Background: Investigation of the internal tissues and organs of a macroscopic organism usually requires destructive processes, such as dissection or sectioning. These processes are inevitably associated with the loss of some spatial information. Recently, aqueous-based tissue clearing techniques, which allow whole-organ or even whole-body clearing of small rodents, have been developed and opened a new method of three-dimensional histology. It is expected that these techniques will be useful tools in the field of zoology, in which organisms with highly diverse morphology are investigated and compared. However, most of these new methods are optimized for soft, non-pigmented organs in small rodents, especially the brain, and their applicability to non-model organisms with hard exoskeletons and stronger pigmentation has not been tested. Results: We explored the possible application of an aqueous-based tissue clearing technique, advanced CUBIC, on small crustaceans. The original CUBIC procedure did not clear the terrestrial isopod, Armadillidium vulgare. Therefore, to apply the whole-mount clearing method to isopods with strong pigmentation and calcified exoskeletons, we introduced several pretreatment steps, including decalcification and bleaching. Thereafter, the clearing capacity of the procedure was dramatically improved, and A. vulgare became transparent. The internal organs, such as the digestive tract and male reproductive organs, were visible through sclerites using an ordinary stereomicroscope. We also found that fluorescent nuclear staining using propidium iodide (PI) helped to visualize the internal organs of cleared specimens. Our procedure was also effective on the marine crab, Philyra sp. Conclusions: In this study, we developed a method to clear whole tissues of crustaceans. To the best of our knowledge, this is the first report of whole-mount clearing applied to crustaceans using an aqueous-based technique. This technique could facilitate morphological studies of crustaceans and other organisms with calcified exoskeletons and pigmentation. Keywords: Tissue clearing, Advanced CUBIC, Crustacea, Isopoda, Decapoda Background have limited resolution compared to light microscopy, Biological structures are three-dimensional (3D). It is and are much less accessible to most zoologists. generally difficult to observe the 3D structures and spatial Another strategy to observe internal structures is to relationships of internal organs in opaque organisms. make opaque organisms transparent. Although the con- Traditionally, this limitation was overcome using 3D re- cept of tissue clearing is over 100-years-old , its use construction from serial sections [1, 2]. However, serial has been relatively limited to the field of osteology. sectioning is usually painstaking, time-consuming, and Recently, advances in genetically encoded fluorescent limited to small specimens. Advanced imaging tech- markers and the advent of various optical sectioning mi- nologies, such as magnetic resonance imaging and croscopies have stimulated the development of new computed tomography , are powerful tools for im- aqueous-based tissue clearing techniques [6, 7]. We con- aging internal structures; however, these instruments sidered that these novel techniques have the potential to reform current experimental designs and advance our understanding on the morphology of a wide range of or- * Correspondence: email@example.com ganisms. However, most of the new tissue clearing tech- Department of Medical Spectroscopy, Hamamatsu University School of niques are designed and optimized for the soft tissues of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu-City, Shizuoka-Pref small rodents, and their applicability to hard tissues or 431-3192, Japan © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Konno and Okazaki Zoological Letters (2018) 4:13 Page 2 of 8 other organisms has scarcely been explored. A recent Pretreatment of samples for clearing study reported that mouse bony tissues could be cleared Samples were gently agitated on a shaker during all using an aqueous-based method coupled with the decal- washing and incubation steps. The fixed animals were cification and decoloration of heme . Here, we tested rinsed several times in PBS, and decalcified in 0.2 M the possible application of aqueous-based tissue clearing EDTA (pH 8.0) at 4 °C for 24–48 h, with one change of on crustaceans. Since crustaceans have a hard exoskel- the EDTA solution. Decalcified specimens were washed eton and strong pigmentation, which hamper the obser- in PBS at RT. To minimize deformation during the pro- vation of internal structures, successful application of cedures, samples were fixed again in 4% PFA/0.2 M PB tissue clearing techniques would facilitate morphological (pH 7.4) overnight at 4 °C. Decalcified specimens were and histological studies of this taxon. then bleached in hydrogen peroxide (H O )/PBS. To 2 2 In this study, we attempted whole-body tissue clearing avoid vigorous reaction with H O , A. vulgare samples 2 2 of small crustaceans using an aqueous-based technique, were first incubated in 0.03% H O /PBS at 