into the same clade and support they are potential orthologs
across bug species which was consistent with Hull's assumption
(Hull et al., 2014). Selective pressure assess by calculation of
dN/dS or ω = 0.295 (Figure 1) also indicates that genes in
this cluster are under purifying selection and would perform
conserved functions (Qiao et al., 2009; Zhou et al., 2010;
Vandermoten et al., 2011). Meanwhile, we found the AlinOBP11
was predominately expressed at mouthparts similar to tissue
expression profiles of previous reported LylinOBP19 (Hull et al.,
2014) and our further studies of AfasOBP11 and AsutOBP11
(data not shown here). However, Hua et al. (2012) suggested
that AlucOBP36 (named as AlucOBP3 in their study) was
antennae-biased expressed. This could be explained by different
genetic relationships and evolutionary processes of these bugs.
A. lineolatus, A. suturalis, and A. fasciaticollis belong to the same
genus Adelphocoris, while A. lucorum belongs to the other genus
Apolygus. Notably, the in vitro functional studies of antennae
expressed AlucOBP36 resembled our results of AlinOBP11,
which also showed better binding abilities to non-volatile host
plant secondary compounds of rutin hydrate, but not to quercetin
and gossypol (Hua et al., 2012), and this could be attributed to the
mutations of several amino acids in these two proteins' binding
pockets.

In conclusion, this study characterizes a mouthparts enriched OBP11 protein in A. lineolatus which preferentially binds to non-volatile plant secondary compounds; to our current knowledge, AlinOBP11 represents the first physiological function of mouthparts highly expressed OBP in Hemiptera species. As putative orthologous genes probably exhibited conserved physiological function, orthologous OBP11 could be involved in mirid bug feeding behaviors and serve as potential molecular targets for the development of eco-friendly pest management strategies against mirid bugs' outbreaks.

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AUTHOR CONTRIBUTIONS

LS and YZ conceived and designed the experimental plan. LS, XM, and YX preformed the experiments. LS, YW, DZ, YZ, XY, QX, and YG analyzed the data. LS and DZ drafted the manuscript.

ACKNOWLEDGMENTS

This work was supported by China National “973” Basic Research Program (2012CB114104), the National Natural Science Foundation of China (31272048, 31321004, 31471778, and 31501652) and Research Foundation of State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF201514).

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found
online at: http://journal.frontiersin.org/article/10.3389/fphys.
2016.00201

**Figure S1 | SDS-PAGE analyses of AlinOBP11 expression and purification.**
Protein markers are shown in the left side; —, crude bacterial extract before induction with IPTG; + crude bacterial extracts after induction with IPTG; Sup, supernatant of disrupted PET/AlinOBP11; Pel, inclusion body of disrupted PET/AlinOBP11; P/His-tag, purified AlinOBP11 protein with His-tag; P, finally purified AlinOBP11 protein obtained after two rounds of purification.

**Figure S2 |** The relative transcript levels of *AlinOBP11* at different developmental stages and adult tissues of both sexes evaluated by qRT-PCR with *AlinElongation factor* (GenBank No.AEY99651) as internal control. The results clearly showed *AlinOBP11* was strongly expressed at adult mouthparts.

**Table S1 |** The primers used in this article.

**Table S2 |** The protein names, GenBank accession numbers, and references of OBPs used in the phylogenetic analysis.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Sun, Wei, Zhang, Ma, Xiao, Zhang, Yang, Xiao, Guo and Zhang. *This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*
<figure><img src="image_144.png" /></figure>

# Flight control and landing precision in the nocturnal bee *Megalopta* is robust to large changes in light intensity

Emily Baird¹*, Diana C. Fernandez², William T. Wcislo³ and Eric J. Warrant¹

¹Department of Biology, Lund University, Lund, Sweden, ²Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, Canada, ³Smithsonian Tropical Research Institute, Panama City, Republic of Panama

