Comparing micro-CT results of insects with classical anatomical studies: The European honey bee (Apis mellifera Linnaeus, 1758) as a benchmark (Insecta: Hymenoptera, Apidae)

article
Javier Alba-Tercedor and Ignacio Alba-Alejandre, Department of Zoology, Faculty of Sciences, University of Granada, Campus de Fuentenueva s/n, 18071-Granada, Spain
 
Biography
Javier Alba-Tercedor, received his MSc in 1977 and PhD in zoology in 1981 from the University of Granada, Spain. Conducted research in many different countries around the world. He is a full professor of zoology – since 1988 – at the university and responsible for a research group on the biology and ecology of lotic freshwater ecosystems, with over 200 publications. His interests have been mainly focused on freshwater ecology, especially on aquatic macroinvertebrates and the biomonitoring of running water. Now he is an active enthusiast of the potential of X-ray tomography, in both research and education. More details in: mendeley.com/profiles/javier-alba-tercedor/
 
Ignacio Alba-Alejandre was awarded his veterinary degree in from the University of Cordoba, Spain in 2005. In 2009, he graduated with a masters in biological agriculture and aquaculture. In 2015 he graduated in food safety, and 2016 in surgery and medicine of exotic animals. Since 2017 he’s been working on his PhD on micro-CT to study functional anatomy of insects.
 
Abstract
Many papers mention advantages of the micro-CT because it does not damage the samples, and because the images are comparable with low magnifications SEM images. In this article we have  undertaken a detailed micro-CT study of the anatomy of the European honey bee (Apis mellifera Linnaeus, 1758), species well known to the general public and about which much has been published. We compare the micro-CT image results with a classical anatomical study published by Snodgrass at the beginning of the twentieth century, still considered as the more complete compendium about the anatomy of the honey bee and in general it represents some sort of bible about the knowledge of the insect anatomy. We decided in this paper to show details of the external and internal anatomy of the whole body of worker bees, comparing the micro-CT volume renderings images with the Snodgrass’ drawings and find a complete correspondence when comparing results of the detailed study of the anatomy by the modern micro-CT technique and the classical anatomical study by Snodgrass.
 
Acknowledgements
To Bruker-Skyscan staff for fast and effective support, their patience and effectiveness, and their constant improvements to the software and in implementing new options we requested. Also for their kindness in providing the senior author fast and effective suggestions and answers to queries. We are especially indebted to: Alexander Sasov, Stephan Boons, Xuan Liu, Phil Salmon, Jeroen Hostens, and Vladimir Kharitonov. To Dr. Nikolaus Nestle from BASF, SE, Ludwigshafen am Rhein in Germany who let us know about the use of Basotec® for micro-CT and provided us with the first sample of it, and to Luisa Kari and Stephanie Thum from the BASF company who kindly sent us additional material. This paper benefited  of the project: “Developing an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Disease”, 2015 (extended in 2017), lead by Dr. S.Brown, Kansas State University and funded by the USDA’s National Institute of Food and Agriculture through the Specialty Crops Research Initiative/Citrus Disease Research & Extension.USDA NIFA Award No.2015-70016-23028. As well of the project granted by the Spanish government MIMECO: “Life history strategies to cope with human-induced rapid environmental changes”(CGL2013-47448-P), lead by Dr. Daniel Sol. 
 
Corresponding author
Prof. Dr. Javier Alba-Tercedor: jalba@ugr.es
Telephone +34958244015   Fax +34958243238
Microscopy and Analysis 33(1): 12-15 (EU), February 2019
 
INTRODUCTION
 
Many papers mention the advantages of micro-CT because it does not damage the samples, and because the images are comparable with others we can get using scanning electron microscopy (SEM). There is little discussion about it, and there is a general consensus on this point,2-5 in fact many papers obtained renderings that are of high quality, supporting the argument that micro-CT is more than a complementary technique.4,6,7 Previously we have published different studies of small animal anatomy pointing out the incredible results that can be obtained not only in the field of the anatomy,5, 8–18 but also results of relevant importance to elucidate adaptive survival strategies of insects.19, 20
 
