 1, e.g. Machida et al. 1990, nal structures (e.g. tips of legs) is often unavoidable. 1994a; Tojo & Machida 1998; Ikeda & Machida 2001; Deformations almost always occur, even though they are Uchifune & Machida 2005; Jintsu et al. 2010; Mashimo minimized if proper resins are used. The production of et al. 2011). Additionally, Machida developed an high-quality serial sections requires special training and automatic vacuum infiltrator which greatly improved the entire process takes at least two weeks. This excludes the infiltration efficiency and minimized deformation the detailed screening of a large taxon sampling from the (Machida et al. 1994b; R Machida, pers. comm., 2013). economic perspective. Data can be acquired much more efficiently using micro-computer tomography. Neverthe- Scanning electron microscopy less the very high optical resolution and reliable and fast Scanning electron microscopy (SEM) is an approach discrimination of tissues using semi-thin sections are still frequently used by entomologists since the 1970s.

Samples for this technique are relatively easy to handle stub using carbon adhesive pads. A very useful tool is a and yield excellent results for documenting surface rotatable specimen holder developed by Pohl (2010) structures at very high resolution. It is conceivable that (Fig. 2b). The insect is fixed on a pin on the rotatable the intensive use of SEM in the last decades has contrib- arm of this device. The precisely oriented insect can be uted to the decline of insect anatomy (e.g. musculature), rotated through 360°, which makes it possible to obtain as attractive results can be obtained with a limited all standard views (usually dorsal, lateral and ventral amount of training. The visualization of internal soft views) with a single specimen. Moreover, the hollow parts with SEM is possible in principle but clearly basal part of the brass specimen holder absorbs elec- limited. Sagittally sectioned specimens dried at the criti- trons passing the specimen. This results in a homo- cal point can be examined but this approach yields only genous black background and distinctly reduces charg- a general overview of internal structures. Even histologi- ing, which is often a problem, especially when a dense cal sections can be studied by SEM after the embedding vestiture of setae is present (e.g. with Hymenoptera). resin has been removed (Tsutsumi & Machida 2004). Usually the dried specimens are sputter coated with a SEM images are clearly insufficient for detailed anatomi- very thin layer of a conductive element (e.g. gold, plati- cal reconstructions. Aside from this, coloration, degree num, carbonate). of sclerotization, and structures below tissues such as A novel approach sometimes used in entomology is membranes, which would be transparent in visible light, environmental scanning electron microscopy (ESEM). can not be visualized using SEM. However, in some This technique makes it possible to examine samples cases structures just below the cuticle can be made under environmental pressure using specialized electron visible by irradiation of high emission current, i.e. detectors and pumping systems differing from those 20–30 kV (H Pohl, pers. obs., 2013). of the normal high vacuum SEM mode. This has the Before scanning, specimens should be cleaned if the advantage that moist or even live insects can be exam- cuticle is covered with secretions, soil particles, food ined with low vacuum without conductive coating. The substrates or other materials. Especially in the mouth- resolution is lower than in high vacuum but charging of part region, structural details can be obscured by adher- the unprocessed specimens is minimized. The functional ing substrates. An efficient cleaning method is by principle and merits vs demerits of ESEM compared to ultrasonic sound. However, this can result in the loss standard SEM are discussed in Kirk et al. (2009). ESEM of appendages or disintegration in delicate insects, also allows examining museum specimens (including especially larvae or in insufficiently fixed specimens. type material), as the surface is not affected. Surface contamination of strongly encrusted specimens SEM is popular among Japanese taxonomists and (e.g. larvae using substrates for camouflage) can be systematists, especially those associated with a university removed using KOH (about 1–5%), cleaning agents or museum. In most cases it is used to investigate surface (e.g. Triton) or diluted sodium hypochlorite solution structures. Adhesive sheets sometimes used for mounting (4% solution, i.e. antiformin, commercially available) specimens can emit small particles, which are reflected by for a few minutes prior to ultrasonic treatment. the electron beam and can cause a contamination of the After cleaning, specimens are dehydrated in an detector. This can be avoided with the holder established ethanol series (absolute ethanol, acetone or isoamyl by Pohl (2010), aside from other advantages (see above). acetate as final medium). Air drying can be sufficient in Samples that have to be spread out (e.g. membranous the case of strongly sclerotized insects (e.g. beetles), but parts of genitalia) can be placed on a small piece of cover drying at the critical point or freeze-drying using t-butyl glass, which is then mounted on a stub using carbon alcohol is usually necessary to minimize shrinkage arti- adhesive pads. Gaps between the piece of the glass and facts. Good results can also be obtained by gradually the carbon pad should be covered with an electrically transferring specimens to distilled water and then conductive adhesive to minimize electronic charging. freeze-drying them. Alternatively, chemical drying with Recently Takaku et al. (2013) established a new hexamethyldisilazane (HMDS) yields good results in method for observing live animals with a high vacuum short time (Brown 1994). Specimens are dehydrated up SEM setup called “nano-suite”. This is used to investi- to pure ethanol and kept in pure HMDS two times for gate physical interactions between small organisms or 30 min. Finally they are placed in an open Petri dish between organisms and substances. It is a new approach under a laboratory hood, allowing the fluid to vaporize. to interpret functions of organisms and underlines the Drying at the critical point is comparatively time- future potential of the “classical” SEM. consuming, but in most cases yields the best results. An attractive project using SEM is presently carried After drying, specimens are mounted on a specimen out by M. Ôhara (Hokkaido University Museum) in holder (e.g. a thin wire) with glue or nail polish or on a collaboration with engineers and physiologists (e.g.

