Mark Greco

The alternative Pharaoh approach: stingless bees mummify beetle parasites alive

Workers from social insect colonies use different defence strategies to combat invaders. Nevertheless, some parasitic species are able to bypass colony defences. In particular, some beetle nest invaders cannot be killed or removed by workers of social bees, thus creating the need for alternative social defence strategies to ensure colony survival. Here we show, using diagnostic radioentomology, that stingless bee workers (Trigona carbonaria) immediately mummify invading adult small hive beetles (Aethina tumida) alive by coating them with a mixture of resin, wax and mud, thereby preventing severe damage to the colony. In sharp contrast to the responses of honeybee and bumblebee colonies, the rapid live mummification strategy of T. carbonaria effectively prevents beetle advancements and removes their ability to reproduce. The convergent evolution of mummification in stingless bees and encapsulation in honeybees is another striking example of co-evolution between insect societies and their parasites.

A new technique for monitoring Trigona carbonaria nest contents, brood and activity using X-ray computerized tomography

SUMMARY Brood status in the Australian stingless bee Trigona carbonaria is difficult to monitor non-invasively as splitting the hive damages the nest, and the involucrum prevents observations of internal structures and evidence of queen activity. In addition, increases in hive weight, also used as a measure of colony health, can be attributed to increased pollen and nectar collection by foragers in times of colony stress rather than improved brood status. To assess brood status and queen activity, we performed helical two-dimensional and three-dimensional X-ray computerized tomography (CT) to measure brood chamber volume in a T. carbonaria hive. All previously reported nest structures including larval cells, pupal cocoons, involucrum, cerumen and batumen layers were clearly identifiable. In addition, the on-screen linear callipers enabled accurate estimates of brood chamber volume to be made. A batumen bridge was found that may secure the brood chamber to the base of the hive box; this structure has not been reported before. CT could be used to follow the life cycle of stingless bees, track the development of natural nests and observe nest morphology to distinguish between species of Trigona. In addition, sequential scans will be useful in non-invasively assessing changes in brood status.

Description of an ancient social bee trapped in amber using diagnostic radioentomology

The application of non-invasive imaging technologies using X-radiation (diagnostic radioentomology, ‘DR’) is demonstrated for the study of amber-entombed social bees. Here, we examine 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. The study permits the accurate reconstruction of features otherwise obscured or impossible to visualize without destroying the sample and allows diagnosis of the specimen as a new species, Proplebeia adbita Greco and Engel.

X‐ray computerized tomography as a new method for monitoring Amegilla holmesi nest structures, nesting behaviour, and adult female activity

Nest structures of the Australian blue‐banded bee, Amegilla holmesi Rayment (Hymenoptera: Apidae), are difficult to monitor non‐invasively, because the bee is a fossorial species that constructs tunnels and natal cells within the soil. Visualization of nest structures requires physical separation of the structures from the soil substrate, which destroys the nest. Using helical, two‐dimensional (2D) X‐ray computerized tomography (CT), and three–dimensional (3D) volume rendering software on 13 artificial mud‐brick nests, we were able to view all previously reported nest structures and cell contents without damaging the nests. We used three volume rendering algorithms (sum along ray, scatter HQ, and false colour), two of which (scatter HQ and false colour) have not previously been used to visualize nest architecture. Each algorithm has a particular advantage for visualizing specific nest structures. The 2D CT images provided the greatest spatial resolution within and between nest structures (capture pixel matrix size 512 × 512; voxel size 0.449 mm3), and for the first time we were able to visualize a meniscus within natal cells that contained liquid provisions, which is a valuable characteristic in detecting the earliest stage of juvenile development once a natal cell is sealed. We also recorded and attributed Hounsfield units to nest structures and their contents that can be utilized in future studies. This new method also enabled us to count and measure natal cells using on‐screen linear callipers, visually observe and record stages of larval and pupal development, and visually gauge the incidence of cell parasitism without damaging the nest. Artificial nests can be transported easily to research facilities that have CT scanners; each scan including image processing takes less than 30 min per nest and currently costs less than AUD 20.00, which is within the reach of apiculturists. X‐ray CT could be used to follow the life cycle of blue‐banded bees and other cryptic species and also to monitor their health.

Combining X-ray micro-CT technology and 3D printing for the digital preservation and study of a 19th century cantonese chess piece with intricate internal structure

Laser scanning has been widely adopted for the digital preservation and study of cultural heritage artifacts. However, surface scans alone are not sufficient for a variety of intricate objects such as the Cantonese chess pieces that were delicately carved from ivory throughout the 19th Century. These pieces incorporate a puzzle ball base, which is comprised of several balls, one inside the other. In this project we explore the use of x-ray micro-CT technology to scan the piece with a voxel separation of less than 9 μm, By using masking and editing software we generate 3D surface models of the separate parts to enable close inspection of both exterior and interior sections. Visualizing the dataset at a much larger scale enables the viewer to appreciate the fine surface details imperceivable in the original piece. Finally, 3D scaled versions are printed where the small details are also easily observed and the inner balls within the puzzle ball base move freely. The work flow demonstrated in this article has important possibilities in digital preservation, documentation, and presentation.

