The Modified Pharaoh Approach: Stingless bees mummify beetle parasites alive

Social insect colonies usually live in nests, which are often invaded by parasitic species^1^. Workers from these colonies use different defence strategies to combat invaders^1^. Nevertheless, some parasitic species are able to bypass primary colony defences due to their morphology and behaviour^1-3^. In particular, some beetle nest invaders cannot be killed or removed by workers of social bees^2-5^, thus creating the need for alternative social defence strategies to ensure colony survival. Here we show, using Diagnostic Radioentomology^6^, that stingless bee workers _Trigona carbonaria_, immediately mummify invading destructive nest parasites _Aethina tumida_ alive, with a mixture of resin, wax and mud, thereby preventing severe damage to the colony. In sharp contrast to the responses of honeybee^7^ and bumblebee colonies^8^, the rapid live mummification strategy of _T. carbonaria_ effectively prevents beetle parasite advancements and removes their ability to reproduce. The convergent evolution of live mummification by stingless bees and social encapsulation by honeybees^3^ suggests that colonies of social bees generally rely on, secondary defence mechanisms when harmful nest intruders cannot be killed or ejected easily. This process is analogous to immune responses in animals

Diagnostic Radioentomology

Apart from the Neotropical flesh eating Trigona species, all existing bees are pollen feeding. Approximately 5% of these form colonies. In honeybees, colony health is evaluated by measuring seasonal hive weight increases and by visual inspections. However, rather than indicating good colony health, hive weight increases can be attributed to increases in stores from foragers feeding precociously during times of colony stress. Additionally, the subjective nature of these methods, leads to large errors. Visual inspections with stingless bee colonies are particularly invasive. Many bees die during inspections because they drown in spilt honey. Re-sealing the hive also kills bees, and the queen risks being squashed. Nevertheless, studies on bees continue as new, improved methods emerge to replace the old. Diagnostic Radioentomology is an innovative, non-invasive, imaging method for studying insects. Since development, it has been adopted by universities, synchrotron facilities and CT scanners to study morphology, physiology and behaviour of insects and has been hailed as the ‘Gold Standard’ for honeybee monitoring. In 2008, it was described as an emerging non-invasive technique for behavioural, evolutionary and classical biologists who choose to study animals without harming them. This chapter describes methods and includes examples of research conducted using Diagnostic Radioentomology

Rapid assessment report: Upper Rocky Arroyo restoration project

The Upper Rocky Arroyo Restoration Project (URAR) is located on the Lakeside Ranger District, Apache-Sitgreaves National Forest (ASNF). The URAR project area covers a landscape of approximately 30,860 acres and is one of the ASNF's "Bridge the Gap" Projects identified in 2013 to help accelerate restoration treatments, to: continue to provide restoration treatments on large, "at risk" landscapes, provide wood fiber from tree thinning activities for wood-products industries in the White Mountain area, help "bridge the gap" of treating large landscapes to reduce the threat of unwanted wildfire or other disturbances, while the larger 4FRI analysis is being completed, and ensure watershed function and integrity is not adversely affected. The project area is dominated primarily by ponderosa pine (Pinus ponderosa), with Gambel oak (Quercus gambelii), alligator juniper (Juniperus deppeana), Utah juniper (Juniperus osteosperma), one-seed juniper (Juniperus monosperma), pinon pine (Pinus edulis), white fir (Abies concolor), Douglas-fir (Pseudotsuga menziesii), and aspen (Populus tremuloides) present as lesser species. The landscape is predominately relatively flat terrain interrupted by distinctive ephemeral drainages generally flowing northward in the Silver Creek Watershed with several volcanic cinder cones across the area. Generally, three Potential Natural Vegetation Types (PNV) are represented on the URA area, which include ponderosa pine, Madrean pine-oak, and pinon-juniper. The Ecological Restoration Institute (ERI) was invited by the ASNF to collect site-specific historical ecological data for the URAR Project area to establish site-specific reference conditions (forest conditions that were in place 130-140 years ago when frequent fire was still a dominant component of the ecological system). These reference conditions would be used by the interdisciplinary team (IDT) as a point of reference for forest restoration project planning. To meet this need, ERI worked with the Lakeside Ranger District Ranger and staff to identify priority areas where data would be collected to establish reference conditions. ERI placed 49 individual study plots within the project area (Figure 1). The entire plot data collection was completed through a Rapid Assessment process and is documented in Appendix C of this report (Plot Data Summary Appendix C). Data on other ecological conditions were not collected as part of this effort; however, some of these data, such as fire history, are available from other sources (see Historical Context, Appendix A) and included in this report

Larson forest restoration project historic range of variation (HRV reference conditions) assessment report. Special report to the Apache-Sitgreaves National Forests.

The Larson Forest Restoration Project (Larson Project) is located on the Apache-Sitgreaves National Forest (A-S N.F.), Black Mesa Ranger District. The project covers a landscape area of approximately 30,000 acres, dominated primarily by ponderosa pine (Pinus ponderosa), with some dry mixed conifer on the north facing slopes and an increase in alligator juniper (Juniperus deppeana) component on the dryer sites in the north portion of the project. The A-S N.F. asked the Ecological Restoration Institute (ERI) to help collect site-specific historical ecological data for the Larson Project area to establish site-specific reference conditions (forest conditions that were in place 130-140 years ago when frequent fire was still a dominant component of the ecological system). These reference conditions would be used by the interdisciplinary team (IDT) as a point of reference for forest restoration for project planning

Radio-frequency electromagnetic field exposure of Western honey bees

Radio-frequency electromagnetic fields (RF-EMFs) can be absorbed in all living organisms, including Western Honey Bees (Apis Mellifera). This is an ecologically and economically important global insect species that is continuously exposed to environmental RF-EMFs. This exposure is studied numerically and experimentally in this manuscript. To this aim, numerical simulations using honey bee models, obtained using micro-CT scanning, were implemented to determine RF absorbed power as a function of frequency in the 0.6 to 120 GHz range. Five different models of honey bees were obtained and simulated: two workers, a drone, a larva, and a queen. The simulations were combined with in-situ measurements of environmental RF-EMF exposure near beehives in Belgium in order to estimate realistic exposure and absorbed power values for honey bees. Our analysis shows that a relatively small shift of 10% of environmental incident power density from frequencies below 3 GHz to higher frequencies will lead to a relative increase in absorbed power of a factor higher than 3

Imaging live bee brains using minimally-invasive diagnostic radioentomology

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

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 parasite