Figure 2 Preparation and preservation of samples for SEM and an example image. (a) Permanent preservation of samples in 70% ethanol, (b) rotatable specimen holder and sample attached to an insect pin, (c) preservation of samples in plastic cases, (d) SEM image taken using the holder; Diasemopsis comoroensis (Diptera: Diopsidae).

M. Shimomura, Tohoku University and T Shimozawa, below. Specific literature should be used by researchers Hokkaido University). It is based on an extensive col- intending to use TEM; and satisfying results can not be lection of SEM micrographs of insects deposited in the obtained without appropriate training. museum. In regular meetings the images are screened, As penetration of the material is essential for its effi- analyzed and discussed with respect to possible func- cient fixation, the structures to be examined (e.g. gland tions of structures of the scanned insects (Fig. 3). It is tissue) should be isolated. Small insects can be fixed as planned to establish an online database in the near a whole, but opening the body cavity (e.g. by removing future. This interdisciplinary project may lead to impor- appendages) is necessary to improve the penetration. tant new perspectives in insect morphology, functional Glutaraldehyde (2.5%) is usually applied as primary morphology and biomechanics. fixative to cross-link proteins. For larger specimens paraformaldehyde can be used. Both are prepared in Transmission electron microscopy a phosphate or sodium cacodylate buffer to maintain Transmission electron microscopy (TEM) (Fig. 4) is the pH and, if necessary, with various additives (e.g. essential for studies at the ultrastructural level. It has sucrose) to control osmolarity. After one to four hours played a crucial role in the investigation of the structure of primary fixation the specimens are washed overnight and functions of cells and tissues (Stirling & Woods in buffer. This is followed by secondary fixation in 2002). Even though its role in insect systematics is osmium tetroxide (2%) for one to four hours to preserve limited, it is indispensable for the study of specific and stain lipids (Hayat 1981) and finally washing in character systems such as sperm ultrastructure (e.g. buffer for a few minutes. Dallai et al. 2012). Epoxy resins (e.g. Spurr, Araldite, Epon) are used as The crucial advantage of TEM is the tremendously embedding materials. As they are immiscible with water, increased resolution compared to light microscopy careful dehydration is necessary. A graded ethanol series (Woods & Stirling 2002). A disadvantage is the compli- is used but the time of dehydration should be kept cated and time-consuming preparation of specimens, as brief as possible to minimize the risk of extracting which also requires the use of toxic substances. A brief cellular constituents (Woods & Stirling 2002). Gradual description of the fixation and preparation is given infiltration of the resin is required, beginning with a

(a) (b)

Figure 3 (a,b) Regular meetings held by M. Ôhara (Hokkaido University Museum) to analyze and discuss possible functions of structures of the scanned insects based on SEM images.

Reynolds 1963). Uranyl acetate is applied to stain proteins and nucleic acids, and lead citrate is used to contrast cytoplasm, membranes and glycogen-rich structures. After drying the sections can be examined. The three-dimensional applicability of the technique is limited. Slot grids have to be used and the preparation of continuous section series is difficult. Nevertheless, there is no alternative for ultrastructural investigations below the cellular level. Down to the cellular level serial block-face scanning electron microscopy (SBFSEM) can be used as an alternative (see next section). Even though most Japanese researchers are familiar with TEM, it is only rarely used in entomology. Notable exceptions are the laboratories of R. Machida (Univer- Figure 4 TEM in R Machida’s laboratory (Sugadaira Montane Research Center, University of Tsukuba). Image by K Sekiya. sity of Tsukuba), T. Tsutsumi (Fukushima University) and S. Niitsu (Tokyo Metropolitan University), where TEM is used routinely (e.g. Machida et al. 1990, 1994a; 50 : 50 mixture of resin and solvent. Finally the speci- Ikeda & Machida 2001; Niitsu 2001; Tsutsumi et al. men is transferred into fresh pure resin in a separate 2005; Uchifune & Machida 2005; Niitsu & Kobayashi silicon mold. Polymerization takes place in a heating 2008; Jintsu & Machida 2009; Mashimo et al. 2011; cabinet. Niitsu et al. 2011). Obviously the application of this The polymerized resin block is tightly trimmed as a demanding technique depends on the aim of the pyramid around the black object. For sectioning the research. It is only recommended if extremely small specimen is fixed in an ultra-microtome. Glass knives objects are under investigation. or diamond knives are used for ultrathin sectioning. The former have to be replaced frequently, whereas the latter are very durable and therefore much better suited Serial block-face scanning electron microscopy for continuous sectioning. The sections of about 70 nm This innovative technique combines SEM surface thickness are transferred to small round cupric holders. analysis with ultra-microtomy similar to that used Mesh grids provide the highest stability, but the bars for TEM (Denk & Horstmann 2004). Ultrathin serial of the mesh may hide structures of interest. A slot grid sections perfectly suitable for 3D reconstruction are has a large single opening, which allows examining produced (Zankel et al. 2009). Advantages are minimal the whole section. The slot is coated with an extremely artifacts, very high 3D resolution and almost auto- thin and fragile foil (e.g. formvar, pioloform) to carry matic data acquisition. The maximum magnification is the sections. lower than that of TEM. A disadvantage is the limita- To enhance the contrast in the electron beam, speci- tion to very small specimens (every dimension below mens are treated with two heavy metal agents (see 1 mm; Hörnschemeyer et al. 2012).