37 °C until 2 2 advanced CUBIC . We found that the original proto- the formation of fine bubbles stopped (~ 24 h, but with col did not clear the terrestrial isopod, Armadillidium significant variation among individuals). Since Philyra vulgare. Therefore, we introduced some pretreatment sp. did not bubble vigorously, this step was skipped. steps, including decalcification and bleaching. After opti- Then, A. vulgare and Philyra sp. samples were bleached mizing the pretreatment, clearing efficiency was dramat- in 3% H O /PBS for 12–48 h at 37 °C. The containers 2 2 ically improved and most of the body parts became were not tightly closed to allow for the release of bub- transparent. The same procedure was also effective for bles. The samples were then washed several times in the marine crab, Philyra sp. The internal anatomy of PBS. When bubbles formed inside the gut, samples were cleared specimens was easily observed using stereomi- transferred to an airtight container filled with degassed croscopy. Further characteristics of some of the cellular PBS at RT. Then it was capped without introducing air arrangements were revealed using fluorescent nuclear and kept at 4 °C to dissolve the bubbles. staining. Our approach provides a useful tool for the morphological study of crustaceans, and possibly other Whole-mount clearing animals with calcified body parts and/or pigmentation. Whole-mount clearing was performed with the advanced CUBIC protocol . Briefly, delipidation and refractive Methods index (RI) matching were conducted with reagent-1 Reagents [25% (w/w) urea, 25% Quadrol, 15% (w/w) Triton X-100 All reagents were purchased from Wako Pure Chemical in distilled water] and reagent-2 [25% (w/w) urea, 50% Industries (Osaka, Japan), except for the following: ethyl- (w/w) sucrose, 10% (w/w) 2,2′,2″-nitrilotriethanol enediaminetetraacetic acid (EDTA) (Dojindo Laboratories, (triethanolamine) in distilled water], respectively. Decal- Kumamoto, Japan), N,N,N′,N′-tetrakis(2-hydroxypropy- cified and bleached samples were incubated in 1/2 l)ethylenediamine (Quadrol) (Tokyo Chemical Industry, reagent-1 (reagent-1:H O = 1:1) for 6 h to overnight and Tokyo, Japan), Triton X-100 (Sigma-Aldrich, St. Louis, then in 1× reagent-1 at 37 °C until they became trans- MO, USA), and propidium iodide (PI) (Thermo Fisher parent. The samples were washed several times in PBS Scientific, Waltham, MA, USA). and treated with 1/2 reagent-2 (reagent-2:PBS = 1:1) for more than 3 h. Then, samples were transferred to 1× Animals reagent-2 and incubated until the solution became Common pill bugs, A. vulgare, were collected in homogeneous. All steps were performed on a shaker at Hamamatsu City, Japan. They were starved for about RT, except for the incubation in 1/2 and 1× reagent-1 at 24 h to empty the gut and were then fixed in 4% 37 °C. paraformaldehyde (PFA)/0.1 M phosphate buffer (PB) Nuclear staining with PI was performed after the (pH 7.4). As immersion in the fixative causes them to reagent-1 treatment. Following several PBS washes, sam- roll up into a ball, we sandwiched them between ples were incubated in PBS containing 20 μg/ml PI over- stainless steel meshes in stretched form, and fixed night at 4 °C. After PI staining, samples were treated them at 4 °C for at least 48 h. Marine crabs, Philyra with reagent-2, as described above. sp., were collected in Shimoda Bay, and were fixed immediately in 10% formalin/sea water. They were Observations kept in the fixative at room temperature (RT) until Macroscopic images were photographed with a digital use. A hornet, Vespa analis, was collected in Hamamatsu camera (Optio WG-2, Pentax, Tokyo, Japan). Stereomi- City, Japan, and stored as a dry specimen. Before use, the croscopic images were obtained with an INFINITYHD hornet was rehydrated in PBS, and fixed in 4% PFA/ camera (Luminera Corporation, Ontario, Canada) under 0.1 M PB (pH 7.4) at 4 °C for 24 h. oblique illumination. Fluorescence images of PI were Konno and Okazaki Zoological Letters (2018) 4:13 Page 3 of 8 obtained with WRAYCAM-SR130M camera (Wraymer, pump was not effective, so samples were transferred into Osaka, Japan) with a filter for RFP. Both were mounted on degassed PBS at RT and then the temperature was low- an SZX16 stereomicroscope (Olympus, Tokyo, Japan). ered to 4 °C to further increase the solubility of gas. After this treatment, bubbles were completely dissolved Results within one day. Optimization of pretreatments for whole-mount clearing After optimizing the preclearing steps, the tissue clear- We first assessed the applicability of the aqueous-based ing efficiency of the advanced CUBIC protocol was dra- tissue clearing technique to crustaceans using the com- matically improved and most of the body parts became mon pill bug, A. vulgare. We tested the advanced transparent (Fig. 1b). Males and females were distin- CUBIC  method due to its high tissue clearing cap- guishable by the relatively low transparency of the testes acity and the simplicity of the procedure [10, 11]. This and vas deferens  (Figs. 1 and 2). Stereomicroscopic method consists of two steps: (1) delipidation, decolora- observations