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. 2025 Jan 20;533(1):e70017. doi: 10.1002/cne.70017

Comparing microCT Staining and Scanning Methodology for Brain Studies in Various Sizes of Spiders

Vanessa Penna‐Gonçalves 1,✉, Nikolas J Willmott 2, Michael B J Kelly 1,3, Jay R Black 4, Elizabeth C Lowe 1,5, Marie E Herberstein 1,6,7
PMCID: PMC11937621  PMID: 39833126

ABSTRACT

Recent advances in microCT are facilitating the investigation of microstructures in spiders and insects leading to an increased number of studies investigating their neuroanatomy. Although microCT is a powerful tool, its effectiveness depends on appropriate tissue preparation and scan settings, particularly for soft, non‐sclerotized tissues, such as muscles, organs, and neural tissues. As the application of microCT in spiders is only in its infancy, published protocols are often difficult to implement due to substantial size variation of the specimens. The present study was initiated to determine how to account for this variation. Our work builds on previous methods using microCT to image spider brains, with the aim to consolidate current knowledge and reduce time spent troubleshooting appropriate methodology, thereby facilitating future studies of spiders and their central nervous systems (CNS). We tested three different preparation and imaging techniques based on published protocols with minor modifications using 216 spiders with prosoma lengths ranging from 1.25 mm (small spiders) to 13.33 mm (large spiders). We compared the efficacy of the various specimen preparations, staining methods, and scan settings by categorizing the quality of dorsal and lateral microCT scans. We observed that only the phosphotungstic acid (PTA) staining agent resulted in complete staining of the prosoma and the CNS, allowing the CNS structures to be distinguished for small, medium, and large spiders. The use of image averaging, increased number of projections, image exposure timing, and detector binning did not greatly affect image quality for small and larger spiders but reduced noise. These settings did help improve image quality for medium spiders in conjunction with higher resolutions and an aluminum filter. We discussed the suitability of methods concerning spider size, effort, chemical risk, and image quality.

Keywords: brain, central nervous system, microCT, spiders, staining solution RRID:NCBITaxon_74971

This study refines microCT techniques for imaging spider neuroanatomy, focusing on size variation. The phosphatetungstic acid (PTA) staining agent was most effective for staining spider CNS structures. The research provides optimized protocols, reducing troubleshooting time, and improving image quality, thereby advancing the study of spider central nervous systems.

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1. Introduction

As the rise of microscopic anatomical studies in the 17th century (Araki 2017), new technologies have provided ever‐clearer biological images at ever‐decreasing scales. Although conventional microscopic techniques rely on visible light, applying shorter wavelength x‐rays has facilitated more powerful imaging techniques. Computed tomography (CT) scans reconstruct three‐dimensional (3D) structures based on projections of x‐rays through the sample at different rotation angles. CT scans generate clear 3D images of both external and internal structures. Importantly, tissue preparation for CT scanning is considered noninvasive and nondestructive, making it highly suitable for rare and valuable specimens (Stauber and Müller 2008; Faulwetter et al. 2013; Hall, Sherlock, and Sykes 2015; Keklikoglou et al. 2021) or fossils enclosed in sediments or other materials (Ghoshal et al. 2021; Penney et al. 2011). Depending on how tissues are prepared, biological samples may still be used for post‐scan DNA extractions, which can be important for taxonomic identification (Hall, Sherlock, and Sykes 2015; Martínez‐Sanjuán et al. 2022). Additionally, enhancements in microCT technology have produced higher resolution images of increasingly smaller structures, allowing the detection of diseases based on subtle shifts in anatomy (Ritman 2011; Smith et al. 2016). With proper implementation, microCT can be used to image musculature to explain biomechanical processes (Das et al. 2018; Stamm and Dirks 2022), explore the structure and function of respiratory systems (Franz‐Guess et al. 2016), and describe small neural structures and their connectivity in 3D space to infer neural function (Rother et al. 2021; Smith et al. 2016). Finally, rapid technological improvements have reduced financial costs and scanning time, making microCT a method of choice for many biological questions. There are other methodological frameworks for imaging biological specimens, including synchrotron facilities, that generate high resolution 3D images from a high intensity x‐ray beam with often very short scan durations, allowing a large number of samples to be processed (e.g., Larrue et al. 2011). However, access to synchrotron faculties can be limited, and thus, here we focus on microCT equipment that can be found in most universities or research facilities.

Although microCT is a potent tool, its efficacy hinges on proper tissue preparation and scan configurations, especially when dealing with soft, non‐sclerotized tissues like muscles and neural tissues. Additionally, appropriate tissue staining is necessary to ensure accurate delineation between anatomical tissues (Buytaert et al. 2014; Rivera‐Quiroz and Miller 2022). However, trade‐offs exist between different staining techniques. For example, iodine is commonly used as a stain to enhance contrast because of its short staining time and fast tissue penetration, but to achieve high contrast, iodine staining usually involves a drying process (e.g., critical point drying [CPD]) that often damages the tissue (Rivera‐Quiroz and Miller 2022; Sombke et al. 2015; Henne et al. 2017). Furthermore, iodine staining can inhibit the polymerase chain reaction (PCR), making those samples unsuitable for post‐scan molecular investigation (Hall, Sherlock, and Sykes 2015). Conversely, phosphotungstic acid (PTA) results in good contrast without the need for drying (Rivera‐Quiroz and Miller 2022) but is slow to penetrate and can dissolve calcified structures (Keklikoglou et al. 2019). In summary, choosing a tissue preparation technique is important to preserve tissue structure while maintaining sufficient contrast (Buytaert et al. 2014).

Following sample preparation, scan settings need to be optimized to improve image reconstruction and reduce noise, and as with staining, there are trade‐offs. Optimal settings depend on the properties of the sample and the desired scan qualities, requiring expert knowledge (du Plessis et al. 2017). For example, scan time is a function of multiple settings: increasing the number of rotational steps during the scan (image projections) and averaging multiple projections per rotation can reduce noise, but increasing both of these settings also increases scan time.

MicroCT has been extensively used to image the morphology of vertebrates and invertebrates including insects (e.g., Metscher 2009; Wipfler et al. 2016; Spitzner et al. 2018; Spahr et al. 2023; Moraes et al. 2023) but less so in spiders, despite several promising studies. For example, high quality microCT images of trap‐jaw spider musculature have been used to explain their remarkable jaw speed (Kallal, Elias, and Wood 2021) and to identify key morphological and anatomical traits of taxonomic importance, such as pedipalp structures (Michalik and Ramírez 2013; Lipke, Hammel, and Michalik 2015; Dederichs et al. 2019). MicroCT has also been used to describe the anatomy and function of genitalia in fossilized spiders (Penney et al. 2011; Rix et al. 2021), including in cryofixed mating pairs (Poy et al. 2023) and to map hemolymph vascular systems in spiders (Huckstorf et al. 2013).

Recently, there has been an increasing interest in using high‐resolution microCT to visualize spider brains to address a range of biological and neurological questions. For example, it has been implemented to test the effects of social and environmental complexity on neural development (Steinhoff et al. 2018), to examine trade‐offs associated with morphological ant mimicry (Kelly & Penna‐Goncalveset al. 2024) and to describe visual systems in different spider families (Stafstrom, Michalik, and Hebets 2017; Steinhoff et al. 2020). Fast and accurate volumetric analyses using microCT images could also be applied to questions like the social brain hypothesis in social spiders as it has been done in insects (O'Donnell et al. 2015) or testing the effects of pollution on neural investment (Moaraf et al. 2020; Monchanin 2021).