Specimen preparation is similar to what is described for TEM (see section above) but contrasting has to be applied prior to resin embedding (block contrasting). Primary fixation in glutaraldehyde (2.5%) is followed by a secondary treatment with osmium tetroxide (1–2 h). As for TEM, uranyl acetate and lead citrate are used for contrast but are applied longer to gene- rate high contents of heavy metals. Additionally substance binding especially to membranes (like thiocarbohydrazide (TCH)) should be applied. Since all the contrasting is done on a comparatively large piece of tissue the ability of contrasting agents to penetrate the tissue is an extremely important factor. To get homogenous contrast throughout the specimen, extreme care should be taken to cut it as small and early as possible in the contrasting process (2 mm in every direction should be the maximum). Heavily sclerotized parts of arthropods are especially problematic, because most contrasting agents penetrate sclerotized cuticle only very slowly or not at all. In such cases it is inevitable to cut away as much of the cuticle as possible, without damaging the structures that are to be investigated. Careful contrasting with heavy metals is not only necessary to distinguish between different types of tissues but also to reduce charging artifacts during the scanning procedure (Hörnschemeyer et al. 2012). The contrasted specimens are dehydrated in an ethanol series, transferred to two stages of pure acetone and finally embedded in resin. Durcupan has been shown to be most resistant to the electron beam, but other resins such as Araldite can also be used. However, soft- ening of the resin during high-resolution scans may result in artifacts. The polymerized resin blocks are trimmed to fit into the ultra-microtome in the SEM chamber. In an automatic process the microtome cuts ultrathin sections from 25 to 100 nm, which are discarded. After every section an image of the surface of the block is automatically recorded (Hörnschemeyer et al. 2012). The fully aligned image stack is perfectly suitable for 3D reconstruction. This novel technique has not yet been applied in insect morphology and phylogeny in Japan. Cardona et al. Figure 5 SBFSEM and images example pictures. (a,b) (2010) emphasized using it to visualize the 3D anato- SBFSEM, Institut für Zoologie und Anthropologie der mical configuration of the brain of Drosophila on the Universität Göttingen. (c–e) Stylops sp., first instar larva level of individual neuronal processes and synapses. (Strepsiptera: Stylopidae); rendered pictures of a head and a Although this technique has strong size-related limita- part of prothorax in lateral view (sagittal plane shown in (c) tions (Table 1), it has a great potential to investigate and entire view shown in (d)); (e) SEM image of the surface of the block; the region is shown in (c). anatomy of very small insects (Fig. 5c–e; e.g. first instar larvae of strepsipterans; H Pohl, pers. obs., 2013) and other organisms with a resolution distinctly surpassing that of classical histology.