Like their diurnal relatives, *Megalopta genalis* use visual information to control flight. Unlike their diurnal relatives, however, they do this at extremely low light intensities. Although *Megalopta* has developed optical specializations to increase visual sensitivity, theoretical studies suggest that this enhanced sensitivity does not enable them to capture enough light to use visual information to reliably control flight in the rainforest at night. It has been proposed that *Megalopta* gain extra sensitivity by summing visual information over time. While enhancing the reliability of vision, this strategy would decrease the accuracy with which they can detect image motion—a crucial cue for flight control. Here, we test this temporal summation hypothesis by investigating how *Megalopta's* flight control and landing precision is affected by light intensity and compare our findings with the results of similar experiments performed on the diurnal bumblebee *Bombus terrestris*, to explore the extent to which *Megalopta's* adaptations to dim light affect their precision. We find that, unlike *Bombus*, light intensity does not affect flight and landing precision in *Megalopta*. Overall, we find little evidence that *Megalopta* uses a temporal summation strategy in dim light, while we find strong support for the use of this strategy in *Bombus*.

**Keywords:** flight control, light intensity, neural summation, *Megalopta*, *Bombus*

**OPEN ACCESS**

**Edited by:**
Sylvia Anton,
Institut National de la Recherche
Agronomique, France

**Reviewed by:**
Natalie Hempel De Ibarra,
University of Exeter, UK
Jake Socha,
Virginia Tech, USA

*Correspondence:
Emily Baird
emily.baird@biol.lu.se

**Specialty section:**
This article was submitted to
*Invertebrate Physiology, a section of the journal Frontiers in Physiology*

**Received:** 21 July 2015
**Accepted:** 12 October 2015
**Published:** 28 October 2015

**Citation:**
Baird E, Fernandez DC, Wcislo WT and Warrant EJ (2015) Flight control and landing precision in the nocturnal bee *Megalopta* is robust to large changes in light intensity. *Front. Physiol.* 6:305. doi: 10.3389/fphys.2015.00305

## INTRODUCTION

As light intensities fall, visual information becomes increasingly unreliable and nocturnal animals compensate for this by having eyes that are extremely sensitive to light (Warrant, 2008a,b). Many nocturnal insects, for example, possess superposition compound eyes, a design that greatly increases light capture compared to the apposition compound eye, which is better suited to fast vision in bright environments and is therefore more typical of diurnal insects (Land, 1981). Nonetheless, the nocturnal neotropical sweat bee *Megalopta genalis*, which relies heavily on visual information to control flight (Baird et al., 2011) and locate its nest stick (Warrant et al., 2004) in dim light, possesses apposition compound eyes. So how are these insects able to see at night? *Megalopta* elevate their photon capture by having unusually wide, light-sensitive rhabdoms, and very large facet lenses (Warrant et al., 2004; Greiner et al., 2004a). Although this increases the sensitivity of their eyes quite significantly, theoretical calculations indicate that it does not allow them to capture enough light to reliably control flight and to locate a small nest stick under the dense rainforest canopy at night (Warrant et al., 2004; Greiner et al., 2004a). Anatomical investigations suggest
that *Megalopta* most likely enhances visual reliability in dim light by neurally summing visual information in the spatial domain (Greiner et al., 2004b). In addition, theoretical analyses also suggest that they may also sum this information in the temporal domain (Theobald et al., 2006), although neither possibility has been tested behaviorally.

While improving the reliability of visual information,
temporal neural summation comes at the cost of decreasing
sensitivity to image motion (Sponberg et al., 2015), a crucial
requirement for flight control and landing in many flying insects
(for a review, see: Taylor and Krapp, 2007). To maintain the
precision of flight control in dim light despite a loss of temporal
resolution, the insect would need to reduce the overall speed of
image motion by flying slower as light levels decline. This strategy
has been observed in hornets (Spiewok and Schmolz, 2006),
honeybees (Menzel, 1981), and bumblebees (Reber et al., 2015).
Interestingly, *Megalopta* does not seem to change ground speed
in response to decreasing light intensities but instead appears
to sacrifice flight performance: when returning to their nest in
dim light, they fly with significantly more convoluted trajectories
than when returning in brighter light, sometimes even making
unsuccessful approach and landing attempts (Theobald et al.,
2007). One explanation for the increased tortuosity in their flight
paths is that the bees are losing temporal resolution without
making any compensatory decreases in speed, something that
would likely limit the precision with which *Megalopta* could
control its flight and land. If *Megalopta* does indeed use temporal
summation to enhance visual reliability in dim light without
flying slower, we would expect flight control and landing accuracy
to be significantly compromised as light levels fall. But is this
the case? Here, we aim to answer this question by investigating
experimentally the effect of light intensity on position control and
landing in *Megalopta* and compare this with similar experiments
performed on the diurnal bumblebee, *Bombus terrestris*, which
most likely uses temporal summation in dim light (Reber et al.,
2015).