Imaging and studying the brain structures of spiders using microCT are in its infancy with substantial studies published only as recently as 2017 (Steinhoff et al. 2017; Stafstrom, Michalik, and Hebets 2017). The physiology and anatomy of spiders present numerous unique challenges. Like insects, imaging spider brains requires high resolution and strong contrast between tissues, as described above. However, unlike insects, which have a head capsule containing primarily the central nervous system (CNS), spiders have a prosoma (fused head and thorax, also termed cephalothorax), which also includes the muscles that control movement in the legs, pedipalps, and chelicerae. Spiders also have comparatively highly pressurized internal fluids due to their hemolymph‐based hydraulic system for movement (Hill 2018; Menda et al. 2014), which makes dissections much more technically difficult, as opening the prosoma causes pressure shifts that can reposition internal structures. Additionally, spider nervous systems are intertwined with other structures like muscles and the digestive tract, potentially making neural structures harder to delineate (Long 2021; Lin, Lopardo, and Uhl 2021; Rivera‐Quiroz and Miller 2022; Steinhoff et al. 2023). Challenges also arise when studying spiders of different sizes, because the methods required for effective staining of the entire prosoma vary with prosoma size. This can be an issue when comparing species of disparate sizes, but also when comparing males and females of different sizes in sexually dimorphic spider species. Here, we combine the experience we accumulated via three separate projects that aimed to visualize spider CNS, where we trialed several techniques and scan settings for optimal image quality. We summarize our combined efforts to provide best practice guidelines for tissue preparation and scanning of spider brains of a range of sizes. By building on previous method explorations (Long 2021; Rivera‐Quiroz and Miller 2022; Steinhoff et al. 2023), our study aims to inform researchers and reduce the time spent troubleshooting appropriate methodology, thereby facilitating efficient future microCT studies of spider brains.

2. Methods

There is currently no consolidated protocol for imaging the brains of spiders from various sizes. We tested several staining and scanning techniques for the spider CNS to develop standard methods that can be used to investigate a range of research questions. Answering research questions relating to differences in spider brain structure across different species, or large intraspecific size differences, requires microCT scans that are suitable for segmentation that delimitates the structures and allows the calculation of volumes. In this article, we are only concerned with the quality of the microCT scans and not the processes of segmentation and volume calculation, which are described in detail elsewhere (Yushkevich et al. 2006; Kelly & Penna‐Goncalves et al. 2024).

2.1. Spiders

In this study, we sampled spiders from four different families (Araneidae, Salticidae, Sparassidae, and Thomisidae, Spiders: RRID:NCBITaxon_74971), representing a considerable range in size from 1.25 to 12.02 mm in total prosoma length. We categorized the spiders as small (1.25–3.09 mm), medium (6.5–8.13 mm), and large (8.34–13.33 mm). Mature and subadult females of small spiders—Xysticus bimaculatus, Tharrhalea evanida, Myrmarachne luctuosa, Myrmarachne smaragdina, Astia hariola, and Helpis sp.; medium spiders—Hortophora biapicata; and large spiders—Delena cancerides, Isopeda villosa, and Heteropoda jugulans were collected on public land of Victoria, New South Wales, and Queensland in Australia (see Table S1 for details). These spiders were used in several studies testing how overall CNS volume and the volume of structures within (mushroom body, arcuate body, visual neuropils, and the ventral nerve cord) varied with extreme morphological phenotypes (Kelly & Penna‐Goncalves et al. 2024), sociality (Penna‐Gonçalves et al., unpublished data), and exposure to artificial light at night (Willmott et al. 2024).

All collected spiders were taken to the laboratory at Macquarie University in Sydney (except H. biapicata which were taken to a laboratory at the University of Melbourne), and kept in individual plastic vials, or upturned plastic cups, of varying sizes (4–9 cm in diameter and 6.5–13 cm in height), depending on the species. Vials and cups contained a moist cotton bud allowing access to water ad libitum and a small piece of smooth bark to provide a substrate for the spiders. Additionally, the cups were sprayed with water three times a week to provide suitable humidity. Spiders kept for more than a week were fed vinegar flies (Drosophila melanogaster) or crickets (Acheta domesticus) at regular intervals. Room temperature was 25°C with 60%–80% humidity, and a 12 h light: 12 h dark lighting cycle.

2.2. Specimen Preparation

Spiders were kept alive in the laboratory from 1 to 10 weeks because some individuals were immature at the time of collection, and some were used for laboratory experiments. Live individuals were weighed using a Mettler Toledo Analytical Balance (Model: ML204T/00) to the nearest 0.001 g. Afterward, spiders were anesthetized in a −4°C (−20−C for H. biapicata) freezer (3–15 min, depending on size). The anesthetized spiders were imaged with a Cannon EOS 80D camera mounted to a Motic SMZ‐171 microscope. From the generated image, we measured the prosoma length in millimeters using ImageJ v1.51 software (Schneider, Rasband, and Eliceiri 2012; https://imagej.nih.gov/ij/). The chelicerae were excluded from the prosoma length measurements.

2.3. Dissection, Fixation, and Staining

We removed the legs (at the coxa or mid‐coxa), pedipalps, chelicerae, and abdomen (opisthosoma) to facilitate stain penetration. For a few samples, we additionally removed the dorsal (carapace) or ventral section (sternum) of the prosoma (see Table S2). We performed three different techniques based on published protocols, but with minor modifications (Figure 1). After euthanizing spiders, they were kept in fixation and/or staining solution for 2 to 164 days because the methodology required weeks or months of staining. The modifications we implemented are described below; however, they do not include the steps from the original protocols that remained unchanged:

FIGURE 1.

FIGURE 1

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Specimen preparation for microCT scans including spider dissection, fixation, ethanol dehydration series, staining, ethanol washes, drying process, and scan medium (created with Biorender.com); (a) mounted specimen in liquid medium inside the MicroCT. CPD, critical point drying; PTA, phosphotungstic acid.

(1) Bouin‐iodine‐critical point drying: We replicated the method published by Steinhoff et al. (2020) using Bouin solution as the fixative (Sigma‐Aldrich, Missouri, USA) instead of Dubosq–Brazil solution (following the recommendation from Philip Steinhoff). Samples were dissected submerged in the fixative solution, fixed for 4 days, then followed by phosphate‐buffered saline (PBS, pH 7.4) washes, an ethanol dehydration series (80, 90, 96, and 3 × 99.8% ethanol), and staining in 1% iodine solution (iodine, resublimated in 99.8% ethanol; Sigma‐Aldrich, Missouri, USA) for 48 h. Samples were then subjected to 2 h of CPD (Leica EM CPD300) in slow CO2 admittance with a delay of 120 s, 30 exchange cycles, slow heating process, and slow gas discharge (following Steinhoff et al. 2020).

(2) 4% Paraformaldehyde–iodine‐CPD: Instead of Bouin solution, we dissected the samples submerged in the fixative solution, then fixed the sample using 4% paraformaldehyde (more details in Steinhoff et al. 2017) for 4 days, followed by ethanol dehydration series, staining in 1% iodine (iodine, resublimated in 99.8% ethanol; Sigma‐Aldrich, Missouri, USA) solution for 48 h, and then subjected to 2 h of CPD (Leica EM CPD300) in slow CO2 admittance with a delay of 120 s, 30 exchange cycles, slow heating process, and slow gas discharge (following Steinhoff et al. 2020).