confirmed good transparency of the cleared tion, and hyperhydration in reagent-1 solution, followed pill bugs, except for the jaw and respiratory structures in by (2) refractive index (RI) matching in reagent-2 solu- pleopods , as well as the male reproductive organs tion. Despite its powerful clearing capacity for various (Fig. 2a). The largest compartment of the digestive tract rodent tissues, this technique only rendered slight color in pill bugs is the hindgut . In cleared samples, the change and no transparency in A. vulgare (Fig. 1a). ordered lattice-like structure of the hindgut wall, a pair Most of the recently developed techniques have only of typhlosole channels on the dorsal side of the anterior been tested on tissues that lack hard components or pig- hindgut, and the junction between anterior and posterior ments, except for heme and its derivatives. Therefore, hindguts were easily observed through the dorsal scler- we reasoned that the calcified exoskeleton and body pig- ites (Fig. 2b). At higher magnification, muscle striation mentation were barriers to effective tissue clearing in in the legs was also observed (Fig. 2c). the isopod and introduced the decalcification and These results indicate that the aqueous-based tech- bleaching steps. After decalcification with EDTA, the so- nique enabled whole-mount tissue clearing of small lution turned slightly brownish, and the isopods were crustaceans after calcified deposits and pigments were softened and changed in color. The same treatment had removed. no effect on the hornet cuticle, which lacks calcium de- posits (Additional file 1: Figure S1). Then A. vulgare Visualization of internal structures with fluorescent samples were fixed again and bleached in H O solution. nuclear staining 2 2 Some individuals unexpectedly showed explosive bub- We used fluorescent nuclear staining to visualize ana- bling upon contact with 3% H O and their bodies were tomical structures in the cleared specimens (Fig. 3). PI 2 2 frequently broken apart. We resolved this problem by staining revealed internal organs, especially the male re- treating the samples with dilute (0.03%) H O first. After productive system, of A. vulgare. The male reproductive 2 2 the formation of fine bubbles stopped, they were safely system was similar to that of other terrestrial isopods transferred to a higher concentration of H O solution . Each of a pair of male reproductive organs is com- 2 2 for bleaching. Another problem we encountered was the posed of three testis follicles, a seminal vesicle, and a vas formation of bubbles inside the gut of some individuals. deferens (Fig. 3a and b). Testis follicles , which were This appeared not to damage tissues in the samples pre- not visible in unstained samples (Fig. 2a), were clearly treated with dilute H O , but hampered microscopy observed after PI staining (Fig. 3a and b). In the anterior 2 2 after clearing. Degassing the samples using a vacuum hindgut, a characteristic array of cells was observed at a Fig. 1 Whole-mount clearing of the terrestrial isopod, A. vulgare, with advanced CUBIC protocol. a A. vulgare cleared with advanced CUBIC protocol without any pretreatment. b Clearing after pretreatment, including decalcification and bleaching. Grid = 5 mm Konno and Okazaki Zoological Letters (2018) 4:13 Page 4 of 8 Fig. 2 Stereoscopic examination of a cleared A. vulgare. a Dorsal view of a cleared male (left) and female (right). Most of the body parts became transparent, except for the sperm vesicle (Sv), vas deferens (Vd), mandibles (M), and pseudotrachea (Pt). Scale bars = 2 mm. The boxed region on the female is shown in b at higher magnification. b Junction of anterior (Ah) and posterior (Ph) hindgut. A pair of typhlosole channels (Tc) run along the dorsal midline of the anterior hindgut. Scale bar = 1 mm. c Muscle striation in a leg. The boxed region in the left panel is enlarged in the right panel. Scale bar = 500 μm (left), 200 μm (right). All observations were performed under appropriate oblique illumination higher magnification. Large cell nuclei were arranged in the arrangement and relationships of internal organs in ordered rows. The four dorsalmost rows, possible typh- non-model organisms. losole channel cells, were particularly conspicuous by their large, laterally elongated nuclei (Fig. 3c). Whole-mount clearing of a small decapod These data suggest that the combination of whole-mount Finally, we tested whether our procedure could be ap- tissue clearing and fluorescent nuclear staining can reveal plied to another crustacean using the marine crab, Fig. 3 Propidium iodide (PI) staining of a cleared A. vulgare male. a Top view of the stained specimen. Dotted lines indicate the contour of male reproductive organs. Legs are removed. Scale bar = 2 mm. b Side view of the middle body part. Scale bar = 1 mm. Testis follicles (Tf), sperm vesicle (Sv), vas deferens (Vd). c Close-up image of the dorsal anterior hindgut. Large cell nuclei aligned in ordered rows. Scale bar = 500 μm Konno and Okazaki Zoological Letters (2018) 4:13 Page 5 of 8 Fig. 4 Whole-mount clearing of the marine crab, Philyra sp. a