**1. Introduction**

Nearly all existing species of bee are pollen feeding, aculeate (with a stinger) Hymenopterans (membranous wings). There are only three species that do not collect or eat pollen and these are the necrophagic (eat dead or decaying flesh) Neotropical, Trigona species [1–4]. Approximately 5% of all bees are highly social. The highly social bees include species of bumble bees, honey bees and stingless bees. The other species are either semi-social, which occur in aggregations or are solitary. For a full description of these terms, see [5, 6]. With honeybees, colony health has been traditionally evaluated by simple visual inspections and/or by measuring changes in hive weights over time. However, these methods are estimates and subjective. Typically, beekeepers and scientists look for behavioural signs which indicate healthy individuals or colonies, where foragers are regularly bringing in resources. In contrast, they look for weak colonies, where there are usually fewer foragers. These foragers typically exhibit a more lethargic and less purposeful behaviour. However, there are also situations where increases in hive weights can be attributed to increases in pollen and nectar stores due to hyper-collection by foragers that exhibit precocious feeding during times of colony stress [7].

These colonies give the false impression that all is well. In these situations, an increase in hive weight can be misinterpreted as a sign of good colony health yet the colony could be under considerable stress from infection or disease rather than being in good health.

Visual inspections on colonies of stingless bees is particularly invasive because of the central location of the brood and many species are less than 3 mm in size. In stingless bees, colony health can be assessed by manually splitting the hive box apart to view internal structures and any evidence of queen activity [8]. Opening the hive for such inspections invariably damages honey storage pots. This causes honey to spill and many hundreds of bees die because many species are diminutive and will drown in their own spilt honey. Closing the hive after visual inspection also kills bees, and places the queen at risk of being harmed because they can be squashed in the process.

Therefore, the subjective nature of visual inspections and hive weight estimations often leads to errors when assessing colony health. Issues such as these were not so important in previous decades however, with the continued pressure from large scale agriculture, loss of bee habitat and the global increase in bee pathogens and pests [9] it has become paramount that new and more accurate methods are developed.

It is vital that behavioural, morphological and physiological studies on bees continue. However, because they have propensities to live in cavities and traditional methods are often invasive and prone to large errors, new methods for studying them are emerging. These new methods will add accuracy to current estimates on individual bees and colony health parameters which will, in turn, enable better solutions for scientists and beekeepers to improve bee health globally.

This chapter describes one new method termed 'Diagnostic Radioentomology' and includes examples of research conducted using this method.

#### **1.1 Non-invasive imaging**

On the 8th of November 1895, Wilhelm Conrad Röntgen (accidentally) discovered an image cast from one of his cathode ray generators. He later repeated the experiment by taking an X-ray photograph of his wife Anna Bertha Ludwig's hand **Figure 1**, which revealed the bones in her hand and her wedding ring on one of her fingers.

The photograph initiated great scientific interest in the new found radiation and because Röntgen did not know what type of radiation it was, he called it 'X-radiation', hence the modern term, X-rays. In general, non-invasive imaging is associated with X-rays or medical imaging, which is a non-invasive method for evaluating anatomy and physiology. Although it is now known that X-rays can be invasive (and can damage biological tissues) at the higher energies, the term non-invasive is based on the fact that, at the lower energies that are used in modern imaging methods, X-rays do not create any damaging biological effects.

#### **1.2 Techniques available for non-invasive imaging**

As a field of scientific investigation, non-invasive imaging constitutes a subdiscipline of biomedical engineering, medical physics or medicine depending on the context. Methods such as nuclear medicine use radioactive materials to diagnose or treat various pathologies and are generally considered to be invasive. Many of the techniques developed for non-invasive imaging such as X-rays, nuclear magnetic resonance imaging (MRI) and ultrasonography (U/S) also have industrial applications, although the energies used in industrial applications are extremely high

**155**

Scanners).

**Figure 1.**

*[public domain].*

*Diagnostic Radioentomology*

*DOI: http://dx.doi.org/10.5772/intechopen.89005*

have much faster image capture times.