(3) 70% ethanol‐1% PTA: Here, we follow the method established by Rivera‐Quiroz and Miller (2022) using 70% ethanol as fixative, whereas spiders were dissected. The sample was then dehydrated in an ethanol graded series and finally stained with 1% PTA‐70% ethanol. Some samples were kept in a medium vacuum gas chamber to optimize the time for the staining between 9 and 164 days, whereas other samples were not submitted to the vacuum system (Table S2). During this process, fresh PTA solution was replaced weekly. After this period, some samples were washed in 96% ethanol and placed in a fresh tube containing 96% ethanol, whereas other samples did not receive ethanol washes and were stored in fresh PTA medium until microCT scanning was performed (modification from the original Rivera‐Quiroz and Miller (2022) protocol). Methods 1 and 2 required 4 days for fixation and 2 days for staining. In Method 3, the fixation duration varied from 2 to 5 min depending on the spider size, and the staining duration varied between 9 and 164 days.

2.4. MicroCT Setting and Scanning

MicroCT scanning was performed at the University of Melbourne with a Phoenix Nanotom M (Waygate Technologies, Germany) micro‐/nano‐CT scanner, operated using xs control and phoenix datos|x acquisition software (Baker Hughes Digital Solutions). Parameters varied in scan time, tube voltage (kV) and current (mA), resolution (µm), and the use of image averaging at each projection, depending on the size of the specimen (see Table S2).

2.5. Method Comparison

We compared the efficacy of the various specimen preparations, staining methods, and scan settings by examining the resolution of the dorsal and lateral views of the regions of interest. We used the following (non‐mutually exclusive) categories to describe the quality of microCT scans: (1) complete staining of prosoma (Table 1: Prosoma +): when the entire prosoma was fully stained; (2) incomplete staining or shrinkage of prosoma (Table 1: Prosoma−): when some areas of the prosoma were unresolved and black; (3) complete staining of CNS (Table 1: CNS +): when the entire CNS was fully stained; (4) incomplete staining or shrinkage of the CNS (Table 1: CNS −): when some areas of the CNS were black; (5) all CNS structures distinguishable and the edges between the structures were traceable (Table 1: CNS Structures +); (6) most or some CNS structures indistinguishable and the edges of the structures could not be traced (Table 1: CNS Structures −); (7) total image blurry (Table 1: yes), when the entire image was not clear. Unresolved black areas in the image may have been due to a lack of staining or tissue shrinkage, but we were not able to determine the exact cause.

TABLE 1.

Summary of staining method, species, size, dissection method, staining and scan duration, scan settings, image outcomes and image reference.

Fixative/Staining method Family Species Id number Spider size Staining duration (days) Scan medium Scan duration (min) Image averaging Exposure (ms) Resolution (µm) Detector binning Number of images Filter Prosoma CNS CNS Structures Blurry Figure reference
Bouin‐iodine‐CPD Salticidae Maratus (Hypoblemum) griseus griseus 74 Small 2 Air 10 1 500 2.7 2 × 2 1199 None — — — Yes S1B
Astia hariola 60 Small 2 Air 114 1 500 3.4 2 × 2 1199 None — + + Yes S1D
Myrmarachne smaragdina 90 Small 2 Air 114 2 2000 3.4 2 × 2 1600 None — + + No 2A
Sparassidae Heteropoda jugulans 47 Large 2 Air 10 1 2000 9.6 2 × 2 1600 None — — — Yes S1W
Delena cancerides 111 Large 2 Air 114 2 2000 9.6 2 × 2 1600 None — + — Yes 4C
4% Paraformaldehyde‐iodine‐CPD Salticidae Helpis sp. 7 Small 2 Air 10 1 500 2.6 None 1199 None — — — Yes 2B
Myrmarachne luctuosa 30 Small 2 Air 126 3 500 3.7 2 × 2 1199 None — — — No S1G
70% Ethanol‐1%PTA Salticidae Helpis sp. 117 Small 9 Ethanol 10 1 500 2.7 none 1199 None + + + Yes S1H
Thomisidae Xysticus bimaculatus 196 Small 22 PTA 10 1 500 4.0 2 × 2 1199 In Al tube + + + Yes 5A
Tharrhalea evanida 267 Small 46 PTA 10 2 2000 2.9 2 × 2 1800 In Al tube + + + No S1I
Araneidae Hortophora biapicata 1D14 Medium 59 Ethanol 10 1 500 2.9 None 1199 0.5 mm Al + + — Yes 5C
Hortophora biapicata 1D14 Medium 59 Ethanol 131 2 2000 2.9 2 × 2 1800 0.5 mm Al + + + No 5D
Hortophora biapicata 1L21 Medium 70 Ethanol 128 2 2000 9.0 None 1800 0.5 mm Al + + — Yes 3B, S1J
Hortophora biapicata 1L21 Medium 70 Ethanol 128 2 2000 9.0 None 1800 0.5 mm Al + + — No 3A
Hortophora biapicata 1D14_S04 Medium 59 Ethanol 30 1 2000 2.9 None 750 0.5 mm Al + + + No 3C
Sparassidae Isopeda villosa 144 Large 64 PTA 61 2 1000 10.0 2 × 2 1600 0.5 mm Al + + + No 6F
Isopeda villosa 114 Large 75 Ethanol 10 1 500 6.0 None 1199 None + + + No S1S
Delena cancerides 235 Large 91 PTA 10 1 500 6.0 None 1199 None + + + No S1O
Isopeda villosa 238 Large 92 PTA 10 1 500 8.0 None 1199 None — + + No S1R
Isopeda villosa 172 Large 37 Ethanol 10 1 500 6.0 None 1199 None + + + No S1P
Isopeda villosa 123 Large 12 Ethanol 10 1 500 7.0 None 1199 None — + + No 5C
Delena cancerides 282 Large 56 PTA 10 1 500 6.0 None 1199 None n/a + + No 5B
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Notes: The examples listed are a subset (n = 22) of samples from Table S2 (n = 35) chosen to represent the diversity in the staining method and scan methods applied. For the full range of specimen and methods, please see Table S2. Outcome: Prosoma/CNS (complete (+), incomplete (−)); structures CNS (all distinguished (+), most undistinguished (−)). Small size (1.25–3.09 mm), medium (6.5–8.13 mm) and larger (8.34–13.33 mm). Scan medium: refer to the solution in which the samples were scanned (air/ethanol/PTA).

Abbreviations: CNS, central nervous systems; PTA, phosphotungstic acid.

Due to the limited sample sizes for some species, statistical analyses were not feasible. Rather, we discuss the suitability of methods with respect to spider size, effort, chemical risk, and image quality. When developing staining and scanning techniques, we contacted the authors of previous spider neuroanatomy publications, and they provided very helpful additional details (see Acknowledgements).

3. Results

We processed over 200 individual spiders, representing 11 species from 4 families. We tested three different fixation and staining methods (Bouin‐iodine, paraformaldehyde–iodine, and ethanol‐PTA), different fixation and staining durations, and several microCT scan settings (see Table 1 and Table S2). In general, we observed that only the ethanol‐PTA method resulted in complete staining of the prosoma and the CNS and allowed the CNS structures to be distinguished with a high enough contrast for small, medium, and large spiders (Table 1).

3.1. Fixation and Staining Methods

The fixation durations for Bouin and paraformaldehyde were kept constant for 4 days, and the staining in iodine was also constant for 2 days for all samples. Thus, we cannot comment whether the duration was suboptimal. However, for the ethanol‐PTA method (which combines fixation and staining), we varied the duration.