Focused ion beam/scanning electron microscopy Focused ion beam/scanning electron microscopy (FIB/ SEM) is mainly used in material sciences but was also applied in insect morphology recently (e.g. Schmitz et al. 2007; Di Giulio et al. 2012). Dual beam SEMs are equipped with an additional, strongly focused ion beam (e.g. helium, gallium), which allows removing precisely defined parts of the specimen to examine structures below the surface (Knott et al. 2008) or to open small compartments (e.g. surface receptors) (e.g. Stavenga et al. 2004). FIB/SEM samples must be embedded in resin, and specimen preparation is similar to that described for TEM. Due to the highly precise milling Figure 6 CLSM, Friedrich-Schiller-Universität, Jena, Germany. process structures can be removed layer by layer (Knott et al. 2008; Di Giulio et al. 2012). The freshly created surfaces are recorded using the electron beam. without clearing the cuticle. Suitable agents for When FIB/SEM is used for milling specific struc- bleaching are hydrogen peroxide (35% H O ) (Stüben tures for observing materials whose covering structure 2 2 & Linsenmair 2008), methyl salicylate, lactic acid was removed (e.g., Stavenga et al. 2004), specimen (Michels 2007) and Murray’s clear (=BABB; 1 part preparation is identical to standard SEM-based inves- benzyl alcohol + 1 part benzyl benzoate) (Zucker 2006; tigations. Proper fixation (e.g. glutaraldehyde) and an McGurk et al. 2007). Specimens are kept in the solution ultrastructure-preserving drying process (e.g. critical for one hour to several days. Soft parts are not drasti- point drying) are necessary. cally affected by this procedure and can be visualized K. Ohta (Kurume University School of Medicine) after the process (Deans et al. 2012a). and co-workers introduced a FIB/SEM facility in Depending on the thickness of the specimen, the Japan. They greatly improved the technique to produce number of scan channels and the settings of the con- high-resolution images for biological tissues, equivalent focal microscope, the recording of high-quality scans to a resolution obtained with TEM (Ohta et al. 2012). can take several hours. It is important to fix the speci- An introduction in Japanese is available at: http:// men properly during the scan. Even very slight move- www.med.kurume-u.ac.jp/med/anat2/. As FIB/SEM can ments strongly affect the image stack. Scans can be provide a larger volume of information than TEM, it is obtained with a minimum of preparation time by fix- foreseeable that it will be more widely used in our dis- ing the specimen in a drop of glycerine between a slide cipline in the future. and a high-precision cover glass supported by small spacers (e.g. wax, self-adhesive rings). Ethanol, buffer Confocal laser scanning microscopy and distilled water are also suitable, but evaporation Even though this technique allows for a very effi- can induce movements of the specimen. For longer cient investigation of structural details, confocal laser scans, embedding media with higher viscosities are scanning microscopy (CLSM) (Fig. 6) is rarely used in recommended. a phylogenetic context. Without staining (e.g. immuno- Agarose (1%), glycerine jelly and mowiol are useful, staining), results can be obtained using the autofluo- but traditional media such as Canada balsam or Euparal rescence of the cuticle and unsclerotized body parts (e.g. are also useful (e.g. Schawaroch & Li 2007). They also muscles). No specific fixation is necessary. Specimens facilitate the controlled adjustment of the specimen preserved in 70% ethanol are suitable. Using combined without deformation. lasers with different excitation wavelengths, soft and Autofluorescence of insect cuticle is induced by a hard parts can be easily differentiated. Even the degree laser wavelength of 488 nm (e.g. Michels 2007). The of sclerotization and the resilin content can be visualized emitted light is recorded in two separate spectra: (e.g. Michels & Gorb 2012). green (about 500–570 nm) and red fluorescence (about Very small or flat insects and small isolated parts (e.g. 580–690 nm). The overlay of both channels allows genitalia) can be visualized completely (e.g. Schawaroch for detailed imaging of the grade of sclerotization et al. 2005; Klaus & Schawaroch 2006; Michels 2007). of the cuticle, with membranes appearing green and Deeper layers of larger specimens can not be detected sclerites brown (e.g. Deans et al. 2012a). Additionally,

resilin-rich structures can be visualized by applying UV with superglue or fixed within a small container (e.g. radiation (405 nm) and recording the emitted blue light Eppendorf tube, pipette tip). To ensure maximum (420–480 nm; e.g. Michels & Gorb 2012). The applica- spatial resolution, the rotation axis should correspond tion of glutaraldehyde as a fixation agent increases the to the longitudinal axis of the specimen. If drying is not fluorescence at 488 nm. An efficient fluorescence stain- possible, scanning in alcohol or distilled water is an ing of the integument is Congo red, which is excited alternative. Even living organisms can be used, if they do at a wavelength of 561 nm (emission spectrum 570– not move during scans (e.g. Lowe et al. 2013). However, 670 nm; Michels & Büntzow 2010). the similar electron density of water or alcohol and the Data obtained with CLSM are mainly useful for insect tissues greatly reduces the contrast. To enhance volume rendering. Maximum projections of the image the contrast, reversible staining can be applied. The best stack usually result in lower-quality visualizations. The results are obtained with alcoholic iodine solution (I2E; reconstruction of surface-based 3D models is possible 1% iodine in pure ethanol) for one to five days followed for flat or small isolated objects (see above). by washing in alcohol (e.g. Metscher 2009). The stained specimens have to be firmly fixed to avoid movement Bleaching caused by circulation of the liquid medium. Cotton and Bleaching is often mentioned in the context of CLSM foamed plastic are suitable materials for this purpose. but is also useful for other investigations, such as analy- The iodine can be removed by washing the specimen in sis of the genital fitting (Kamimura & Mitsumoto alcohol. 2011a). Kamimura and Mitsumoto (2011a,b) adopted Specimens prepared for TEM investigations can be BABB solution to bleach Drosophila spp. couples stabi- documented using μ-CT. The osmium tetroxide– lized by an agarose block (the method was described in stained and resin-embedded samples show good con- Kamimura & Mitsumoto 2011b). trast between tissues and embedding media. This is usually not the case if samples embedded for microtome μ Micro-computer tomography ( -CT) sectioning are used without specific staining. However, This highly efficient anatomical technique was such specimens can be scanned with equipment opti- established in insect morphology about ten years ago mized for phase contrast. The technique is also an excel- (Hörnschemeyer et al. 2002). The improved hardware lent tool for investigating amber fossils (e.g. Tafforeau and software of modern desktop scanners yield high- et al. 2006; Pohl et al. 2010). Prior to scanning a dis- resolution scans and make the technique increasingly pensable piece of amber should be tested to estimate attractive for the anatomical study of small and medium possible darkening of the material. However, this effect sized insects (e.g. Wipfler et al. 2012). The maxi- can be reversed by placing the specimen under a UV mum resolution is presently about 0.5 μm. High-end lamp for some time. A disadvantage of phase contrasted scanners produce data with a resolution below 0.1 μm μ-CT scans is that different tissue types can barely be (nanotomography). distinguished. The greatest advantage of μ-CT is the highly acceler- Recently two teams successfully established this ated (compared to histology) and non-destructive acqui- technique in Japan (Fig. 7). The first team is composed sition of almost artifact-free anatomical image stacks. of Shun-ichi Kinoshita, Osamu Sasaki (Tohoku Uni- The data are ideal for fast and precise 3D reconstruc- versity), Yoshiaki Hashimoto (University of Hyogo/ tion. The specimens can be used for SEM or histological Museum of Nature and Human Activities, Hyogo), sectioning after the μ-CT scans are obtained. This allows Katsuyuki Eguchi (Tokyo Metropolitan University) and for a very detailed morphological documentation with Takuji Tachi and Shingo Hosoishi (Kyushu University). a minimum of material (see below under “work flow”). Sample images are shown in Figure 7b–d and their activ- As a non-invasive technique, μ-CT can also be applied to ities are described on http://webdb2.museum.tohoku very rare species or even type material. High-density .ac.jp/e-foram/indexj.html and http://www.museum resolution based on the specific absorption of different .tohoku.ac.jp/press_info/news_letter/index.htm. The tissue types (e.g. skeleton, muscles) is usually recorded second team comprises Yukihiro Nishikawa (Kyoto with low beam energy, and facilitates the discrimination Institute of Technology) and Kyohei Watanabe of structures (e.g. Friedrich et al. 2008). (Kanagawa Prefectural Museum of Natural History). The preparation of specimens is simple. Usually They used μ-CT not only for anatomical research, but they are dehydrated in an alcohol series and dried at also for improving the usage of museum specimens, in the critical point. Thus a high contrast between the ecological studies and in education. A study describing tissues and the surrounding medium (air) is generated. their activities will be published in the near future The specimen can be mounted directly on a holder (Watanabe et al., unpubl. data, 2013).

