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Series: Advances in Insect Physiology

The x-rays are converted to visible light by a scintillator screen, and the resulting image is recorded by a CCD image sensor. Yellow material is Kapton, used to provide an x-ray transparent window to the animal. Internal chamber volume is 0. Video image quality as a function of x-ray energy and sample-detector distance. Data are from an ant head Camponotus pennsylvanicus using a Cohu video camera. Within each column, the absorbed x-ray dose on the insect is constant. Image quality versus TTRS. At least three trials were performed per data point.

TTRS measurements as a function of animal mass showed no correlation for the mass range 8. X-ray energy is 25 keV. Field of view is 1. Head and thoracic air sacs and leg trachea can be clearly seen. These images are taken with our new camera Cohu , which is twice as sensitive as the camera used in the major part of this study.

Although we subjectively consider iv to be a high quality image, usable images can be obtained using lower beam intensities. A major concern in using synchrotron x-rays to study physiological processes in small animals is the effect of the x-rays on the animal. Radiation causes molecular damage, including protein and lipid oxidation and gene transmutation; however, the effects depend on dose [ 9 ]. Previous studies show that fruit flies Drosophila melanogaster [ 10 ] and wasps Habrobracon and Bracon hebetor [ 11 , 12 ] temporarily lose motor control after a dose of about 1—2 kGy, but recover to normal behavior within minutes [ 9 ] or hours [ 12 ].

At exposures greater than 2. Feeding patterns are affected after D. In one study of D. However, in most prior studies of radiation effects on insects concerned primarily with insect control [ 14 ] and ageing [ 15 ] , animals have been subjected to full body irradiation; the few studies that examined localized x-rays have used low levels of radiation [ 16 — 19 ].

Thus it is unknown how insects are affected by intense, targeted radiation — such as in a synchrotron x-ray beam — on specific parts of the body. Furthermore, previous studies focused primarily on effects that occur on a relatively long time scale, usually days after irradiation, and few studies have examined immediate radiation effects.

This study strives to answer two questions: what combination of x-ray beam parameters optimizes image quality while minimizing damage to the animal? And under these conditions, how much time is available before the insect is negatively impacted? We varied x-ray parameters and used both CO 2 emission patterns and motor behaviors as proxy indicators to assess physiological damage in four insect species.

In addition, we demonstrate the range of studies that can be addressed using this technique by showing examples of high-resolution still imagery and real-time movement of food during ingestion and digestion. There is a trade-off between image quality and survivorship: higher quality images require greater exposures to radiation, which result in greater harm to the animal. With these settings, insects exhibited no negative behavioral effects for a period of about 5 minutes.

X-rays on the insect's head or thorax caused major changes to the respiratory pattern by about 17 minutes 2. With the beam on the abdomen, no significant changes were observed on the respiratory pattern throughout the full 2-hr trials No thermal effects of the x-rays were observed. Food transport and gut structures could be clearly seen using labeled food Figure 1e—l. In cases where tracking food transport was more important than maximizing the clarity of internal anatomy, Although not explicitly tested, the shorter wavelength of the x-rays at this setting results in lower absorption and therefore lower impact on the animals.

We observed insect feeding under irradiation for more than 30 minutes, depending on species and location of the x-rays on the insect. Figure 3 demonstrates the advantage of phase-contrast imaging over conventional absorption-based imaging. At a fixed energy, increasing d clearly increases the image contrast, as predicted by Equation 1. Measurements from both the thermocouple and the infrared camera showed no change in temperature to the irradiated insect.

The effect of x-ray radiation dosage on metabolic rates was quantified by examining the effect of incident beam flux density on the ratio of mean CO 2 emission rate during the 2 minutes after 'beam on' divided by the mean CO 2 emission rate during the 2 minutes before 'beam on' R 2 min all measurements at 25 keV; Figure 5. Except for grasshoppers, the data show a slight but significant increase in average CO 2 emission immediately after 'beam on'.

This small increase is probably the result of movement of the insect seen in the x-ray videos as it tried to move away from the beam. However, increasing beam intensity to four-times nominal values had little effect on R 2 min , suggesting that, although insects appear to sense the beam, there is a considerable safety margin in the capacity to absorb x-rays before major physiological damage occurs during the initial minutes of exposure. R 2 min as a function of incident beam power density. R 2 min is the ratio of mean CO 2 emission rate during the 2 minutes after 'beam on' divided by the mean CO 2 emission rate during the 2 minutes before 'beam on'.