For the smaller sized spiders (e.g., X. bimaculatus, Helpis sp. and Myrmarachne spp.), the Bouin‐iodine‐CPD (Figure 2A) and the paraformaldehyde–iodine‐CPD (Figure 2B) methods produced incomplete staining of the prosoma and many of the samples also had incomplete staining of the CNS with shrinkage of tissue and blurry images (Table 1; Figure S1A–G). The ethanol‐PTA method with a staining duration of just 9 days produced images with the prosoma and CNS fully stained (Table 1; Figure S1H). But for other samples, it took longer staining periods (e.g., 22–46 days, Table 1; Figure 2C; Figure S1I).

FIGURE 2.

FIGURE 2

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Small spiders: microCT scans of total prosoma sagittal view, with a zoomed‐in CNS and prosoma dorsal view: (A) Myrmarachne smaragdina; (B) Helpis sp.; (C) Xysticus bimaculatus. Scale bars: 1 mm. CPD, critical point drying; PTA, phosphotungstic acid.

For medium sized spiders such as H. biapicata (prosoma length: 6–9 mm), the vacuum‐assisted PTA staining resulted in effective penetration of the entire prosoma (Figure 3A–C; Figure S1J–M); other methods were not performed. Complete staining of the prosoma took between 40 and 70 days. Again, there was no relationship between staining duration and scan quality, apart from longer staining periods required to stain larger prosoma completely.

FIGURE 3.

FIGURE 3

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Medium spiders: microCT scans of Hortophora biapicata, showing the effects of scan duration on scan quality. (A) Scan focused on whole prosoma, (B) Scan focused on whole prosoma with zoomed‐in CNS and (C) Scan focused on the brain. A 0.5 mm aluminium filter has been applied to all imaging. Views, from left to right, are sagittal view of the CNS; dorsal view at the arcuate body plane; dorsal view at the esophagus plane; and dorsal view at the VNC plane. Scale bar: 1 mm. PTA, phosphotungstic acid.

Finally, for the largest spiders, only the ethanol‐PTA method resulted in complete staining of the prosoma and CNS and produced distinguishable images of CNS structures (Figure 4A). The staining duration varied from 10 to 164 days (Table 1; Table S2; Figure S1N–T). For some specimens, relatively short staining durations (37 days) were successful (Table 1; Figure S1P), but not sufficient for others of similar size (Table 1; Figure S1Q). For a few samples (Figure S1R), the full prosoma was not stained at all. Vacuum assisted ethanol‐PTA staining did not seem to speed up the staining duration (Figure S1S). The Bouin‐iodine‐CPD method was mostly unsuccessful, resulting in incomplete staining of the prosoma, CNS, and indistinguishable CNS structures (Table 1; Figure 4B).

FIGURE 4.

FIGURE 4

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Large spiders: microCT scans of total prosoma sagittal sections, with zoomed‐in CNS and prosoma dorsal view: (A) Isopeda villosa, ethanol‐PTA; (B) Delena cancerides treated with Bouin‐iodine‐CPD. Scale bar: 1 mm. CPD, critical point drying; PTA, phosphotungstic acid.

3.2. Specimen Dissection

Removing the appendages and the abdomen did not damage or distort the CNS tissue. The additional removal of the carapace or sternum for larger specimens in both Bouin‐iodine‐CPD and ethanol‐PTA method did not accelerate or improve the staining (Table 1; Figure 5A–C). The removal of the carapace, however, meant that this structure could no longer be visualized, and carried the risk of damaging the internal organs.

FIGURE 5.

FIGURE 5

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Large spiders: comparison among spiders with dissection of the carapace or sternum. (A) Delena cancerides treated with Bouin‐iodine‐CPD with the sternum removed, (B) D. cancerides treated with ethanol‐PTA with the carapace removed, and (C) Isopeda villosa treated with ethanol‐PTA with the sternum removed. Scale bar: 1 mm. CPD, critical point drying; PTA, phosphotungstic acid.

3.3. Scan Quality

MicroCT image quality was impacted by specimen size and thus the optimized resolution of scans that could be achieved. For the smaller spiders, such as the jumping spider Helpis sp. and the crab spider X. bimaculatus, we were able to distinguish all relevant neuropils in scans at a resolution of 4 μ (with 2 × 2 detector binning) (Table 1; Figure 6A,B).

FIGURE 6.

FIGURE 6

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Comparison of scan quality for short (left side) scans and long scans (right side) of small spiders Xysticus bimaculatus 10‐min scan (A) and 61‐min scan in aluminium tube (B), medium spiders Hortophora biapicata 10‐min scan (C) and 131‐min scan in 0.5 mm aluminium filter (D) and large spiders E: Isopeda villosa 10‐min scan (E) and 61‐min scan in 0.5 mm aluminium filter (F). All specimens were stained with ethanol‐PTA. Scale bar: 0.5 mm.

For medium specimens, such as H. biapicata, short scans (10 min using 1199 projections, 500 ms exposure time, and no image averaging) were collected at coarser resolutions, which were useful for assessing stain penetration and allowed assessment of larger structures such as the ventral nerve cord. At these coarser resolutions, smaller structures were more difficult to distinguish. For instance, for H. biapicata, we could not delineate the mushroom bodies from the surrounding midbrain tissue. Other visual neuropils and the arcuate body, while distinguishable, had blurry, poorly defined edges (Figure 6C). Longer scans (∼130 min using 1800 projections, 2000 ms exposure time, 2× image averaging, and 2 × 2 detector binning) run at higher magnifications focused ROI (region of interest), and thus resolution provided clearer reconstructions, reducing noise in images and providing clearer contrast at the edges of structures (Figures 3A–C and 6D). When used for H. biapicata, the higher resolution scans allowed mushroom bodies to be distinguished, and neuropils in general had more clearly defined edges, providing more precise volume calculations (Willmott et al. 2024). We also observed that instability of the specimen during the scan (slight movement observed between initial projection and final projection after 360‐degree rotation) resulted in blurry reconstructions (Figure S1J), which were not present when the specimen was stable throughout the scan (Figure 3A).

Surprisingly, for the largest specimens (D. cancerides, I. villosa and H. jugulans), a resolution of 6–10 µm also produced high quality (but sometimes blurry) images, from which we were able to delineate small structures such as the visual neuropils, mushroom bodies, and arcuate body (Figure 6E). However, running with a 2 × 2 detector binning at a slightly coarser resolution did not drastically improve detectable detail in images, but it did reduce noise (Figure 6F).

In general, higher resolution scans helped to distinguish smaller structures (Figure 6C,D), particularly for H. biapicata visual neuropils. Similarly, performing more image acquisitions per rotation reduced noise in the final scan, enhancing detectable detail. Longer scans were run with 2× image averaging per rotational step as well as a longer exposure time (1000–2000 ms compared with 500 ms for 10 min scans) and an increased number of projections (1600–1800 vs. 1199 for 10‐min scans). These parameters increase scan time and should be used to reduce noise as needed.

4. Discussion

With advancements in microCT enabling the investigation of microstructures in spiders and insects, the number of studies has been increasing—for example, from January 1, 2008, until August 20, 2024, the Web of Science (accessed: August 20, 2024) lists 248 studies for insects and 59 for spiders (search terms: (1) insect AND “microCT” or “micro‐CT” or “microcomputed tomography” (2) “spider” AND “microCT” or “micro‐cCT” or “microcomputed tomography”). For neuro‐anatomy investigations, microCT technology has improved the visualization not only of the entire CNS but also the small neuropils (e.g., Stafstrom, Michalik, and Hebets 2017; Steinhoff et al. 2020; Rivera‐Quiroz and Miller 2021, 2022).