Figure 7 Development of μ-CT in Japan. (a) μ-CT at Museum of Natural History of Tohoku University and S. Kinoshita. (b–d) Sample images taken by S. Kinoshita; (b) Lema coronata, (c,d) Pristomyrmex punctatus, color variation means density variation of material composition; red: high, blue: low. (e,f) μ-CTs in Kyoto Institute of Technology with K Watanabe (Kanagawa Prefectural Museum of Natural History) (e) and Y Nishikawa (Kyoto Institute of Technology) (f).

Computer-based 3-dimensional reconstruction and visualization. Data based on other sources (e.g. images of histological or TEM sections) have to be Three-dimensional reconstructions can be based on aligned, either using speciﬁc functions implemented different types of data sets. Most common sources in commercial software packages such as Amira (FEI are serial sections, μCT image stacks and CLSM data. Visualization Sciences Group, Mérignac Cedex, France) Three-dimensional reconstructions greatly facilitate or AutoAligner (Bitplane Imaris, Zurich, Switzerland) or analyses of the spatial arrangement of morphological using open-source tools (e.g. ImageJ). structures by using virtual section planes computed Volume rendering is an easy way to visualize image by the software. Smaller data sets can be visualized on data and yields results similar to low-magniﬁcation standard desktop computers, but powerful graphic SEM images. An advantage compared to standard SEM workstations are needed for high-resolution data and imaging is the easy assessment of the thickness of the extensive imaging procedures (Fig. 8). body wall (and other structures). Volume models can be Perfectly aligned data as for μ-CT and SBFSEM image used to create 3D images (e.g. for red–cyan goggles) stacks can be immediately used for 3D reconstruction or videos (e.g. with ImageJ). In order to show internal

Matsumura et al. 2013), even though only a few of them study insects, and some of them in collaboration with European researchers. Nagashima et al. (2009) used free software (imageJ) for 3D reconstructions to illus- trate the development of turtles. This technique is frequently used in vertebrate developmental studies (e.g. Nagashima et al. 2009; Adachi & Kuratani 2012; Tokita et al. 2012) and will also be established in the entomological laboratory of R. Machida (University of Tsukuba) in the near future (Y Nakagaki & R Machida, pers. comm., 2013).