Australian stingless bees improve greenhouse Capsicum production

Summary Australian stingless bees contribute to the pollination of some commercially important field crops, but it is unclear whether they can increase crop production reliably in the greenhouse environment. Three 20 week trials were therefore conducted, each using a different hive of Austroplebeia australis Friese and Trigona carbonaria Smith placed in separate glasshouses containing Capsicum annuum L. A third glasshouse contained C. annuum but no bees. In the third week of each trial, the numbers of pollen grains present on stigmatic surfaces and pollen tubes growing along styles were determined. Changes in brood volume were assessed by x-ray computerised tomography at weeks 1, 10 and 20. Additionally, the hives were weighed at these times. At the end of each trial, fruit diameter and length and their fresh and dry weights were measured as were seed fresh and dry weights. Bee behaviour was recorded in the third trial. T. carbonaria foraged less sporadically on C. annuum flowers than did A. australis, and pollination by both bee species showed their potential to increase fruit yield and quality. The effects of pollination by either species were, however, not consistent among the three trials. Hive weights and brood volumes for all colonies increased, so it is considered that both species thrived whilst being able to pollinate the plants. Both species therefore have the potential to improve fruit yield and quality within the greenhouse environment. It was noted that A. australis caused damage to the styles in each trial. This may be attributed to the foraging strategies employed by this species and further work is needed to determine optimum bee to flower stocking rates.

Imaging live bee brains using minimally-invasive diagnostic radioentomology.

Abstract The sensitivity of the honey bee, Apis mellifera L. (Hymeonoptera: Apidae), brain volume and density to behavior (plasticity) makes it a great model for exploring the interactions between experience, behavior, and brain structure. Plasticity in the adult bee brain has been demonstrated in previous experiments. This experiment was conducted to identify the potentials and limitations of MicroCT (micro computed tomograpy) scanning “live” bees as a more comprehensive, non-invasive method for brain morphology and physiology. Bench-top and synchrotron MicroCT were used to scan live bees. For improved tissue differentiation, bees were fed and injected with radiographic contrast. Images of optic lobes, ocelli, antennal lobes, and mushroom bodies were visualized in 2D and 3D rendering modes. Scanning of live bees (for the first time) enabled minimally-invasive imaging of physiological processes such as passage of contrast from gut to haemolymph, and preliminary brain perfusion studies. The use of microCT scanning for studying insects (collectively termed ‘diagnostic radioentomology’, or DR) is increasing. Our results indicate that it is feasible to observe plasticity of the honey bee brain in vivo using diagnostic radioentomology, and that progressive, real-time observations of these changes can be followed in individual live bees. Limitations of live bee scanning, such as movement errors and poor tissue differentiation, were identified; however, there is great potential for in-vivo, non-invasive diagnostic radioentomology imaging of the honey bee for brain morphology and physiology.

3-D visualisation, printing, and volume determination of the tracheal respiratory system in the adult desert locust, Schistocerca gregaria

Here, we describe a single micro‐CT scan with a spatial resolution of 10 μm of a 10‐day‐old adult male Schistocerca gregaria (Forskål) (Orthoptera: Acrididae) and we compare our tracheal volume (VT) determination with published work on the subject. We also illustrate the feasibility of performing non‐invasive ‘virtual dissection’ on insects after performing micro‐CT. These post‐processing steps can be performed using free downloadable 3‐D software. Finally, the values of producing stereo‐lithography (STL) files that can be viewed or used to print out 3‐D models as teaching aids are discussed.

A Preliminary Report on the Use of Bench-Top X-Ray Micro-Computerised Tomography to Study the Malpighian Tubules of the Overwintering Seven Spotted Ladybird Coccinella septempunctata L. (Coleoptera: Coccinellidae)

The application of micro-CT scanning techniques on a small sample of “Seven-spot ladybirds” Coccinella septempunctata, collected in December 2009, identified an accumulation of material with a very high, relative X-ray attenuation value in the malpighian tubules of most but not all of the individuals sampled. The passage of metals such as cadmium in soil through a food chain to finally accumulate in high concentrations in ladybirds and lacewings has been previously reported. The identification of the dense material found in our sample of ladybirds, its origin, and the process by which it accumulates in, and is processed by, the malpighian tubules is the challenge ahead. The authors speculate that a straightforward means of monitoring levels of metallic pollutants in the environment might emerge.

Three-dimensional rendering of otolith growth using phase contrast synchrotron tomography

A three-dimensional computer reconstruction of a plaice Pleuronectes platessa otolith is presented from data acquired by the Diamond Light synchrotron, beamline I12, X-ray source, a high energy (53-150 keV) source particularly well suited to the study of dense objects. The data allowed non-destructive rendering of otolith structure, and for the first time allows otolith annuli (internal ring structures) to be analysed in X-ray tomographic images.