The spider brain is surrounded by muscles, venom glands, and the digestive system (Foelix 2011), so it cannot easily be extracted without damage. Hence, less invasive methods such as microCT scans provide a valuable alternative approach but nevertheless requires careful selection of methodology. Although Rivera‐Quiroz and Miller (2021, 2022) also published an assessment of microCT methods in spiders, our study here focuses on the impact of specimen size on staining and scanning quality. Thus, our work will guide researchers to the most appropriate methods for a range of specimen sizes. For example, studies interested in capturing neurodevelopment should implement a single staining and scanning setting that is suitable for small spiderlings as well as larger adults (Stafstrom, Michalik, and Hebets 2017). Prior to staining, spiders need to be dissected to optimize subsequent stain penetration. To enhance the penetration of a staining solution, we concur with previous studies (Steinhoff et al. 2020; Rivera‐Quiroz and Miller 2021, 2022) and recommend the removal of the appendages. In addition, we tested whether the removal of the sternum and carapace would increase the absorption of the stain, but we did not observe any improvement in staining time or the quality of image. As this dissection technique can also cause some damage to the CNS and prevents usable imaging of muscles and prosoma structures, we do not recommend this approach.

In relation to the preparation of the dissected material, regardless of the stain solution used, we found that CPD caused tissue shrinkage and was not suitable for use in spiders when aiming to investigate small neuropils. In other studies, CPD also shrank soft tissue (e.g., Spitzner et al. 2018), causing a lack of resolution, making it challenging to assess neuroanatomical structures in detail (Henne et al. 2017). It also impacted the position, morphology, and volume of internal structures such as the CNS (Rivera‐Quiroz and Miller 2022) or the microstructure of sensory organs (Sombke et al. 2015). In some studies, CPD resulted in a supraesophageal ganglion volume reduction of between 35% and 70% (Rivera‐Quiroz and Miller 2022).

In some published protocols (e.g., Steinhoff et al. 2020), iodine staining (48 h) was successful for the CNS but less reliable for staining the entire prosoma interior. In our study, iodine staining, irrespective of staining duration, was problematic for visualizing the full prosoma, although it stained the CNS of smaller spiders sufficiently. A helpful extension of our study would be to vary the duration of iodine staining; however, Rivera‐Quiroz and Miller (2022) did reduce the staining duration to only 24 h and found that iodine, especially in combination with CPD, was not the best choice for staining in spiders.

The only method we found to result in complete staining of the prosoma and CNS independent of specimen size was the ethanol‐PTA method. However, this stain varied in staining duration, where some small spiders only took 9 days, but other similar sized species took between 22 and 46 days. It has been suggested that the staining duration could be reduced by applying a vacuum system that removes the air from the sample container and accelerates the stain penetration through the tissue (Rivera‐Quiroz and Miller 2022). However, we did not explore this method with multiple samples and further exploration of the strength of the vacuum and the frequency and duration of its application, whereas avoiding tissue damage is warranted, especially for larger specimens.

The intraspecific variation in how long it took for the ethanol‐PTA method of staining to completely stain the carapace and CNS is intriguing, considering spiders of similar size required different durations for full staining. Even the iodine (Bouin solution‐fixative) was quite variable, with the CNS of some specimens fully stained, whereas others were incomplete despite the same staining duration. Observed variation could be due to age of organism, sex (Li et al. 2015; Swart et al. 2016; Chen et al. 2018), or we argue in the case of spiders, it could also be their physiology, such as the state of the viscosity of the hemolymph‐based hydraulic system at the time of fixation, which may affect how the staining solution is absorbed. Although there are limited empirical tests, Göttler et al. (2021) argue that in jumping spiders the viscosity of the hemolymph varies due to changes in internal pressure and temperature, and this in turn may affect how the stain disperses through the prosoma.

Scan resolution was an important factor for medium sized spiders, resulting in higher‐quality images with improved detectable detail in combination with extra image averaging (2× averaging), extra projections (1600–1800 compared to 1199), and an increased exposure time (2000 compared to 500 ms). These settings led to a noticeable improvement in scan quality in H. biapicata (Figures 3 and 5), where scans were conducted on a focussed ROI within specimens (i.e., full specimen not visible within the field of view of the detector). Interestingly, we did not observe significant improvement in detectable detail for smaller and larger spiders when scanned using similar settings with an intermediate exposure time (1000 ms). As we did not test the higher magnification focused ROI scans with the smaller and larger spiders, we cannot make firm conclusions but recommend the exploration of higher resolution scan settings for future studies.

Image averaging combined with longer exposure times and detector binning improved scan quality but increased scan duration, with other studies having implemented scans of up to 9 h (Rivera‐Quiroz and Miller 2022). However, considerable costs associated with scan duration can be limiting. We found that scans using a limited number of projections (1199), a short exposure time (500 ms), and no image averaging can be sufficient to assess whether the stain has penetrated appropriately or whether shrinkage has occurred. Furthermore, these scans can also provide sufficient information for volumetric estimation of CNS structures using segmentation software (e.g., ITK‐SNAP v3.8.0 (www.itksnap.org); Yushkevich et al. 2006), as long as boundaries between structures are clearly defined. Finally, it is important to consider risk factors associated with some methods, such as the carcinogenic nature of formaldehyde present in both Bouin's solution and paraformaldehyde. On the basis of our risk assessment of these toxins, using the producer's safety datasheet, we decided to severely limit our use of this method. Concerns also exist for other methods not discussed here, such as using osmium tetroxide as a stain, which is extremely dangerous to organisms, including humans (Ribi et al. 2008).

In summary, we identified preferred methodologies for each stage of the sample preparation and microCT scanning process including dissection, fixation, staining, scanning, and filter which can optimize the final scan quality for the CNS of spiders.

We recommend (Table 2) dissecting the appendages at the coxa to increase penetration of the stain but do not recommend opening the carapace or sternum. Further, we recommend PTA staining as a safe and effective stain. The use of a vacuum system is optional and may reduce staining duration (see also Rivera‐Quiroz and Miller 2022). Scan settings need to be optimized for purpose, with coarser resolution scans using a limited number of projections (1199), no image averaging, and a short exposure timing (500 ms) enough to evaluate how well a specimen has been stained and segment CNS structure volumes in some cases. Where boundaries between CNS structures cannot be resolved, it is recommended that higher resolution scans are run with increased exposure timing (2000 ms), some image averaging and detector binning to help reduce noise.

TABLE 2.

Summary of method recommendations for visualizing spider CNS and structures within.

Spider size Dissection Fixation and staining Staining duration (average) Scanning time (min) (minimun) Image averaging Exposure time (ms) (mode) Resolution (µm) (mode) Filter
Small Legs at coxa 70% Ethanol‐1%PTA 24 10 1 500 4 AL tube
Medium Legs at coxa 70% Ethanol‐1%PTA 59 30 2 2000 2.9 0.5 mm AL
Large Legs at coxa 70% Ethanol‐1%PTA 60 10 1 500 6 0.5 mm AL or none
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Abbreviations: CNS, central nervous systems; PTA, phosphotungstic acid.

The present study was initiated because of difficulties encountered with reproducing the protocols of previous studies. This is likely due to a lack of detail in the published protocols and due to the size variation of the spiders we used for our studies. We hope that providing guidance on sample preparation and scanning here will reduce the time spent troubleshooting in future studies.

Author Contributions

Vanessa Penna‐Gonçalves and Nikolas J. Willmott conceived the study and designed the methodology; Vanessa Penna‐Gonçalves, Michael B. J. Kelly, Nikolas J. Willmott and Jay R. Black collected the data; Vanessa Penna‐Gonçalves and Nikolas J. Willmott analyzed the data; Vanessa Penna‐Gonçalves and Marie E. Herberstein led the writing of the manuscript and submission. Lizzy Lowe contributed to the manuscript. Lizzy Lowe, Nikolas J. Willmott and Marie E. Herberstein funded the study. All authors contributed critically to the drafts and gave final approval for publication.