MATERIALS AND METHODS

Animals

*M. genalis* create nests (otherwise referred to as nest sticks) by burrowing holes and tunnels into dead, broken branches, lianas and vines [typically 30–50 mm in diameter (Eickwort, 1969)], in the rainforest understory. The entrance holes to these nest tunnels are ~5 mm in diameter (Eickwort, 1969). *Megalopta* nest sticks were collected and transferred to an experimental site in the rainforest of Barro Colorado Island in Panama. The experiments were conducted in March and April 2013 (with the exception of the natural nest stick landings, which were performed in 2009, see below for details). A light meter (IL1700, International Light, USA) placed at the experimental location (2 m from the nest stick) recorded light intensity (illuminance in lux) at 1 s intervals using an electronic data logger built in-house. The time stamp of the light meter recordings was then carefully matched to the time stamp of the recordings from the camera. Trajectories of *Megalopta* returning to the nest in both experimental conditions

(see below for details) were filmed under infrared illumination at
25 fps (using a Sony Handycam HDR-HC5E, Sony Corporation,
Japan) during their normal foraging times, approximately 40 min
both before sunrise and after sunset. The light intensities at which
the flights were filmed depended on when the bees returned
and were therefore not under experimental control. In some
cases, more than one bee inhabited the nest stick so that two or
more individual flights were recorded per session. Because we
could not identify the individual bees, we therefore report the
approximate number of individuals included in the data set as
well as the absolute number of nest sticks.

B. terrestris experiments used a commercial hive (Koppert, UK) and were performed at Lund University in an indoor flight cage (2.3 m long × 2 m wide × 2 m high) during their peak activity period (between 08:00 and 14:00) at light intensities of either 19 or 190 lux. Bees returning to the nest were recorded in 30 min sessions interspersed with 30 min control periods to allow for habituation to the test condition and light intensity. For both experimental conditions (see below for details) only the first 10 flights to the nest were recorded in each session because they often occurred in quick succession— we could thus be confident that they represented 10 different individuals. Otherwise, experiments were conducted as for *Megalopta*.

For both species, different individuals were used for the two
different experiments (described below).

Statistics

Non-parametric Wilcoxon rank sum tests (z statistic) and Spearman's rank-order correlations at the 5% significance level were used for all statistical comparisons. The *r*<sub>s</sub> statistic of the correlation test indicates the strength and sign of the relationship between -1 (perfect negative correlation), 0 (no correlation), and 1 (perfect positive correlation). Values are reported as the median and 25-75% interquartile range (iqr). Linear regression analyses were performed using the "fitlm" function in Matlab 2015a (Mathworks), which provided the F-statistic vs. constant model value and associated *P*-value.

The Effect of Light Intensity on Flight
Control

The experimental apparatus consisted of a clear acrylic tunnel, 14 cm wide × 14.5 cm high × 50 cm long, mounted 65 cm above the ground (Figure 1A). The nest was placed at an opening in one end of the tunnel at least 2 days before recording began to ensure that the bees were accustomed to flying along the tunnel to exit and enter their nest. The tunnel remained in this position for the duration of the experiment. After 2 days of habituation to the tunnel, all of the bees that flew made direct trajectories through the tunnel to the nest. The nest entrance was covered with a 5 cm diameter white disk (which had a low contrast against the sandblasted Perspex back wall) having a central 1 cm diameter hole aligned with the entrance hole of the nest. The walls of the tunnel were lined with a pattern composed of randomly distributed black and white 3 × 3 cm squares. The top panel of the tunnel was sandblasted. Flights to the nest were recorded at 25 Hz using a camera (Sony Handycam
<figure><img src="image_145.png" /></figure>

<figure><img src="image_146.png" /></figure>

**FIGURE 1** | The experimental apparatus. (A) The experimental tunnel used to investigate the effect of light intensity on flight control in *Megalopta* and *Bombus*. Flights from the tunnel entrance to the nest were recorded using a camera mounted underneath the tunnel. (B) The experimental apparatus used to investigate the effect of light intensity on landing in *Megalopta* and *Bombus*. Approaches to and landings on the disk were recorded using a camera mounted to the side.