We already tested the quality of the images obtained with micro-CT,12–14,17,18,21–25 which have the advantage that the image datasets can be visualized as many times as needed with the possibility to change the observation perspective and the original sample can be returned to the collections or museum where it is displayed without harm. Now that it seems that there are no doubts about the importance of micro-CT as a modern research tool, as it is demonstrated in the increasing number of papers using it.  Because we are involved in a project studying bees, recently we published a general micro-CT study of a mason bee (Osmia rufa),9 and we decided to undertake a detailed micro-CT study of the European honey bee (Apis mellifera Linnaeus, 1758), species well known to the general public and about which much literature has been published. A classical anatomical study was published in the beginning of the twentieth century,26 and is still the more complete compendium about the anatomy of the honey bee, representing some sort of bible about the knowledge of the insect anatomy. Recently, papers have used modern microscopic techniques, including micro-CT, to study different body structures, but mainly the brain (ie 27–34). We decided in this paper to show details of the external and internal anatomy of the whole body of worker bees, comparing the micro-CT volume renderings images with the Snodgrass’ drawings as the most classic representative study.
 
MATERIALS AND METHODS
 
Workers of the the European honey bee (Apis mellifera Linnaeus, 1758) were collected alive when foraging in the Sierra Nevada montains, southern Spain (11-12-2016, 2-I-2017, Pinos Genil, Granada 980 m. a.s.l.). Specimens were killed and preserved in 70% ethanol for five days and then transferrred to a 1% solution of Iodine in 100% ethanol during nine-24 hours, after that they were submerged in Hexamethyldisilazane for nine-24 hours and air dried overnight. The samples were mounted in a piece of BASOTECT® (melamine resin foam, created by the Chemical Company BASF), inside a plastic container to avoid any movement induced by the air refrigerating current during the scan process (see fig. 2). The Basotect’s material is of very low density, and therefore virtually transparent to X-Rays (see fig. 2c), consequently it can be easily eliminated during the segmentation procedure.
 
A SkyScan 1172 high resolution microtomograph, upgraded to have a Hamamatsu 100/250 source and a SHT 11Mp camera was used. The scanning parameters were setup as it follows: Isotropic voxel size = 4.45 µm per pixel; Source voltage=53-55KV, Source current=124-127µA, and image. Rotation step=0.5º, 360º of rotation scan. The daisy flower (Aster sp.) used for the artistic composition of fig.1, was cut from the garden of the faculty campus, immediately fixed with plasticine to the sample holder, and fresh scanned with the following setting parameters: Isotropic voxel size = 23.5 µm per pixel; Source voltage=54KV, Source current=43µA, and image. Rotation step=0.5º, 180º of rotation scan.
 
For primary reconstructions and the ‘cleaning’ process to obtain the datasets of cross-section images (‘slices’) the latest versions of the Bruker micro-CT’s Skyscan software (NRecon, DataViewer, CTAnalyser,) were used. Volume rendering images were obtained with ThermoFisher’s Amira software v.6.2.35 The free Skyscan’s software CTVox was used to get color volume rendering images in figures: 1, 13 and 14. Colors were obtained varying the color transfer function curves, in conjunction with the lighting and shadowing options. For more detailed explanation of the process see the previous paper.1 It is important to point out that Amira’s volume renderings were obtained directly by loading image datasets obtained with the Bruker-microCT Skyscan’s, resulting in mirrored inverted images.
 


Figure 1. Picture composition of volume rendering reconstructions of honey bee workers in their daily activities: foraging and sipping nectar on a daisy flower (Aster sp.). To point out the internal structures the insect and the flower have been “virtually” cut by using the free Skyscan’s software CTVox. Colors were obtained varying the color transfer function curves, conjunction with the lighting and shadowing options within CTVox’s software, as described by1
 
 


Figure 2.
 Mounting and fixation of samples to the sample holder for the scanning process. A prepared honey worker bee, on an excavated piece of Basotect®, inside a plastic container (a, b, d); e: garden daisy flower (Aster sp.), fresh-fixed with plasticine; c: detail of a NRecon’s view of a specimen to show how transparent is the Basotect® material to x-ray.
 
RESULTS
 
Figures 3 to 17 illustrate the results obtained of this study on the anatomy of the honey bee and the respective comparison of the structures as were drawn by Snodgrass in his classic paper from first decade of the twentieth century.26 These images demonstrate the high precision with which the authors did the anatomical studies, because after a thorough comparison only small differences in shapes can be observed. In general Snodgrass’ drawings are somewhat more elongated than in reality, but even when looking at very delicate small structures as the brain and its nerves are in accordance with what we could enhance by using the modern technique of micro-CT.
 