Peer Review

The peer review history for this article is available at https://publons.com/publon/10.1002/cne.70017.

Supporting information

Figure S1 Comparison among Bouin‐iodine, paraformaldehyde–iodine and ethanol‐PTA fixative and staining protocols. Prosoma sagittal, CNS zoomed‐in and dorsal slices of A: Helpis sp., B: Maratus (Hypoblemum) griseus, C: M. griseus, D: Astia hariola, E: Myrmarachne smaragdina, F: Myrmarachne luctuosa, G: M. luctuosa, H: Helpis sp., I: Tharrhalea evanida, J: Hortophora biapicata unstable specimen, K: H. biapicata stable, L: H. biapicata, M: H. biapicata, N: : Delena cancerides, O: Heteropoda jugulans, P: Isopeda villosa, Q: I. villosa, R: I. villosa, S: I. villosa, no vaccum system, T: I. villosa, U: D. cancerides. Note that magnified scans (K, L, M) do not have total prosoma sagittal views. Scale bar: 1 mm.

CNE-533-e70017-s001.pdf (745.1KB, pdf)

Table S1 Location where spiders were collected in Australia, including species names, GPS coordinates, collection dates and maturation stage.

CNE-533-e70017-s002.xlsx (18.1KB, xlsx)

Table S2 Additional information about the specimens, staining methodology, and scan settings.

CNE-533-e70017-s003.xlsx (24.3KB, xlsx)

Acknowledgments

We acknowledge the traditional custodians of the Macquarie University (RRID:SCR_017596) land, the Wattamattagal clan of the Darug nation where the fieldwork and studies were conducted. We also acknowledge the traditional custodians of the Melbourne city land, the Wurundjeri Woi‐wurrung and Bunurong / Boon Wurrung peoples of the Kulin nation, the traditional custodians of the Brisbane, the Jagera people and the Turrbal people as the Traditional Custodians of Meanjin and the traditional custodians of the Townsville land, the Wulgurukaba of Gurambilbarra country and the Bindal of Thul Garrie Waja country where the fieldworks for this study were conducted. Thanks to the University of Melbourne (RRID:SCR_000999) TrACEES (Trace Analysis for Chemical, Earth and Environmental Sciences) Platform for access to the microCT scanner. Thanks to Sue Handley and Arthur Chien for the support and guidance in preparing samples for microCT scanning. Thanks to Philip Steinhoff and F. Andres Rivera Quiroz for their help in providing further information about their protocols. Thanks to Louis O'Neill, Jess Herbert, Jim McLean, Marilia Erickson, Carson Kerins, Hayden Eyles, Caitlin Selleck, Stephen Tobin, and Laura Spooner for help collecting spiders. Thanks to Macquarie University for providing scholarships for the first author of this paper. This work was supported by research grants from the Association for the Study of Animal Behaviour, Holsworth Wildlife Research Endowment and Environmental Microbiology Research Initiative.

Funding: Association for the Study of Animal Behaviour Holsworth Wildlife Research Endowment Environmental Microbiology Research Initiative.

Data Availability Statement

The data that support the findings of this study are available in the supporting information of this article.