HDR-HC5E, Sony Corporation, Japan) mounted beneath the tunnel. The trajectories were analyzed over the first 25 cm of the tunnel to avoid including landing maneuvers at the nest. Fifty-one flights from 19 individuals from 11 nest sticks were recorded for *Megalopta* and 51 flights (23 flights at 19 lux and 28 flights at 190 lux) from approximately 20 individuals were recorded for *Bombus*.

Ground speed was calculated as the average of the two-dimensional distance traveled between successive frames divided by the time step between the frames (0.04 s). Accuracy of position control was calculated by finding the average lateral distance from the midline of the tunnel as well as the variance in lateral position (the iqr of lateral positions) for each flight.

## The Effect of Light Intensity on Landing

In these experiments, the flights of bees landing on either patterned disks or natural nest sticks (*Megalopta* only) were recorded (**Figure 1B**). Black-and-white concentric ring or radial patterns (*Megalopta* only) were printed on paper and attached to plastic disks, 10 cm in diameter with a 1 cm diameter hole at the center. The radial pattern provided strong expansion cues for bees approaching the disk while these cues were minimized

in the sector pattern. In these experiments, the entrance to the nest was not placed in a tunnel but was surrounded by clear space. The disks were fitted over the nest entrance such that bees returning to their nest would have to approach and land on them. A camera mounted to the side of the disks, parallel to the trajectories of the bees, recorded the landings. Leg extension was defined as the moment when the bees began to extend their front or middle legs prior to making contact with the disk (depending on which came first). Time to contact (TC) was calculated as the time between leg extension and contact with the disk. Sixty-one landings (27 for the ring pattern, 34 for the radial pattern) from 10 individuals from four nest sticks were recorded for *Megalopta*, 58 landings from approximately 20 individuals were recorded for *Bombus*.

The natural nest stick landings for *Megalopta* were performed in March and April 2009. In these experiments, light intensity measurements (recorded in cd/m²) were made every 5 min using a Kodak 18% gray card reflecting incident downwelling daylight and a light meter (IL1700, International Light, USA), at a location about 2 m from the nest (as for the other *Megalopta* measurements). The nest sticks sticks were between 30 and 50 mm in diameter with a ~5 mm diameter hole that has approximately 72% contrast with the surrounding wood (Warrant et al., 2004). For the purpose of consistency and ease of comparison, these light intensity measurements were exchanged for careful intensity measurements in lux that were taken under similar conditions and at the same time of year for identical pre- and post-sunset times in later years. It is important to note that we performed statistical tests using both units and they both indicated that light intensity had no significant effect on landing precision in this experiment.

## RESULTS

### Changes in Light Intensity Affect Flight Control in *Bombus* But not in *Megalopta*

To investigate the effect of light intensity on flight control in *Megalopta* and *Bombus*, we recorded the trajectories of bees flying through an experimental tunnel at different light intensities. Flights of *Megalopta* were recorded over a range of light intensities between 0.0014 and 40.4 lux (this could not be experimentally controlled as it was determined by when the bees chose to return to their nest after a foraging trip). Despite the four log units of difference, light intensity did not have a strong effect on ground speed ($r_s = 0.24, P = 0.09, n = 51$; **Figure 2A**) nor the absolute lateral position ($r_s = -0.06, P = 0.69$; **Figure 2B**). There is a suggestion, however, that the within-flight variance of lateral position is weakly affected by light intensity ($r_s = 0.26, P = 0.06$; **Figure 2C**). This result is also reflected in the linear regression analyses of the data and the statistical comparison with a constant relationship between the variables (details of these analyses are provided in each subplot of **Figure 2**). In contrast to *Megalopta*, the ground speed of *Bombus* flying at higher light intensities of 190 or 19 lux (the bumblebees were reluctant to fly in lower light intensities) was significantly affected by light intensity ($r_s = 0.46, P < 0.001, n = 50$; **Figure 2A**), although
<figure><img src="image_147.png" /></figure>