Focus stacking of digital images Figure 8 Computer-based three-dimensional reconstruction. Tracing materials of pictures of histological sections by RG Focus stacking is a technique for extending the depth of Beutel (Friedrich-Schiller-Universität, Jena). focus (Fig. 9). Due to the special optical conditions the area of the object rendered in optimal focus is extremely narrow when taking photographs of very small objects. features, cutting planes can be applied to the volume Thus focus stacking procedures are very helpful in rendering. This procedure is fast, but limited in its macro or micro photography. However, the techniques potential to visualizing complex internal configurations. can be applied whenever images similar to photographs To obtain more specific 3D reconstructions (e.g. of the are available, for example, SEM micrographs. skeleto–muscular or nervous systems), segmentation The focus stacking procedure follows a simple prin- of the data set is necessary. Structures of interest can ciple: as many differently focused photographs should be manually outlined or semi-automatically labeled be taken as are necessary to get well-focused images of throughout the stack using commercial software pack- all areas of the object that should be visualized in the ages (FEI VSG Amira, Bitplane Imaris, VGStudio MAX) final picture. Then only the focused areas from each or open source tools (e.g. Reconstruct). The prepared photograph is selected and combined in the final image. data can be used to produce segmented volume render- This shows much more of the object in perfect focus ings of selected structural complexes, which allows for than any combination of photographic parameters easy coloration of volume data. would produce (e.g. lens or aperture). It is possible to The segmented image stacks can also be used for the carry out all steps of this procedure manually using automatic creation of surface objects of discrete struc- any software for cutting certain areas from an existing tures. Unlike volume renderings, surface objects are digital picture and pasting them into another file. As hollow and represent only the outline of the structures, this is an arduous and error-prone process, it is recom- usually resulting in a more or less simplified 3D model. mendable to use specific software for combining the The main advantages of this visualization technique images. are the potential for multiple modifications (e.g. sim- Most of the larger microscope manufacturers offer plification, minimizing artifacts), coloration, illumina- special systems, for example microscopes where the tion and animation of anatomical structures using focus is operated through software, automatically gen- software (e.g. Autodesk, Maya, Luxology Modo and erating microscopic images with an extreme depth of open-source Blender). Surface models can be used to focus. These systems are expensive, but reasonably calculate the volume of structures, to carry out finite priced or even free alternatives producing final images of element analyses (FEA) and print magnified, solid comparable quality are available (see below). models of the structures by rapid prototyping. Further- M. Maruyama (Kyushu University) is one of few more, surface-based 3D models can be included in experts producing high quality images using this tech- scientific presentations and publications using the nique in Japan (e.g. Ballerio & Maruyama 2010; common pdf file format, allowing for easy exchange of Maruyama 2010, 2012). Maruyama uses the software 3D contents. Combine ZM, which is freely available from Alan This technique is not widely used in Japan, but some Hadley (http://hadleyweb.pwp.blueyonder.co.uk/), to morphological studies were published by Japanese produce stacks of photos. The procedure is described in authors (e.g. Nagashima et al. 2009; Kaji et al. detail at: https://sites.google.com/site/myrmekophilos/ 2011; Adachi & Kuratani 2012; Tokita et al. 2012; czm. A commercial but reasonably priced alternative is

Figure 9 Sepsis fulgens (Diptera: Sepsidae). (a) Partly focused image taken with a digital SLR equipped with a macro lens. (b) Focus stacked image of 142 partly focused images.

Helicon Focus (http://www.heliconsoft.com/) or Zerene shape were explored. In the 1980s, the nature of data Stacker (http://zerenesystems.com/cms/stacker), which gathered and analyzed changed fundamentally, with a allows for a very comfortable work flow. With Helicon focus on the coordinates of landmarks and the geo- Focus it is even possible to remotely operate a SLR- metric information about their relative positions. The camera and stepper motors to adjust the camera’s posi- developing novel approach was referred to as geometric tion relative to the object, making semi-automatic image morphometrics (Adams et al. 2004). capture and processing possible. The multivariate part of geometric morphometrics is The images produced by focus stacking are ideal, usually carried out in a linear tangent space to the non- for example, for documenting type specimens, show- Euclidean shape space in the vicinity of the mean shape. ing stunning pictures of specimens in publications or Traditionally, morphometrics was mainly focused on printing sharp pictures of small or large specimens for size, which plays an important role. However, geometric exhibitions. morphometrics is better suited to assess information about shape. More generally, it is the class of morpho- Geometric morphometrics metric methods that preserves complete information Geometric morphometrics is a collection of approaches about the relative spatial arrangements of the data for the multivariate statistical analysis of Cartesian coor- throughout an analysis. As such, these methods allow dinate data, usually limited to landmark point locations. for the visualization of group and individual differences, Shape is the geometrical information that remains when sample variation, and other results in the space of the location, scale and rotational effects are filtered out from original specimens. an object (Kendall 1977). The analysis of shape is a The crucial first step is the selection of specimens fundamental element of biological research (Bai & Yang suitable for the addressed scientific question. The second 2007). step is to collect photos (using a camera, or based on Morphometrics in a traditional sense is the applica- drawings, SEM micrographs, etc.) based on the same tion of multivariate statistical analyses to sets of quan- direction rule, which ensures the comparability of shape. titative variables such as width, length, depth, volume Although in principle there is no limitation for the photo and area (Bookstein 1998). This approach was limited size, it should be as small as possible (as long as it is still in different ways, for instance by the difficulty to assess clear enough). The third step is to build a data file (such the homology of linear distances. Another inherent as a .tps file) which links all photos involved. The fourth problem is that the geometric relationships among the step is to gather landmark or semi-landmark data when variables are not preserved. Consequently, alternative the photos are loaded in software, such as TPS-Dig methods of quantifying and analyzing morphological (Rohlf 2006). The procedure for 3D data collection is