**1.3 Diagnostic Radioentomology**

and would be considered to be highly invasive for biological samples. In the case of U/S, the probe emits the beam which consists of ultrasonic pressure waves that return echoes from the various tissue interfaces. The echoes show details of the internal structures. U/S waves do not travel through large interfaces or air and thus limits its use in biological tissue. In the cases of X-rays and MRI, either X-radiation or a magnetic field respectively pass through the tissues to identify, separate and quantify different tissue types such as bone, cuticle, muscle or fat. In general, MRI is the best modality for discerning muscle or fat, is non-invasive and has very long image capture times whereas X-rays are better for discerning smaller structures and

*First medical X-ray by Wilhelm Röntgen of his wife Anna Bertha Ludwig's hand. Wilhelm Röntgen* 

It is the ability to see smaller structures and the fast capture times that led to the development of Diagnostic Radioentomology (DR) to study insects non-invasively. DR is performed on insects using X-ray Computer Tomography Scanners (CT

The term 'Diagnostic Radioentomology' first came to be used in 2003 during a pollination experiment on the behaviour of the Australian stingless bees *Tetragonula carbonaria* and *Austroplebeia australis* [10]. The term was used because the new method is diagnostic, it uses X-radiation (radio) and is used for studying insects

Therefore, DR became the term for an innovative method for studying insect morphology, physiology and behaviour using non-invasive imaging. Since its development, DR has been adopted by the Museum of Natural History in London,

In 2008, DR was described by The International Bee Research Association (IBRA) as an emerging non-invasive technique for behavioural, evolutionary and

(entomology). **Video 1** gives a brief overview of the methods.

Universities, synchrotron facilities and research associations globally.

classical biologists who need to study insects without harming them.

#### **Figure 1.**

*Modern Beekeeping - Bases for Sustainable Production*

being in good health.

squashed in the process.

**1.1 Non-invasive imaging**

developed.

fingers.

These colonies give the false impression that all is well. In these situations, an increase in hive weight can be misinterpreted as a sign of good colony health yet the colony could be under considerable stress from infection or disease rather than

Visual inspections on colonies of stingless bees is particularly invasive because of the central location of the brood and many species are less than 3 mm in size. In stingless bees, colony health can be assessed by manually splitting the hive box apart to view internal structures and any evidence of queen activity [8]. Opening the hive for such inspections invariably damages honey storage pots. This causes honey to spill and many hundreds of bees die because many species are diminutive and will drown in their own spilt honey. Closing the hive after visual inspection also kills bees, and places the queen at risk of being harmed because they can be

Therefore, the subjective nature of visual inspections and hive weight estimations often leads to errors when assessing colony health. Issues such as these were not so important in previous decades however, with the continued pressure from large scale agriculture, loss of bee habitat and the global increase in bee pathogens and pests [9] it has become paramount that new and more accurate methods are

It is vital that behavioural, morphological and physiological studies on bees continue. However, because they have propensities to live in cavities and traditional methods are often invasive and prone to large errors, new methods for studying them are emerging. These new methods will add accuracy to current estimates on individual bees and colony health parameters which will, in turn, enable better

This chapter describes one new method termed 'Diagnostic Radioentomology'

On the 8th of November 1895, Wilhelm Conrad Röntgen (accidentally) discovered an image cast from one of his cathode ray generators. He later repeated the experiment by taking an X-ray photograph of his wife Anna Bertha Ludwig's hand **Figure 1**, which revealed the bones in her hand and her wedding ring on one of her

The photograph initiated great scientific interest in the new found radiation and because Röntgen did not know what type of radiation it was, he called it 'X-radiation', hence the modern term, X-rays. In general, non-invasive imaging is associated with X-rays or medical imaging, which is a non-invasive method for evaluating anatomy and physiology. Although it is now known that X-rays can be invasive (and can damage biological tissues) at the higher energies, the term non-invasive is based on the fact that, at the lower energies that are used in modern

As a field of scientific investigation, non-invasive imaging constitutes a subdiscipline of biomedical engineering, medical physics or medicine depending on the context. Methods such as nuclear medicine use radioactive materials to diagnose or treat various pathologies and are generally considered to be invasive. Many of the techniques developed for non-invasive imaging such as X-rays, nuclear magnetic resonance imaging (MRI) and ultrasonography (U/S) also have industrial applications, although the energies used in industrial applications are extremely high

imaging methods, X-rays do not create any damaging biological effects.

**1.2 Techniques available for non-invasive imaging**

solutions for scientists and beekeepers to improve bee health globally.

and includes examples of research conducted using this method.

**154**

*First medical X-ray by Wilhelm Röntgen of his wife Anna Bertha Ludwig's hand. Wilhelm Röntgen [public domain].*

and would be considered to be highly invasive for biological samples. In the case of U/S, the probe emits the beam which consists of ultrasonic pressure waves that return echoes from the various tissue interfaces. The echoes show details of the internal structures. U/S waves do not travel through large interfaces or air and thus limits its use in biological tissue. In the cases of X-rays and MRI, either X-radiation or a magnetic field respectively pass through the tissues to identify, separate and quantify different tissue types such as bone, cuticle, muscle or fat. In general, MRI is the best modality for discerning muscle or fat, is non-invasive and has very long image capture times whereas X-rays are better for discerning smaller structures and have much faster image capture times.