Specimen before (top) and after (bottom) clearing. Dark parts seen through the carapace are the gut content. Grid = 5 mm. b, c Nuclear staining with propidium iodide (PI). Dorsal view through the carapace and the right cheliped. Scale bars = 1 mm Philyra sp. (Fig. 4). Its transparency was increased by de- organisms are almost transparent when they have a simi- calcification alone (Additional file 1: Figure S1C). After lar RI to water . Therefore, a general strategy for tissue bleaching and subsequent CUBIC procedure, this species clearing is the removal of pigments and the matching of was also successfully cleared. In this species, direct RIs. For mammalian organs, the contribution of pigments immersion in 3% H O did not cause vigorous bubbling, to opacity is relatively small, except in some heme-rich or- 2 2 and treatment with dilute H O was omitted. Since they gans. Conversely, strong pigmentation is a significant bar- 2 2 were not starved before fixation, the gut content was rier to whole-mount tissue clearing of small invertebrates. observed through the carapace (Fig. 4a). Fluorescent In addition, calcified exoskeletons can hinder the effective nuclear staining with PI revealed the hepatopancreas penetration of clearing reagents into tissue components. (Fig. 4b) and muscular architecture (Fig. 4c). Indeed, the advanced CUBIC method failed to clear A. We conclude that tissue clearing with advanced vulgare. To resolve these problems, we introduced the CUBIC method after decalcification and bleaching is ef- pretreatments of decalcification with EDTA and bleaching fective for various crustaceans. with H O . 2 2 Decalcification has already been shown to be effective Discussion in clearing mammalian bony tissues [8, 17]. This step In this study, we developed a whole-mount clearing should help subsequent clearing processes by facilitating method for use in crustaceans. To the best of our know- the penetration of clearing reagents. In addition, EDTA ledge, this is the first report of an aqueous-based tissue treatment alone improved the translucency of the calci- clearing technique successfully applied to non-model in- fied exoskeleton, especially in the crab (Additional file 1: vertebrates. Although a previous unique study has de- Figure S1). This phenomenon is likely due to the re- scribed a transparent composite prepared from the crab duced heterogeneity of RI caused by the removal of cal- shell , this approach requires the complete removal cium deposits with high RI. This direct clearing effect of of non-chitin components and is not suitable for histo- EDTA was not apparent in A. vulgare, probably because logical applications. In comparison, the tissue clearing of its stronger pigmentation. EDTA solution turned method described here could be subjected to various im- slightly brownish during decalcification of the crusta- aging analyses. ceans, suggesting that some pigments, most likely the ones strongly associated with mineralized structures, are Optimization of clearing steps liberated. EDTA treatment did not have a visible effect The main causes of tissue opacity are the presence of on the exoskeleton of the hornet, suggesting that the im- pigments and inhomogeneous RIs among cellular com- proved transparency of crustaceans after EDTA treat- ponents and the medium . Non-pigmented aquatic ment is purely caused by decalcification. In larger Konno and Okazaki Zoological Letters (2018) 4:13 Page 6 of 8 crustaceans, decalcified samples may become deformed it is often difficult to make use of genetically encoded after the loss of structural support. In this case, partial marker proteins or good commercial antibodies. There- dissection may be required. Alternatively, hydrogel em- fore, the exploration of chemical probes, which is com- bedding methods, such as PACT [8, 17], may provide patible with tissue clearing, is important. Small chemical extra mechanical support. probes also have an advantage of fast penetration into We observed destructive bubbling in samples of A. large specimens. Fluorescent nuclear staining is a popu- vulgare during the bleaching step. Some A. vulgare bub- lar technique used in aqueous-based tissue clearing to bled vigorously upon contact with 3% H O . We over- visualize the architecture of tissues and organs [9, 20]. 2 2 came the problem by immersing samples in 0.03% H O We confirmed that nuclear staining with PI was useful 2 2 first. We also introduced a second fixation step after de- and sometimes essential to observe the internal structures calcification, based on the expectation that the removal of cleared whole-mount crustaceans (Figs. 3 and 4). The of calcium deposits would unmask reactive groups that non-biased visualization of cellular organization in were not accessible during the first fixation. Although whole-mount specimens using this type of staining might the cause of the different intensities of bubbling was un- also facilitate the discovery of overlooked morphological clear, variation in peroxidase activity during the molting characteristics. cycle might be responsible. Indeed, several studies have Various staining methods are