References

  1. Araki, T. 2017. “The History of Optical Microscope.” Mechanical Engineering Reviews 4, no. 1: 1–8. 10.1299/mer.16-00242. [DOI] [Google Scholar]
  2. Buytaert, J. , Goyens J., De Greef D., Aerts P., and Dirckx J.. 2014. “Volume Shrinkage of Bone, Brain and Muscle Tissue in Sample Preparation for Micro‐CT and Light Sheet Fluorescence Microscopy (LSFM).” Microscopy and Microanalysis 20, no. 4: 1208–1217. 10.1017/S1431927614001329. [DOI] [PubMed] [Google Scholar]
  3. Chen, K. C. , Arad A., Song Z. M., and Croaker D.. 2018. “High‐Definition Neural Visualization of Rodent Brain Using Micro‐CT Scanning and Non‐Local‐Means Processing.” BMC Medical Imaging 18: 1–13. 10.1186/s12880-018-0280-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Das, R. , Yadav R. N., Sihota P., Uniyal P., Kumar N., and Bhushan B.. 2018. “Biomechanical Evaluation of Wasp and Honeybee Stingers.” Scientific Reports 8, no. 1: 1–13. 10.1038/s41598-018-33386-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dederichs, T. M. , Müller C. H., Sentenská L., Lipke E., Uhl G., and Michalik P.. 2019. “The Innervation of the Male Copulatory Organ of Spiders (Araneae)—A Comparative Analysis.” Frontiers in Zoology 16: 1–14. 10.1186/s12983-019-0337-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. du Plessis, A. , Broeckhoven C., Guelpa A., and le Roux S. G.. 2017. “Laboratory X‐Ray Micro‐Computed Tomography: A User Guideline for Biological Samples.” GigaScience 6, no. 6: 1–11. 10.1093/gigascience/gix027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Faulwetter, S. , Dailianis T., Vasileiadou A., and Arvanitidis C.. 2013. “Contrast Enhancing Techniques for the Application of Micro‐CT in Marine Biodiversity Studies.” Microscopy and Analysis 27, no. 2: S4–S7. [Google Scholar]
  8. Foelix, R. F. 2011. Biology of Spiders. Oxford: Oxford University Press. [Google Scholar]
  9. Franz‐Guess, S. , Klußmann‐Fricke B. J., Wirkner C. S., Prendini L., and Starck J. M.. 2016. “Morphology of the Tracheal System of Camel Spiders (Chelicerata: Solifugae) Based on Micro‐CT and 3D‐Reconstruction in Exemplar Species From Three Families.” Arthropod Structure and Development 45, no. 5: 440–451. 10.1016/j.asd.2016.08.004. [DOI] [PubMed] [Google Scholar]
  10. Ghoshal, S. , Bhowmick P., Chakrabarti A., Sur‐Kolay S., Chakravorti S., and Sengupta D.. 2021. “3D Reconstruction From Micro‐CT Slices for Non‐Destructive Viewing inside a Fossil.” Paper presented at International Conference Image and Vision Computing New Zealand, Tauranga, New Zealand, December 09–10, 2021. 10.1109/IVCNZ54163.2021.9653270. [DOI]
  11. Göttler, C. , Amador G., van de Kamp T., et al. 2021. “Fluid Mechanics and Rheology of the Jumping Spider Body Fluid.” Soft Matter 17, no. 22: 5532–5539. 10.1039/D1SM00338K. [DOI] [PubMed] [Google Scholar]
  12. Hall, A. C. , Sherlock E., and Sykes D.. 2015. “Does Micro‐CT Scanning Damage DNA in Museum Specimens?” Journal of Natural Science Collections 2: 22–28. [Google Scholar]
  13. Henne, S. , Friedrich F., Hammel J. U., Sombke A., and Schmidt‐Rhaesa A.. 2017. “Reconstructing the Anterior Part of the Nervous System of Gordius aquaticus (Nematomorpha, Cycloneuralia) by a Multimethodological Approach.” Journal of Morphology 278, no. 1: 106–118. 10.1002/jmor.20623. [DOI] [PubMed] [Google Scholar]
  14. Hill, D. E. 2018. “The Jumping Behavior of Jumping Spiders: A Review (Araneae: Salticidae).” Peckhamia 8526: 45–72. [Google Scholar]
  15. Huckstorf, K. , Kosok G., Seyfarth E. A., and Wirkner C. S.. 2013. “The Hemolymph Vascular System in Cupiennius salei (Araneae: Ctenidae).” Zoologischer Anzeiger 252, no. 1: 76–87. 10.1016/j.jcz.2012.03.004. [DOI] [Google Scholar]
  16. Kallal, R. J. , Elias D. O., and Wood H. M.. 2021. “Not So Fast: Strike Kinematics of the Araneoid Trap‐Jaw Spider Pararchaea alba (Malkaridae: Pararchaeinae).” Integrative Organismal Biology 3, no. 1: obab027. 10.1093/iob/obab027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Keklikoglou, K. , Arvanitidis C., Chatzigeorgiou G., et al. 2021. “Micro‐CT for Biological and Biomedical Studies: A Comparison of Imaging Techniques.” Journal of Imaging 7, no. 9: 172. 10.3390/jimaging7090172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Keklikoglou, K. , Faulwetter S., Chatzinikolaou E., et al. 2019. “Micro‐Computed Tomography for Natural History Specimens: A Handbook of Best Practice Protocols.” European Journal of Taxonomy 522, no. 522: 1–55. 10.5852/ejt.2019.522. [DOI] [Google Scholar]
  19. Kelly, M. B. , Penna‐Goncalves V., Willmott N. J., et al. 2024. “Small Brains: Body Shape Constrains Tissue Allocation to the central Nervous System in Ant‐Mimicking Spiders.” Journal of Comparative Neurology 532, no. 11: e25680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Larrue, A. , Rattner A., Peter Z. A., et al. 2011. “Synchrotron Radiation Micro‐CT at the Micrometer Scale for the Analysis of the Three‐Dimensional Morphology of Microcracks in Human Trabecular Bone.” PLoS ONE 6, no. 7: e21297. 10.1371/journal.pone.0021297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li, Z. , Clarke J. A., Ketcham R. A., Colbert M. W., and Yan F.. 2015. “An Investigation of the Efficacy and Mechanism of Contrast‐Enhanced X‐Ray Computed Tomography Utilizing Iodine for Large Specimens Through Experimental and Simulation Approaches.” BMC Physiology 15: 1–16. 10.1186/s12899-015-0019-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lin, S. W. , Lopardo L., and Uhl G.. 2021. “Diversification Through Gustatory Courtship: An X‐Ray Micro‐Computed Tomography Study on Dwarf Spiders.” Frontiers in Zoology 18: 1–33. 10.1186/s12983-021-00435-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lipke, E. , Hammel J. U., and Michalik P.. 2015. “First Evidence of Neurons in the Male Copulatory Organ of a Spider (Arachnida, Araneae).” Biology Letters 11, no. 7: 20150465. 10.1098/rsbl.2015.0465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Long, S. M. 2021. “Variations on a Theme: Morphological Variation in the Secondary Eye Visual Pathway Across the Order of Araneae .” Journal of Comparative Neurology 529, no. 2: 259–280. 10.1002/cne.24945. [DOI] [PubMed] [Google Scholar]
  25. Martínez‐Sanjuán, J. , Kocot K., García‐Álvarez Ó., Candás M., and Díaz‐Agras G.. 2022. “Computed Microtomography (Micro‐CT) in the Anatomical Study and Identification of Solenogastres (Mollusca).” Frontiers in Marine Science 8: 1–21. 10.3389/fmars.2021.760194.35273967 [DOI] [Google Scholar]
  26. Menda, G. , Shamble P. S., Nitzany E. I., Golden J. R., and Hoy R. R.. 2014. “Visual Perception in the Brain of a Jumping Spider.” Current Biology 24, no. 21: 2580–2585. 10.1016/j.cub.2014.09.029. [DOI] [PubMed] [Google Scholar]
  27. Metscher, B. D. 2009. “MicroCT for Developmental Biology: A Versatile Tool for High‐Contrast 3D Imaging at Histological Resolutions. Developmental Dynamics: An Official Publication of the.” American Association of Anatomists 238, no. 3: 632–640. 10.1002/dvdy.21857. [DOI] [PubMed] [Google Scholar]
  28. Michalik, P. , and Ramírez M. J.. 2013. “First Description of the Male of Thaida chepu Platnick, 1987 (Araneae, Austrochilidae) With Micro‐Computed Tomography of the Palpal Organ.” ZooKeys 352: 117–125. 10.3897/zookeys.352.6021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moaraf, S. , Vistoropsky Y., Pozner T., et al. 2020. “Artificial Light at Night Affects Brain Plasticity and Melatonin in Birds.” Neuroscience Letters 716: 134639. 10.1016/j.neulet.2019.134639. [DOI] [PubMed] [Google Scholar]
  30. Monchanin, C. 2021. “Impacts of Metallic Pollutants on Honey Bees: From the Colony to the Brain.” Doctoral diss., Université Paul Sabatier‐Toulouse III; Macquarie University, Sydney, Australia. https://theses.hal.science/tel‐03591721.
  31. Moraes, S. S. , Söderholm M. S., Aguiar T. M., Freitas A. V., and Sihvonen P.. 2023. “Micro‐CT Imaging in Species Description: Exploring Beyond Sclerotized Structures in Lichen Moths (Lepidoptera: Erebidae, Arctiinae, Lithosiini).” PeerJ 11: e15505. 10.7717/peerj.15505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Donnell, S. , Bulova S. J., DeLeon S., Khodak P., Miller S., and Sulger E.. 2015. “Distributed Cognition and Social Brains: Reductions in Mushroom Body Investment Accompanied the Origins of Sociality in Wasps (Hymenoptera: Vespidae).” Proceedings of the Royal Society B: Biological Sciences 282, no. 1810: 20150791. 10.1098/rspb.2015.0791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Penney, D. , McNeil A., Green D. I., et al. 2011. “A New Species of Anapid spider (Araneae: Araneoidea, Anapidae) in Eocene Baltic Amber, Imaged Using Phase Contrast X‐Ray Computed Micro‐Tomography.” Zootaxa 66, no. 2742: 60–66. 10.11646/zootaxa.2742.1.4. [DOI] [Google Scholar]