It is the ability to see smaller structures and the fast capture times that led to the development of Diagnostic Radioentomology (DR) to study insects non-invasively. DR is performed on insects using X-ray Computer Tomography Scanners (CT Scanners).

### **1.3 Diagnostic Radioentomology**

The term 'Diagnostic Radioentomology' first came to be used in 2003 during a pollination experiment on the behaviour of the Australian stingless bees *Tetragonula carbonaria* and *Austroplebeia australis* [10]. The term was used because the new method is diagnostic, it uses X-radiation (radio) and is used for studying insects (entomology). **Video 1** gives a brief overview of the methods.

Therefore, DR became the term for an innovative method for studying insect morphology, physiology and behaviour using non-invasive imaging. Since its development, DR has been adopted by the Museum of Natural History in London, Universities, synchrotron facilities and research associations globally.

In 2008, DR was described by The International Bee Research Association (IBRA) as an emerging non-invasive technique for behavioural, evolutionary and classical biologists who need to study insects without harming them.

**Figure 2.** *The first DR image of a bee colony (*Tetragonula carbonaria*) in a wooden box [11].*

Nowadays, synchrotron beamlines can completely scan and reconstruct 3D images in a matter of seconds and CT scanners can complete a 1-cm scan in as little as one-third of a second, and recent techniques have been developed to enable scanning software to produce 3D images such as in **Figure 2** and 4D movies and physical 3D models which can be downloaded at this address: http://www.radioentomology.com/.

It is generally accepted that for DR studies, the term MacroCT applies to the CT scanning of large items using human body CT scanners and that MicroCT applies to laboratory or Synchrotron CT scanners to study small items at the microscopic level. In recent years, DR has been adopted to visualise macroscopic characteristics of insects and their behaviour [11–15]. Also, with the improvements in spatial resolution and tissue differentiation that are occurring with MacroCT, conventional micro-focus and synchrotron based MicroCT, new methods for the non-invasive imaging of insects are emerging. For an overview of these methods see [16–22].

Historically, traditional methods for colony health, bee behaviour and the morphological classification of bees have been conducted on apiary hives and with the aid of observation hives and dissecting light microscopes. These techniques are, understandably, limited. The inspection of apiary hives disrupts normal bee behavior, observation hives offer only a view of one side of one frame within an entire hive, dissection obviously kills the bee and the use of light microscopy when used for amber inclusions [23–32], particularly with specimens preserved in opaque amber pieces [33, 34] are grossly limited by the specimens opaqueness. In [35] the authors attempted to address methods of examining insect inclusions within pieces of opaque amber and to supplement traditional light microscopy studies of transparent amber. Those researchers and [36–38] produced traditional radiographs which provided the first, albeit limited, steps toward enhanced visualisation of cryptic bee behaviour and fossil material. More recently, detailed information for the study of bees has been obtained with the use of scanning electron microscopy (SEM) as in [39, 40] and transmission electron microscopy (TEM) as in [41]. While SEM and TEM studies currently provide the highest level of detail, sample preparations are laborious and are often invasive to completely destructive [41]. SEM and TEM can be used for the investigation of amber inclusions as in [39–42] however, these methods are generally not suitable because they require destruction of the material. The development of non-invasive imaging methods such as DR, therefore, offers promise to scientists and beekeepers who need to preserve their specimens or observe behaviour non-invasively.

In 2013, DR was hailed as the 'Gold Standard' for honeybee monitoring [43] and the non-invasive path detailed in the following sections will demonstrate that DR is an ideal method which can be used to study bees and other insects in the most natural of settings. It is also important to mention that the results from the following

**157**

*Diagnostic Radioentomology*

**2.1 Materials and methods**

*2.1.1 About the bee*

global bee health.

*DOI: http://dx.doi.org/10.5772/intechopen.89005*

experiments can be directly applied to beekeeping husbandry to enhance modern beekeeping methods and enable beekeepers to play a more active role in improving

To help demonstrate the non-invasive, diagnostic advantages of DR it is worthwhile detailing the following experiment on one of the oldest bees known, *Proplebeia adbita*. In the following experiment, we examined the external and internal morphology of an Early Miocene (Burdigalian) stingless bee (Apinae: Meliponini) from the Dominican Republic using non-destructive X-ray microtomography analysis (MicroCT). The study shows the accurate reconstruction of features otherwise obscured or impossible to visualise without destroying/damaging the sample and enables diagnosis of the specimen as a new species of bee [44].