applied to samples reported the involvement and cyclical expression of cleared with aqueous-based procedures. For example, peroxidases during ecdysozoan cuticular biosynthesis successful in situ hybridization was reported using [18, 19]. Since the bubbling of Philyra sp. was much CLARITY . Some detergent-free clearing protocols, gentler, post-fixation and incubation with dilute H O including SeeDB , FRUIT , and one of the ScaleS 2 2 was not necessary. Therefore, this process would be sim- variants , are compatible with lipophilic dyes [25, 26]. plified, depending on the species and lifecycle stage. Very recently, Golgi-Cox staining for cleared brain sam- After bleaching, we encountered the problem of re- ples was reported . Although generalized protocols are moving the bubbles formed in the lumen of the digestive not yet available for most of these techniques, it is worth tract. The bubbles do not hinder the latter clearing steps testing their application in non-model organisms in fu- but can disturb the observation of cleared specimens. ture studies. Degassing using a vacuum pump did not remove the bubbles and even damaged tissues. We found that the Selection of tissue clearing techniques for zoologists immersion of samples in a degassed buffer-filled con- For researchers planning their first tissue clearing ex- tainer and storage at a lower temperature removed the periment, it is not easy to choose a suitable method bubbles. When no pump is available, immersion of spec- from the many currently published clearing techniques imens in a warm buffer and lowering the temperature [6, 7]. There is no gold standard, as every method has its would also be effective. own advantages and disadvantages. For zoologists, the Tissue clearing with advanced CUBIC protocol be- first step is to test whether the organism of interest can came very effective after the pretreatments. In the be cleared, irrespective of the extent, since virtually no cleared pill bugs, various organs were observed in situ. non-model organisms have been cleared. We believe Some structures, such as part of the male reproductive that the advanced CUBIC method  is a good choice system and pseudotrachea, were not effectively cleared. for preliminary experiments with various organisms. Although the reason for this was unclear, the RI of the First, chemicals used in the procedure are non-toxic and immersion medium might not be sufficiently high for inexpensive, and most are general reagents found in these structures. An immediate understanding of 3D many laboratories. Second, it is a relatively easy method anatomical structures in cleared whole-mount samples requiring only sequential changes in solutions. Finally, is one of the powerful and unique advantages of this clearing using this method is faster than most of the procedure. Currently, tissue clearing techniques are other aqueous-based methods. The superior clearing used in combination with fluorescent reporter proteins capacity has also been reported in several comparative and advanced microscopies. However, our results illus- studies [10, 11]. One of the disadvantages of the tech- trate that whole-mount clearing of small animals can nique is the temporary expansion of tissues during incu- provide plenty of information, even when using a basic bation in reagent-1. Since the expansion was offset stereomicroscope. during the washing and RI matching steps, this was not a problem in our experiments. The extent of expansion Staining of cleared samples might be reduced with a modified CUBIC procedure Although the internal anatomy of cleared A. vulgare (Reagent-1A protocol. http://cubic.riken.jp), where small could be observed without staining, specific staining amounts of NaCl are added to reagent-1 at a final con- methods made the technique more versatile. In zoology, centration of ≥25 mM. In general, samples undergo Konno and Okazaki Zoological Letters (2018) 4:13 Page 7 of 8 expansion during delipidation with a high concentration Funding This work was supported by a donation from Hamamatsu Photonics K.K. detergent. ScaleS , SeeDB , and FRUIT , which arereportedtohavelittleeffect on sample size,might be Availability of data and materials suitable when tissue expansion is not acceptable. For fragile The dataset supporting the conclusions of this article is included within the specimens, hydrogel-embedding using PACT [28, 29] article. might be useful; however, this approach also causes tissue Authors’ contributions expansion. Recently, another CUBIC protocol, CUBIC-L/R AK performed all experiments and drafted the manuscript. SO organized the was published . Its RI is the highest (RI = 1.52) among research and evaluated the results. Both authors reviewed and approved the all aqueous-based clearing techniques and might improve final manuscript. the final transparency of cleared samples. Ethics approval and consent to participate Not applicable. Possible applications of tissue clearing coupled with high Competing interests through-put imaging The authors declare that they have no competing interests. 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Published: Jun 6, 2018