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 our video camera and beamline
configurations, we found a satisfactory comprise between image quality
and survivorship by using 25 keV x-rays at 80 μW/mm2 flux density (2 × 1010 ph/s/mm2)
and 1 m sample-detector distance (hereafter referred as 'nominal'
settings). 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 (2.4 kGy). With the beam on the abdomen, no
significant changes were observed on the respiratory pattern throughout
the full 2-hr trials (17.3 kGy), or even in two trials that were
extended to 4 hrs (34.5 kGy). 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, 33.2 keV x-rays were
successfully used to visualize iodine-laced food. 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 d =
5 cm, where the phase effects are minimal, image contrast is poor for
all energies, consistent with the fact that the absorption is small.
For d = 5 cm and E = 15 keV, although some
differences due to absorption can be seen, it is the small
phase-contrast edge enhancements that make the features easily
discernible. At a fixed energy, increasing d clearly increases the image contrast, as predicted by Equation 1. A careful comparison of the image at d = 100 cm with d = 5 or 50 cm shows that the spatial resolution of the d = 100 cm image is poorer: the line widths at the edges of the air sacs are broader in the d = 100 cm image.
Thermal effects due to x-ray irradiation
Measurements from both the thermocouple and the infrared camera
showed no change in temperature to the irradiated insect. This is not
surprising, because the nominal power absorbed by the insect is, in the
most extreme case, only about 20% of its unirradiated metabolic rate
Table 1. Measured no-beam metabolic rates and absorbed powers under x-ray irradiation (25 keV, 80 μW/mm2) for the four species studied.
X-ray irradiation effects on CO2 emission patterns
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 CO2 emission rate during the 2 minutes after 'beam on' divided by the mean CO2 emission rate during the 2 minutes before 'beam on' (R2 min) (all measurements at 25 keV; Figure 5). Except for grasshoppers, the data show a slight but significant increase in average CO2 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 R2 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
Figure 5. R2 min as a function of incident beam power density. R2 min is the ratio of mean CO2 emission rate during the 2 minutes after 'beam on' divided by the mean CO2 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
Although CO2 emission patterns during the first minutes after 'beam on' are similar to those prior to irradiation (Figure 6), a major change in the CO2 emission
pattern (respiratory signal, RS) was observed in all species after
1000–1500 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 ,
even though in our case there were no measurable temperature changes
(0.1 K resolution) in the animal. Shortly after the RS, the CO2 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 , so we interpret the RS as indicating major brain damage.
Figure 6. Representative CO2 emission traces for the four species used in this study. The x-ray beam (25 keV, 80 μW/mm2) was incident on the insect's head. No qualitative changes are seen immediately after beam on. A major change in CO2 emission pattern after 1000–1500 s of x-ray exposure is marked by RS (respiratory signature). The RS was based on CO2 emission patterns and was corroborated with x-ray video behavioral data; the RS is a major change in CO2 release pattern associated with shaking of the head or mouthparts. For the grasshoppers (Schistocerca gregaria), no behavioral change was observed at RS.
Time to respiratory signal (TTRS) varied strongly with incident beam power density (Figure 4a);
higher power densities resulted in lower TTRS for all species. 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.
TTRS dependence on insect mass
Measurements at the nominal beam intensity showed no mass dependence
on the TTRS for grasshoppers (N = 9, 13.3–1473.5 mg, Spearman ρ = 0.23, p = 0.51) and ants (N = 14, 8.4–53.7 mg, Spearman ρ
= -0.24, p = 0.38). One possible explanation for this lack of pattern
is that, in all cases, major portions of the brain were irradiated.
TTRS dependence on x-ray beam location on the insect body
From CO2 emission measurements in ants, there is no
significant difference (Student's t-test, p = 0.4) in TTRS between
having the nominal x-ray beam incident on the head (N = 3, TTRS = 1299
± 177 s) or the thorax (N = 3, TTRS = 1092 ± 249 s) of the animal. In
contrast, no change in CO2 emission pattern was observed with the x-ray beam incident on the abdomen (N = 3, Figure 7a),
even after 4 hrs of exposure at the nominal beam intensity. 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 .
Figure 7. Comparison of x-ray impact on two different regions of the insect body. Representative CO2 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. 36 μW/mm2), it showed no discernible changes in CO2 emission pattern. In contrast, the head-irradiated ant (b) showed dramatic changes in CO2 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.
X-ray irradiation effects on motor function
Using simple behavioral assays, we tested for the presence/absence
of righting behavior, defensive behavior, and locomotor ability after a
fixed duration of x-ray exposure on the head using nominal beam
settings (Table 2).
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.