similar, but different in the 3D photo reconstruction and ecology. This often requires a sophisticated quanti- and landmark gathering. Criteria for selecting land- tative representation of the phenotype that captures the marks and curves are homology, which means that a functional, genetic or developmental attributes that are specific point is considered the “same” in all specimens, biologically important for the hypothesis to be tested, by adequacy of coverage, consistency of relative position, linking the data. As existing coordinate-based geometric coplanarity and repeatability. morphometric methods can not be easily extended to For most practical applications, the parameters 3D data, geometric methods for the analysis of 3D data describing the shapes for a sample of homologous land- is still a developing area. Further improvements are mark configurations are estimated by a Procrustes super- needed to address an even broader field of problems imposition. This procedure is a least-squares oriented with greater sophistication than is possible today. approach involving three steps. The position, scale and Practical reviews and specific studies by Japanese orientation of the specimens are removed, retaining entomologists are also available (Fukudome & only the shape for which the same position, size and Sakamaki 2011; Konuma 2011; Novkovic 2011; orientation is assumed. After this the shapes are placed Takahashi 2011; Tatsuta & Sakamaki 2011). on top of one another. Two alternative methods are used: Procrustes superimposition and Bookstein shape Storage of morphological data in data banks coordinates. Impressive amounts of high quality morphological data The greatest strength of geometric morphometric have been produced in recent years. This increases the methods is that graphical representations of results are need for suitable data storage facilities (e.g. μ-CT raw possible as configurations of landmark points rather data). Moreover, to facilitate the maximum use of the than as customary statistical scatterplots. The analysis information, easy accessibility for members of the scien- of landmark data can be estimated by a superimposition tific community should be guaranteed. A presently avail- procedure, followed by the projection of the aligned able data bank is the Morph·D·Base. It is an online data coordinates on a linear tangent Kendall’s shape space repository for morphological metadata and media files for multivariate analyses, and the graphical visualization and a freely accessible general communication platform of results in terms of the configurations of landmarks for scientists (https://www.morphdbase.de/). (Kendall 1984; Rohlf 1999; Slice 2001). In Kendall’s Optimized storage and accessibility is invaluable for shape space, distances between pairs of points (speci- scientific progress. Nevertheless, standards and rules mens) approximate the Procrustes distances between the for reporting and documenting data are not fully estab- corresponding pairs of landmark configurations. Partial lished yet (e.g. Deans et al. 2012b; Vogt et al. 2013). warps, which are the eigenvectors of the bending energy matrix ordered, from the thin-plate spline plus the Work flow uniform shape components (Rohlf & Bookstein 2003), Depending on the aim of an investigation and the are a convenient set of shape variables that can be inter- available material and facilities, different combinations preted as axes for this space. Scores on these axes can of techniques can be used to produce optimal results then be treated as multivariate data representing shape, with maximum efficiency. Even single specimens can be and can be used in conventional multivariate analyses processed to produce very detailed results. Microscopic (Caldecutt & Adams 1998; Bookstein et al. 1999; drawings from different perspectives would usually Adams & Rohlf 2000; Bai et al. 2010, 2011, 2012; be the first step. This is useful for assessing the major Gharaibeh et al. 2000; Klingenberg & Leamy 2001; external features including properties of the cuticle, Rüber & Adams 2001). Differences in shape among for example membranous vs sclerotized areas, and objects can be described not only as a plot, but also in also internal structures (e.g. muscles) visible through tree form via cluster analysis. Different models, includ- the transparent or semitransparent integument. CLSM ing single linkage (the distance between two units is the imaging in glycerine provides additional information on distance between the two closest members of those external and more or less superficial internal structures. clusters), UPGMA (unweighted pair group method with After critical point drying, SEM can be applied for a arithmetic mean; the distance between two clusters is the detailed documentation of surface structures. Micro- average of the distances between units in one cluster and computed tomography using the same specimen pro- units in the other cluster) and Ward’s (minimizes the vides information on the entire external and internal variance of intra-cluster distances), are used to calculate configuration. Finally, 3D reconstruction based on the similarity among clusters. obtained image stacks is an ideal tool for attractive Geometric morphometrics data can be applied to and informative visualization, including animated 3D many research fields, such as phylogeny, development pdf files, which are increasingly used in morphological