Error bars denote standard deviation; numbers below each data point correspond to sample sizes. The 25 keV x-ray beam was incident on the head. Although CO 2 emission patterns during the first minutes after 'beam on' are similar to those prior to irradiation Figure 6 , a major change in the CO 2 emission pattern respiratory signal, RS was observed in all species after — s of irradiation. The RS was correlated with dorsoventral head-shaking in the ants and beetles, and a quivering proboscis in fruit flies, but was not correlated with any observable behavior in grasshoppers.

We note that for ants, the RS is qualitatively similar to the 'mortal fall' signature observed for ants under thermal stress [ 20 ], even though in our case there were no measurable temperature changes 0. Shortly after the RS, the CO 2 emission pattern became periodic for all ant samples, and for most of the beetle 12 of 15 and grasshopper 9 of 16 samples. For ants, these periodic patterns resemble discontinuous gas exchange DGC reported in decapitated ants [ 21 ], so we interpret the RS as indicating major brain damage.

Representative CO 2 emission traces for the four species used in this study. No qualitative changes are seen immediately after beam on. A major change in CO 2 emission pattern after — s of x-ray exposure is marked by RS respiratory signature. The RS was based on CO 2 emission patterns and was corroborated with x-ray video behavioral data; the RS is a major change in CO 2 release pattern associated with shaking of the head or mouthparts.

For the grasshoppers Schistocerca gregaria , no behavioral change was observed at RS. The one exception was grasshoppers at the highest power density, which showed a higher TTRS than the other species. Figure 4b shows still images taken from the video corresponding to the different incident power densities. Together, Figures 4a and 4b provide a guide for an experimenter to gauge the compromise between image quality and physiological impact. One possible explanation for this lack of pattern is that, in all cases, major portions of the brain were irradiated.

These results are consistent with the fact that in ants, and most insects, most ventilatory activity is controlled by major ganglia of the central nervous system in the head and thorax [ 22 ]. Comparison of x-ray impact on two different regions of the insect body. Representative CO 2 traces are from two different ant specimens Camponotus pennsylvanicus with the x-ray beam targeted on the abdomen a and the head b.

Even though the abdomen-irradiated ant a received a higher x-ray flux 80 vs.

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In contrast, the head-irradiated ant b showed dramatic changes in CO 2 emission, including a decrease in variance leading up to the RS at which point the head stopped moving and a cyclic pattern of release similar to DGC in decapitated ants [25, 26] thereafter. No changes in behavior were observed within the first 5 minutes of exposure. During 6—25 minutes of x-ray exposure, ants, beetles and flies progressively lost motor abilities, starting with leg twitches and ranging to full immobility.

By contrast, after 2 hrs of exposure, the grasshoppers could still right themselves, hop, feed and fly and were later observed to mate and lay eggs. One major difference between the grasshoppers and all other insects studied is that, because of their large size, only a part of the grasshopper's head was irradiated as opposed to the entire head in the other taxa. We note that, consistent with other studies [ 11 , 12 , 23 ], the loss of locomotor abilities observed in the insects at lower dosages was temporary, indicating radiation-induced lethargy.

In many individuals, we observed recovery minutes to hours later, suggesting that radiation damage was at least partially repairable. Our measurements show that a major change in CO 2 emission pattern, probably indicating major damage to the central nervous system, occurred after about 2. No change in CO 2 emission was observed if the x-ray beam was incident on the abdomen. The TTRS was independent of mass and species.

In ants, beetles and juvenile grasshoppers whose entire heads were irradiated, a cyclic or discontinuous gas exchange DGC CO 2 emission pattern [ 24 ] occurred after the RS. Ants have also been shown to exhibit DGC after they are physically decapitated [ 25 , 26 ], supporting the hypothesis that the x-ray treatment caused major brain damage. In cases where the RS was observed in this study, it is likely that the very high, acute dose of radiation caused profound tissue damage, causing such problems as potassium leakage [ 27 , 28 ] and leading to effects akin to the 'central nervous system syndrome' known from mammals [ 29 ].