CONCLUSION
 
The complete parallelism observed in the detailed study of the anatomy by the modern micro-CT technique and the classical anatomical study by Snodgrass confirm the validity of the micro-CT. Resulting not only a valid technique but incredibly reliable for anatomical studies. It permits the study the animals or structures scanned as many times as desired, virtually moving the sample and to explore from any angle/perspective. These points make this approach extremely valuable not only for new samples, but for future studies based on the comparison of stored images datasets. 
 


Figure 3.
 Volume rendering of a honey worker bee. External anatomy in lateral view (a); sagittal cut showing the internal structures and organs (b)
 
 


Figure 4.
 Volume rendering of a honey worker bee showing the external anatomy of the head in a lateral (a) and a frontal view (b), with mouth parts composing the sucking proboscis, and details of the very reduced maxillary pal (see red arrow in a, and c). Ventral view of the tip of the tarsus of first leg (f). Original figures (d, e,) from Snodgrass (1910)26 are included to be able to compare.
 
 


Figure 5.
 Volume renderings of the wings (a) of a honey worker bee showing in b details of the hamuli (a row of hook on the fore margin of the hind wing. These fix and attach it to the fore wing, permitting to synchronize the flight movements). Original figure by Snodgrass26 in 1910 is included for comparison (c).
 
 


Figure 6.
 Volume renderings of the head in a lateral view, showing the external (a) and internal (b) anatomical structures. 
 
 


Figure 7
. Volume renderings of internal anatomy of the head in a lateral view, progressive cuts (c, d) show the internal anatomical structures with a high definition. Observe the correspondence with the original Snodgrass figures (a, b). 
 
 


Figure 8.
 Volume renderings of head in a frontal view. In the colored lower picture the external surface has been slightly cut permitting to evidence the fore glandular complex. 
 
 


Figure 9.
 Volume rendering superficial section of head in a frontal view permitting to see the internal structures, and especially the anterior pharyngeal glands. Original Snodgrass’ figure (b), is included for comparison.
 
 


Figure 10.
 Volume rendering section of the head in an anterior view, evidencing the brain and its main nerves, as well as the eyes structure
 
 


Figure 11.
 Volume rendering section of the head in a posterior view showing the internal structures.
 
 


Figure 12.
 Volume rendering of a front-posterior section of the head showing the detailed structure of the compound eye (b), permitting to compare it with the Philip’s original figure reproduced the classic study by Snodgrass (1910) (a). For details about the use of micro-CT to study the insect’s eye structure see.8
 
 


Figure 13.
 Volume renderings of the internal organs of a honey worker bee in ventro-dorsal (b) and dorso-ventral (c) views, respectively and the comparison with the original figure by Snodgrass (1910). To be able to get these images we used the sphere cutting in the cutting/clipping option within CTVox’s menu. (Dvfm: Dorso-ventral flight muscles, Dv: Dorsal vase –heart-, Hs: Honey stomach, Mt: Malpighian tubules, R: Rectum, Rg: Rectal gland, Si: Small intestine)Colors are artificial and represent the opacity of structures to x-ray, according with the scale. 
 
 


Figure 14.
 Volume rendering of an internal view of the dorsal abdominal region of the honey bee’s worker showing the dorsal vase (heart) and its winged shaped muscles, as well other structures (b). It is included an original figure by Snodgrass (1910) to compare. To be able to get this image we used the sphere cutting options in the cutting/clipping option within CTVox’s menu. Colors are artificial and represent the opacity of structures to x-Ray, according with the scale in figure 13.
 
 


Figure 15.
 Volume rendering of a mid-section of the hind abdominal part of the honey worker bee (b) and its comparison with the original figure by Snodgrass (1910) (a).
 
 


Figure 16
. Volume renderings of sections of the hind abdominal part of the honey worker bee, in a lateral (b) and a dorso-ventral view (c), showing the detailed anatomy of the sting and its accessory structures. For comparison the original figures by Snodgrass (1910)- in a lateral (a) and a dorso-ventral (b) views- have been included.
 
 


Figure 17.
 Volume renderings of section, in dorso-ventral views, of the internal anatomy of the honey worker bee. Most of the internal structures have been removed with software to be able to see the nervous system: the brain in the head, and the thoracic (b), and abdominal (c) ventral ganglionic nerve cord, permitting to compare it with a similar view drawn by Snodgrass (1910) (a)
 
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