publications. Using this or a slightly different protocol, of systematic studies (Bybee et al. 2008) that the use of a minimum of material can yield an immense wealth morphological data in insect phylogenetics loses ground of information without destroying the exemplars. when compared to molecular systematics. However, it If additional specimens are available, a very detailed was demonstrated by Wheeler (2008) that even when documentation of internal details including histological morphological parts of combined matrices are seemingly properties of tissues can be obtained using semi-thin dwarfed by the sheer number of informative sites sectioning. Ultrastructural studies require specifically in extensive molecular data sets (e.g. 775 genes in fixed additional specimens and the use of TEM or Meusemann et al. (2010)), they still can have a substan- SBFSEM. tial impact on the resolution of the deeper nodes of The data obtained with the approach outlined here the trees, which are often insufficiently resolved with are an ideal basis for morphology-based phylogenetic molecular data alone (e.g. von Reumont et al. 2009). evaluations and studies in evolutionary morphology, Extensive molecular data sets including transcriptomes also using molecular phylogenies as a background for and complete genomes are already available for a developing complex evolutionary scenarios. For investi- considerable number of taxa (e.g. Meusemann et al. gations mainly focused on comparisons of shapes, geo- 2010; von Reumont et al. 2012; see also 1KITE http:// metric morphometric analyses are highly recommended, www.1KITE.org). These data sets will possibly yield also in a phylogenetic and evolutionary context. very robust phylogenies without using any morphological information (e.g. Niehuis et al. 2012). However, DISCUSSION mere branching patterns, robust as they may be, provide only limited insights into the evolution of insects or It was demonstrated in a recent project on the phylogeny other groups of organisms. As pointed out in Beutel of Holometabola that an optimized combined applica- et al. (2011), the knowledge of the morphological trans- tion of more traditional and innovative morphological formations is essential for the reconstruction of complex techniques can greatly facilitate and accelerate the evolutionary scenarios. It is the phenotype with its mor- acquisition and documentation of high-quality morpho- phological features which interacts with the environ- logical data. In approximately two years a data matrix ment and which is primarily exposed to natural selection containing 356 characters of different body parts and (Beutel et al. 2009, 2011). The concept of evolutionary developmental stages was compiled. The results (Beutel morphology outlined by Wirkner and Richter (2010) et al. 2011) yielded a single highly resolved cladogram emphasizes the importance of in-depth morphological (parsimony analysis) and were almost completely con- investigations in a context that goes beyond mere phy- gruent with analyses of an extensive molecular data set logenetic branching patterns. This also includes investi- (Wiegmann et al. 2009). gations in a functional context (e.g. Beutel & Gorb An often-raised question is whether modern tech- 2001, 2006, 2008) or the detailed study and documen- niques also lead to the discovery of novel characters. tation of the morphology of different life stages of model The introduction of TEM and SEM doubtlessly revo- organisms such as Drosophila or Tribolium. lutionized investigations on the cellular level and of For several reasons it appears likely that morphology surface structures, respectively, and both techniques will continue to play a vital role in insect systematics have contributed to the exploration of new character and evolutionary biology. The careful investigation of systems (e.g. spermatozoan ultrastructure, Dallai & structures is an essential precondition for understanding Afzelius 1984; Jamieson et al. 1999). To a much lesser the functions of diverse structures, which may have degree new characters can be expected using μ-CT. The played an important role in the evolution of the groups advantage of this technique is greatly accelerated data in question, and are often also helpful in reconstructing acquisition. Likewise computer-based 3D reconstruction relationships (e.g. attachment structures, Beutel & Gorb will rarely reveal new phylogenetically relevant features. 2001, 2006; wing base sclerites, Yoshizawa 2011). Mor- However, this approach has tremendously facilitated phology provides an independent data set for critically the understanding of complex structural constellations re-evaluating the results of molecular studies (and vice and greatly improved the visualization of structures and versa). Analyses exclusively based on molecular data characters. may provide robust phylogenies but do not provide It is apparent that the study of the morphology a complex picture of a group. Without knowing the of insects or other organisms is challenged by the transformations on the phenotypic level it is not possible tremendous progress of molecular analyses in the “age to develop a complex and meaningful evolutionary of phylogenomics” (e.g. Beutel & Kristensen 2012; scenario for insects or any other group with a complex Trautwein et al. 2012). It was shown in a recent review morphology.

A field in which morphology will obviously play an dung beetles (Coleoptera: Scarabaeinae). PLoS ONE 6, exclusive role is paleontology. Fossils will only provide e21600. useful DNA sequences in extremely rare cases, but at Bai M, Yang XK (2007) [Application of geometric least amber fossils can be studied with innovative mor- morphometrics in biological researches]. Chinese Bulletin phological approaches and yield detailed information of Entomology 44, 143–147. (In Chinese.) Ballerio A, Maruyama M (2010) The Ceratocanthinae of (e.g. Pohl et al. 2010). Beutel et al. (2008) demonstrated Ulu Gombak: high species richness at a single site, with that the neglect of fossils can lead to serious misin- descriptions of three new species and an annotated check- terpretations in phylogenetic reconstruction, and it is list of the Ceratocanthinae of Western Malaysia and trivial that the knowledge and interpretation of extinct Singapore (Coleoptera, Scarabaeoidea, Hybosoridae). taxa is essential for understanding the evolutionary ZooKeys 34 (special issue), 49–54. history of any group of organisms. Berlese A (1909) Gli Insetti, Vol. 1. Societa Editrice Libraria, Milano. ACKNOWLEDGMENTS Beutel RG, Friedrich F, Ge S-Q, Yang XK (2013) Insect Mor- phology and Phylogeny. De Gruyter, Berlin. This research was supported by the National Basic Beutel RG, Friedrich F, Hörnschemeyer T et al. (2011) Research Program of China (973 Program) (No. Morphological and molecular evidence converge upon 2011CB302102), the National Natural Science Founda- a robust phylogeny of the megadiverse Holometabola. tion of China (Nos. 31010103913, 31172143), and by a Cladistics 27, 341–355. Humboldt Fellowship (to M.B.) from Alexander von Beutel RG, Ge S-Q, Hörnschemeyer T (2008) On the head Humboldt Foundation, by the VolkswagenStiftung (to morphology Tetraphalerus, the phylogeny of Archoste- R.G.B.), by the DFG Heisenberg grant (HO2306/7-1) of mata and the basal branching events in Coleoptera. the Deutsche Forschungsgemeinschaft (to T.H.), by JSPS Cladistics 23, 1–29. Beutel RG, Gorb S (2001) Ultrastructure of attachment Postdoctoral Fellowships for Research Abroad (to Y.M.). specializations of hexapods (Arthropoda): evolutionary We also thank K Yoshizawa, S Kinoshita, K Watanabe, patterns inferred from a revised ordinal phylogeny. 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