One puzzling result is that although grasshoppers were no different in TTRS at some power densities, they showed a surprising degree of behavioral control after long periods of irradiation, suggesting a greater tolerance of x-rays to the head. For these animals, whose heads were larger than the size of the x-ray beam, the positioning of the x-ray beam may have missed or only partially damaged parts of the central nervous system, including the major ganglia controlling respiratory and motor function. In particular, partial control of motor behaviors such as walking occur in ganglia in the thorax [ 30 — 32 ].

Many of the smaller insects received incidental radiation on the thorax due to geometry during nominal 'head only' trials and exhibited motor loss, lending further weight to this hypothesis. Due to the many factors that contribute to the question of image quality versus survivorship, there is no single set of x-ray parameters that provide an optimal setting.

Generally, one would like a very small source size to minimize image blur, and an efficient detector system so that a less intense x-ray beam can be used to maximize survivorship.

Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function

In practice, for insect physiology, the first question is whether the particular internal dynamic or morphology can be visualized by this technique. Given the particular source and detector that is available, one usually starts with parameters that give superior image quality. Based on our experience with insects, this is usually with an x-ray energy of 10—20 keV and a sample-detector distance of 10— cm.

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After the desired feature is visualized, the experimenter can optimize the system based on the relative importance of image contrast, spatial resolution, and survivorship. For body functions that require shorter exposure times e. However, in many cases the total time needed to record such rapid phenomena will be lower. This improvement should double the working time from 5 to 10 minutes before any x-ray related effect is observed.

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Finally, although this study was targeted specifically at insects, these species were chosen primarily as exemplars to introduce the technique to the biological community. Synchrotron x-ray phase contrast imaging is broadly applicable to any organism with features on the micron scale and above. However, we urge caution when exploring new systems with this technique; it is crucial to understand the effects of the radiation on the organism when making biological interpretations. Synchrotron x-ray phase contrast imaging shows great promise as a powerful new tool for internal visualization in biological and medical research.

This is the only generally applicable technique that has the necessary spatial and temporal resolutions, penetrating power, and sensitivity to soft tissue that is required to visualize the internal physiology of small living animals on a scale from millimeters to microns. The impact of this technique is just beginning to be seen as it is applied to some of the more easily arranged experiments such as those on the respiratory systems of insects, where it has already had a major impact.

The discovery of rhythmic tracheal compressive movements in taxa in which it was previously unknown [ 6 ] has opened whole new areas of research, for example those aimed at determining morphological mechanisms of compression and the role of associated convection in insect physiology and evolution. Another exciting possibility is the visualization of previously unknown, complex circulatory patterns within insects that have only been inferred before from changes in body surface temperature [ 34 ].

Current uses of the technique include the analysis of the rapidly moving internal mouthparts of biting insects and the visualization of fluid motion in the pumping organs of fluid feeding insects such as flies and butterflies. The ability to see inside the animal, including the internal workings of jaws, legs, and wing hinges, may be of significant utility in the exploration of functional diversity.

Although more challenging due to lower density differences, this approach has also yielded impressive x-ray video of insect digestive Figure 1e—l ; see also Additional files 2 and 3 and circulatory system function, including the pumping of the tiny pulsatile organs that maintain the internal pressure of the antennae of ants. The first synchrotron research on living vertebrate musculoskeletal systems has recently begun with successful video of the interior bones of the pharynx and skull during fish respiratory pumping.

The potential for investigation of model systems in genetics and medicine such as fly, zebrafish, and mouse is considerable, as the natural and normal mechanisms of heart, circulatory, digestive, and locomotor systems can be analyzed in new ways and compared to mutants or disease models that may be used to study human health concerns.

Ultimately, the ability to clearly visualize internal functions in small animals will have a large impact in both biology and medicine. A Si double crystal monochromator was used to select the x-ray wavelength. Insects were mounted on top of a remotely controlled stage that enabled precise positioning in the x-ray beam. After passing through the insect, the x-rays were converted to visible light via a cerium doped yttrium aluminum garnet scintillator.

The sample-to-scintillator distance was approximately 1 m; a distance of this magnitude is necessary for obtaining the phase-contrast effect. The field of view was 2. Unlike most prior studies, the size of the x-ray beam was comparable to the size of the insect, and we only exposed parts of the insect to radiation.