Bees have several characteristic morphological attributes such as branched or plumose body setae and broadened metabasitarsi [5, 45]. The highly eusocial stingless bees, the Meliponini are within the corbiculate Apinae for example in [46–49]. In addition to extensive morphological and molecular data such as in [48, 50], the corbiculate apines belonging to a single group has been supported by studies investigating their internal anatomy. For example [39], noted that the proventricular morphology of Euglossini and Bombini consists of long columnar plates, triangular apices in Apini, while the Meliponini have slender and elongated plates. Accordingly, the proventriculus can be used as an important diagnostic structure for bee taxonomy [21], among a suite of other internal anatomical features [45]. The examination of such characters often requires considerable manipulation, dissection, sectioning or even complete destruction of the specimen. Thus, the practical application of such data is at times hampered by the methods employed. In the following experiment, the internal and external morphology of an ancient social bee trapped in amber using non-invasive and non-destructive DR techniques is described in detail.

The bee selected for this experiment was collected from the La Bucara mine in the Dominican Republic. This stingless bee was trapped at the widest end of a semi-

The posterior of the bee is at the extreme periphery of the piece's thick end, and

Age estimates of Dominican amber vary considerably in literature nonetheless, most data indicate that the age of most Dominican amber, including the material in this study, is 16–19 Ma [45–47]. The sample had been polished prior to this study

Traditionally, the morphological classification of bees has been conducted with the aid of dissecting microscopes which use light. The technique is understandably limited when used for amber inclusions, particularly with specimens preserved in opaque pieces. Light microscopy was used in this experiment in an attempt to

clear, brown piece of amber that contained many other inclusions **Figure 3**.

the apices of both forewings have broken away from the sample over time.

and therefore required no extra preparation.

describe its limitations with opaque specimens.

*2.1.2 Non-invasive imaging of the bee*

**2. Describing an ancient social bee in amber using DR**

*Modern Beekeeping - Bases for Sustainable Production*

Nowadays, synchrotron beamlines can completely scan and reconstruct 3D images in a matter of seconds and CT scanners can complete a 1-cm scan in as little as one-third of a second, and recent techniques have been developed to enable scanning software to produce 3D images such as in **Figure 2** and 4D movies and physical 3D models which can be downloaded at this address: http://www.radioentomology.com/. It is generally accepted that for DR studies, the term MacroCT applies to the CT scanning of large items using human body CT scanners and that MicroCT applies to laboratory or Synchrotron CT scanners to study small items at the microscopic level. In recent years, DR has been adopted to visualise macroscopic characteristics of insects and their behaviour [11–15]. Also, with the improvements in spatial resolution and tissue differentiation that are occurring with MacroCT, conventional micro-focus and synchrotron based MicroCT, new methods for the non-invasive imaging of insects are emerging. For an overview of these methods see [16–22]. Historically, traditional methods for colony health, bee behaviour and the morphological classification of bees have been conducted on apiary hives and with the aid of observation hives and dissecting light microscopes. These techniques are, understandably, limited. The inspection of apiary hives disrupts normal bee behavior, observation hives offer only a view of one side of one frame within an entire hive, dissection obviously kills the bee and the use of light microscopy when used for amber inclusions [23–32], particularly with specimens preserved in opaque amber pieces [33, 34] are grossly limited by the specimens opaqueness. In [35] the authors attempted to address methods of examining insect inclusions within pieces of opaque amber and to supplement traditional light microscopy studies of transparent amber. Those researchers and [36–38] produced traditional radiographs which provided the first, albeit limited, steps toward enhanced visualisation of cryptic bee behaviour and fossil material. More recently, detailed information for the study of bees has been obtained with the use of scanning electron microscopy (SEM) as in [39, 40] and transmission electron microscopy (TEM) as in [41]. While SEM and TEM studies currently provide the highest level of detail, sample preparations are laborious and are often invasive to completely destructive [41]. SEM and TEM can be used for the investigation of amber inclusions as in [39–42] however, these methods are generally not suitable because they require destruction of the material. The development of non-invasive imaging methods such as DR, therefore, offers promise to scientists and beekeepers who need to preserve their specimens or

*The first DR image of a bee colony (*Tetragonula carbonaria*) in a wooden box [11].*

In 2013, DR was hailed as the 'Gold Standard' for honeybee monitoring [43] and the non-invasive path detailed in the following sections will demonstrate that DR is an ideal method which can be used to study bees and other insects in the most natural of settings. It is also important to mention that the results from the following

**156**

**Figure 2.**

observe behaviour non-invasively.

experiments can be directly applied to beekeeping husbandry to enhance modern beekeeping methods and enable beekeepers to play a more active role in improving global bee health.