  34. Penna‐Gonçalves, V. , Maclean D. J., Willmott N. J., et al. “Volumetric comparison of overall brain and neuropil size between social and non‐social spiders: exploring the social brain hypothesis.” Integrative Zoology.
  35. Poy, D. , Piacentini L. N., Michalik P., Lin S. W., and Ramírez M. J.. 2023. “MicroCT Analysis Unveils the Role of Inflatable Female Genitalia and Male Tibial Complex in the Genital Coupling in the Spider Genus Aysha (Anyphaenidae, Araneae).” Journal of Morphology 284, no. 5: 1–9. 10.1002/jmor.21586. [DOI] [PubMed] [Google Scholar]
  36. Ribi, W. , Senden T. J., Sakellariou A., Limaye A., and Zhang S.. 2008. “Imaging Honey Bee Brain Anatomy With Micro‐X‐Ray‐Computed Tomography.” Journal of Neuroscience Methods 171, no. 1: 93–97. 10.1016/j.jneumeth.2008.02.010. [DOI] [PubMed] [Google Scholar]
  37. Ritman, E. L. 2011. “Current Status of Developments and Applications of Micro‐CT.” Annual Review of Biomedical Engineering 13: 531–552. 10.1146/annurev-bioeng-071910-124717. [DOI] [PubMed] [Google Scholar]
  38. Rivera‐Quiroz, F. A. , and Miller J. A.. 2021. “Old Brains in Alcohol: The Usability of Legacy Collection Material to Study the Spider Neuroarchitecture.” Diversity 13, no. 11: 601. 10.3390/d13110601. [DOI] [Google Scholar]
  39. Rivera‐Quiroz, F. A. , and Miller J. A.. 2022. “Micro‐CT Visualization of the CNS: Performance of Different Contrast‐Enhancing Techniques for Documenting the Spider Brain.” Journal of Comparative Neurology 530, no. 14: 2474–2485. 10.1002/cne.25343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rix, M. G. , Wood H. M., Harvey M. S., and Michalik P.. 2021. “Micro‐Computed Tomography Reveals a Remarkable Twin Intromittent Organ in Spiders—A Novelty for Arachnids With Direct Sperm Transfer.” Frontiers in Ecology and Evolution 9: 1–7. 10.3389/fevo.2021.794708. [DOI] [Google Scholar]
  41. Rother, L. , Kraft N., Smith D. B., el Jundi B., Gill R. J., and Pfeiffer K.. 2021. “A Micro‐CT‐Based Standard Brain Atlas of the Bumblebee.” Cell and Tissue Research 386, no. 1: 29–45. 10.1007/s00441-021-03482-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schneider, C. A. , Rasband W. S., and Eliceiri K. W.. 2012. “NIH Image to ImageJ: 25 Years of Image Analysis.” Nature Methods 9, no. 7: 671–675. 10.1038/nmeth.2089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Smith, D. B. , Bernhardt G., Raine N. E., et al. 2016. “Exploring Miniature Insect Brains Using Micro‐CT Scanning Techniques.” Scientific Reports 6: 1–10. 10.1038/srep21768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sombke, A. , Lipke E., Michalik P., Uhl G., and Harzsch S.. 2015. “Potential and Limitations of X‐Ray Micro‐Computed Tomography in Arthropod Neuroanatomy: A Methodological and Comparative Survey.” Journal of Comparative Neurology 523, no. 8: 1281–1295. 10.1002/cne.23741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Spahr, E. J. , McLaughlin S. L., Tichinel A. M., Kasson M. T., and Kijimoto T.. 2023. “Staining and Scanning Protocol for Micro‐Computed Tomography to Observe the Morphology of Soft Tissues in Ambrosia Beetles.” Bio‐Protocol 13, no. 1: e4584. http://10.21769/BioProtoc.4584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Spitzner, F. , Meth R., Krüger C., et al. 2018. “An Atlas of Larval Organogenesis in the European Shore Crab Carcinus maenas L. (Decapoda, Brachyura, Portunidae).” Frontiers in Zoology 15: 1–39. 10.1186/s12983-018-0271-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Stafstrom, J. A. , Michalik P., and Hebets E. A.. 2017. “Sensory System Plasticity in a Visually Specialized Nocturnal Spider.” Scientific Reports 7, no. 1: 46627. 10.1038/srep46627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stamm, K. , and Dirks J. H.. 2022. “Semi‐Automated Differentiation of Insect Exo‐and Endocuticle in X‐Ray Microtomography.” Arthropod Structure & Development 66: 101139. 10.1016/j.asd.2021.101139. [DOI] [PubMed] [Google Scholar]
  49. Stauber, M. , and Müller R.. 2008. “Micro‐Computed Tomography: A Method for the Non‐Destructive Evaluation of the Three‐Dimensional Structure of Biological Specimens.” Osteoporosis: Methods and Protocols 455: 273–292. 10.1007/978-1-59745-104-8_19. [DOI] [PubMed] [Google Scholar]
  50. Steinhoff, P. O. , Harzsch S., and Uhl G.. 2023. “Comparative Neuroanatomy of the Central Nervous System in Web‐Building and Cursorial Hunting Spiders.” Journal of Comparative Neurology 532, no. 2: e25554. 10.1002/cne.25554. [DOI] [PubMed] [Google Scholar]
  51. Steinhoff, P. O. , Sombke A., Liedtke J., Schneider J. M., Harzsch S., and Uhl G.. 2017. “The Synganglion of the Jumping Spider Marpissa muscosa (Arachnida: Salticidae): Insights From Histology, Immunohistochemistry and microCT Analysis.” Arthropod Structure & Development 46, no. 2: 156–170. 10.1016/j.asd.2016.11.003. [DOI] [PubMed] [Google Scholar]
  52. Steinhoff, P. O. , Uhl G., Harzsch S., and Sombke A.. 2020. “Visual Pathways in the Brain of the Jumping Spider Marpissa muscosa .” Journal of Comparative Neurology 528, no. 11: 1883–1902. 10.1101/640706. [DOI] [PubMed] [Google Scholar]
  53. Steinhoff, P. O. M. , Liedtke J., Sombke A., Schneider J. M., and Uhl G.. 2018. “Early Environmental Conditions Affect the Volume of Higher‐Order Brain Centers in a Jumping Spider.” Journal of Zoology 304, no. 3: 182–192. 10.1111/jzo.12512. [DOI] [Google Scholar]
  54. Swart, P. , Wicklein M., Sykes D., Ahmed F., and Krapp H. G.. 2016. “A Quantitative Comparison of Micro‐CT Preparations in Dipteran Flies.” Scientific Reports 6, no. 1: 39380. 10.1038/srep39380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Willmott, N. J. , Black J. R., McNamara K. B., Wong B. B. M., and Jones T. M.. 2024. “The Effects of Artificial Light at Night on Spider Brains.” Biology Letters 20, no. 9: 20240202. 10.1098/rsbl.2024.0202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wipfler, B. , Pohl H., Yavorskaya M. I., and Beutel R. G.. 2016. “A Review of Methods for Analysing Insect Structures—The Role of Morphology in the Age of Phylogenomics.” Current Opinion in Insect Science 18: 60–68. 10.1016/j.cois.2016.09.004. [DOI] [PubMed] [Google Scholar]
  57. Yushkevich, P. A. , Piven J., Hazlett H. C., et al. 2006. “User‐Guided 3D Active Contour Segmentation of Anatomical Structures: Significantly Improved Efficiency and Reliability.” Neuroimage 31, no. 3: 1116–1128. 10.1016/j.neuroimage.2006.01.015. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Comparison among Bouin‐iodine, paraformaldehyde–iodine and ethanol‐PTA fixative and staining protocols. Prosoma sagittal, CNS zoomed‐in and dorsal slices of A: Helpis sp., B: Maratus (Hypoblemum) griseus, C: M. griseus, D: Astia hariola, E: Myrmarachne smaragdina, F: Myrmarachne luctuosa, G: M. luctuosa, H: Helpis sp., I: Tharrhalea evanida, J: Hortophora biapicata unstable specimen, K: H. biapicata stable, L: H. biapicata, M: H. biapicata, N: : Delena cancerides, O: Heteropoda jugulans, P: Isopeda villosa, Q: I. villosa, R: I. villosa, S: I. villosa, no vaccum system, T: I. villosa, U: D. cancerides. Note that magnified scans (K, L, M) do not have total prosoma sagittal views. Scale bar: 1 mm.

CNE-533-e70017-s001.pdf (745.1KB, pdf)

Table S1 Location where spiders were collected in Australia, including species names, GPS coordinates, collection dates and maturation stage.

CNE-533-e70017-s002.xlsx (18.1KB, xlsx)

Table S2 Additional information about the specimens, staining methodology, and scan settings.

CNE-533-e70017-s003.xlsx (24.3KB, xlsx)

Data Availability Statement

The data that support the findings of this study are available in the supporting information of this article.

Articles from The Journal of Comparative Neurology are provided here courtesy of Wiley

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