Beetles were collected in the woods at Argonne National Laboratory, and grasshoppers were reared at one of the author's laboratory JH. Insects were housed with free access to food and water prior to experimentation. To determine the length of time that insects could withstand radiation on a particular part of the body head, thorax, or abdomen , insects were monitored for CO 2 release using flow-through respirometry while being observed with x-rays Figure 2b.

Insects were cold anaesthetized and placed individually in custom plexiglass respirometry chambers volumes: 0. Because some insects actively moved away from the beam upon contact, cotton was used to fill in gaps within the chamber to constrain the insect within the field of view.

The chambers were oriented such that the long axis of the body lay perpendicular to the beam path, providing either lateral or dorsoventral views. Chamber washout times were on the order of 6—12 s. Pre-beam CO 2 emission was typically recorded for 5—10 minutes before opening the x-ray shutter. For survivorship trials, insects were exposed to x-rays on the head, thorax, or abdomen until they clearly showed a respiratory signature that we infer to be respiratory function damage; otherwise, trials were ended after 2 hrs.

CO 2 emission was also monitored post-beam for up to 30 minutes. To demonstrate the use of x-rays to visualize internal food movement during ingestion and digestion, beetles Platynus decentis were fed macerated insects mixed with fine particles of CdWO 4 , and butterflies Pieris rapae were fed sugar solutions laced with an iodine contrast agent Isovue, Bracco Diagnostics, NJ, USA.

Animals were held in place by securing the body to a microscope cover slip beetles or by a mounted clamp attached to the wings butterflies. These examples illustrate the use of contrast agents to visualize internal food transport. The fine particles of CdWO 4 had a significantly higher absorption over the entire x-ray energy range used in this study and appeared darker than the surrounding soft tissue.

4. Insect central nervous system

In the case of the iodine solution, differences in x-ray absorption at the nominal setting 25 keV were minimal. To maximize contrast between the iodine and the surrounding anatomy, we used an energy just above the K-absorption edge Because in general the use of higher energy x-rays results in an overall lower contrast for soft tissue Figure 3 , this technique is most applicable for cases where it is more important to track internal movements of food than to visualize clearly the surrounding insect anatomy. The use of simultaneous x-ray images above and below the K-edge to improve visualization of the contrast agent is possible, but would require a more complicated set of x-ray optics.

Furthermore, it would imply a doubling of the x-ray dose to the animal. For contrast agents, iodine is more suitable for fluids whereas CdWO 4 is more suitable for solids. Although we have not investigated the toxicity of iodine versus CdWO 4 in insects, we speculate that iodine is less harmful because Isovue is used for human medical diagnosis, and it is well known that cadmium is toxic [ 35 ]. We chose a cadmium compound for its convenience, but other high electron density materials in powder form such as silica or lead can be used to provide radio-opacity with lower toxicity.

Possible change in temperature in the insect due to the absorption of x-rays was measured separately with two methods. First, an implanted 0. To test if local heating occurred at the site of irradiation, an infrared camera Inframetrics , 0. We used two metrics to quantify the effect of the x-rays on the CO 2 emission patterns.

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To assess immediate effects of the beam, we compared CO 2 emission rates in the 2 minutes before 'beam on' to those 2 minutes after 'beam on' Equation 2. We defined the ratio R 2 min as:. Second, to assess the duration required for damage to occur due to x-rays, we identified a major change in the CO 2 emission pattern within each species, which we refer to as the respiratory signature RS; Figure 6.

The RS was chosen for its repeatability; by the time of the RS, major and likely irreversible damage has occurred. Image quality and the corresponding photon fluxes and average power densities for 25 keV x-rays used in this study are shown in Figure 4. The incident photon fluxes were chosen for an approximate factor of two change in intensity between each setting. Absorbed power for a volume of insect that is irradiated can thus be estimated by Equation X-ray absorbed powers were measured for each species Table 1 ; these values are consistent with the theoretical estimates.

Metabolic rate calculations were based on averages of all available prebeam CO 2 recordings for each of the four species. Conversion from CO 2 output to metabolic rate assumed respiratory quotients RQ of 1. J Near Infrared Spectroscopy. J Insect Sci. Ultrasound Med Biol. Country of ref document : BR. Country of ref document : AU.

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