Application to monitoring of the Selangor River fireflies
The laboratory tests established that flash density in images of group displays was directly related to actual firefly density. In images taken at greater distance in the field, different operators readily identified flashes. Site, sampling period, or camera angle had very little impact on overall image interpretation, but subjectivity in the interpretation of the dimmest spots against background noise led to slight overall differences in counts between operators. The counts of different operators can, however, be standardised against a single operator.
Short exposure times increased variation in counts, depending on the level of synchrony with flash cycles, whereas longer exposure times increased the problem of movement of the fireflies. The choice of 0.5 s for P. tener enabled consistent capture of one or two flashes of the dominant males, which have a flash interval of about 0.27 s (Case, 1980). At this exposure time, double flashes usually registered as single bright spots, except during high winds.
In particle analysis, the green separation layer was better than the red even though red was a significant component of the light produced by P. tener, because there was also a strong red component in the background noise. As particle detection was continuous with no point of inflection in the curve when threshold was decreased, it could be inferred that some of the bright spots in the images contrasted only weakly with the background and no single threshold would reliably predict manual counts. However, manual counts had a linear relationship with particle analysis counts at a range of thresholds, providing overall intensity of bright spots was sufficient for detection. Images that had inadequate spot intensity generally occurred in distant sections of the river where the vegetation is viewed at an extreme angle, and could be recognised visually or by examining the green levels of bright spots in image editing software.
The relationship between manual and particle analysis counts varied with site and sampling period as a result of behavioural, environmental, and physical differences, precluding a universal prediction model. However, within sites and sampling periods, regression could still be applied with a high degree of accuracy to estimate counts based on three-point, zero-intercept regression lines. This greatly reduced handling time. Greater accuracy and less bias in the regression slope were achieved by selecting images with high particle analysis counts from a variety of camera angles. Very slight underestimation is expected as a result of selecting images with the highest counts, which tend to have slightly higher detection rates.
In tests of the comparability of counts from two cameras, the EOS 5DII registered the same flashes as the EOS 5D at shorter camera-subject distances, but registered more flashes than the EOS 5D at greater distances, requiring multiple regression with distance and particle parameters for cross-calibration of the cameras. The greater ability of the EOS 5DII to differentiate and to register subtle differences in flash brightness made manual count prediction from particle analysis possible under a wider range of flash intensities and camera-subject distances than in the EOS 5D. In addition, multiple linear regression could virtually eliminate the need for an estimate of the regression slope for each site each month. As digital camera sensors become more advanced, particle analysis has the potential to replace manual counts, enabling the processing of large numbers of images in a short time.
When used as a tool for monitoring the Selangor River firefly population, the technique provided an index of abundance that could be used to determine population trends, although biased towards the more dominant males, which display more consistently (Case, 1980). The effects of fluctuating flash patterns, which were probably related to behavioural changes in flash intensity and orientation (Case, 1980), were effectively minimised by panning the camera three times so that a time lapse of a minute or more occurred between replicate images. Errors due to flash behaviour, weather, and time of night are unlikely to affect interpretation of population trends because of the large area that could be monitored by this technique and the great differences in firefly abundance observed over time.
Application in the study of other light emitting organisms
The technique can be adapted to monitor a variety of firefly species and other light emitting organisms using the principles demonstrated in this case study. Where clear views from a stationary vantage point are not possible, it may be possible to hand-hold a camera at closer range and minimise effects of camera shake and variation by using a shorter exposure time.
The method described will also be a useful tool for the study of group display behaviour, supplementing conventional tools such as photomultipliers and oscilloscopes and having the advantage of enabling recognition of individual organisms, counts of numbers, and spatial analyses.
Advantages and limitations of the technique
Using this technique, large areas of habitat can be monitored in a short period of time with relatively inexpensive, consumer cameras. Other methods such as sweep netting (e.g. Zaidi et al., 2005) or visual counts through a cut-out window (Nallakumar, 2002) would not be able to monitor such large areas of habitat and are more likely to be affected by patterns of migration and local variation. The technique also enables more objective calibration for differences between operators, and is more quantitatively sensitive than visual scores of abundance.
Although the technique has few limitations, it does require a clear view of the display areas of the light emitting organisms, preferably from a stable platform. It also eventually requires carefully calibrated migration from one camera system to another for continuity.