**FIGURE 2** | The effect of light intensity on flight control in *Megalopta and Bombus*. The effect of light intensity on ground speed (A), median lateral position (B), and variance (inter-quartile range) in lateral position (C) of *Megalopta* (blue stars; 51 flights, 19 individuals, 15 nests—different symbols indicate data from different nests) and *Bombus* [box plots; 23 flights (19 lux), 28 flights (190 lux), ~20 individuals] flying along an experimental tunnel (140 mm wide) at different light intensities. The boxes indicate the 25–75% quartile of the data, the red line indicates the median and the whiskers show the extent of the data. Gray lines indicate a linear regression analysis of the *Megalopta* data; details of the analysis and the statistical comparison (F-value) against a constant model are provided in each plot.

the absolute lateral position ($r_s = 0.07, P = 0.63$; **Figure 2B**) and the variance in lateral position was not ($r_s = 0.05, p = 0.76$; **Figure 2C**).

## Changes in Light Intensity Affect Landing Control in *Bombus* But not in *Megalopta*

To investigate the effect of light intensity on the precision of landing (measured in terms of the timing of the leg extension response), we recorded the final stage of return flights to the nest in *Megalopta* and *Bombus* under different light intensities. *Megalopta* landings on a concentric ring pattern (which provides strong visual expansion cues) were recorded over a range of light intensities between 0.0018 and 3.58 lux (once again, this was not under experimental control but rather determined by when the bees returned to their nest after a foraging trip). Over this range, light intensity did not affect the time between leg extension and

<figure><img src="image_148.png" /></figure>

**FIGURE 3** | The effect of light intensity on the timing of leg extension when landing in *Megalopta* and *Bombus*. (A) The effect of light intensity on the time between leg extension and contact (TC) with a concentric ring pattern (inset) in *Megalopta* (blue stars, 27 landings, 10 individuals, 4 nests—different symbols indicate data from different nests) and *Bombus* [box plots, details as in **Figure 1**; 21 landings (19 lux), 37 landings (190 lux), ~20 individuals]. (B) The effect of visual expansion cues on TC in *Megalopta* [ring pattern (inset): 27 landings, radial pattern (inset): 34 landings]. Box plot details as in **Figure 1** indicate the distance between the lower and upper quartile values, red lines indicate the median, whiskers indicate the entire spread of the data and red crosses indicate outliers. (C) The effect of light intensity on TC for a natural nest stick (inset) in *Megalopta* (23 landings, 4 individuals, 2 nests—different symbols indicate data from different nests). Gray lines indicate a linear regression analysis of the *Megalopta* data; details of the analysis and the statistical comparison (F-value) against a constant model are provided in each plot.

contact with the surface, TC ($r_s = -0.02, P = 0.91, n = 27$; see also details of the linear regression analysis in **Figure 3A**). In contrast, a single order of magnitude change in light intensity from 190 to 19 lux was sufficient to significantly affect TC in *Bombus* landing on the same pattern ($r_s = 0.4, p = 0.0016, n = 58$; **Figure 3A**). Because only light intensity varied in this experiment, this result suggests that visual information is important for initiating the leg extension response in *Bombus* but that it may not play such an important role in *Megalopta*.
To examine if visual cues are used to regulate the timing of the leg extension response in *Megalopta*, we compared TC for a concentric ring pattern, which provides strong visual expansion cues, with TC for a radial pattern, which provides only weak expansion cues over a similar range of light intensities (0.00094–19.94 lux). If the bees use visual expansion cues to initiate a leg extension, we expect that it will be initiated later (that is, TC will be reduced) for the radial pattern because the bees will receive little information about the distance to the surface. A TC lower than that obtained for the ring pattern (which we assume to represent optimal timing for landing) would indicate that landing has become less precise. Our results showed that TC was affected by the visual pattern (Wilcoxon rank sum, *z* = 3.3, *P* < 0.0001, *n* = 61; see also details of the linear regression analysis in **Figure 3B**), with leg extension occurring earlier for the ring compared to the radial pattern (ring: 200 [80] ms; radial: 120 [80] ms, median [iqr]). This decrease in TC for the radial pattern indicates that *Megalopta* becomes less precise when visual expansion cues are removed, suggesting that these cues are important for coordinating the timing of leg extension during landing.

To investigate if TC in *Megalopta* is robust to light intensity under more natural conditions, we filmed landings on unmodified nest over a range of light intensities between 0.0007 and 0.95 lux. Again, we found no effect of light intensity on TC (*r*<sub>s</sub> = −0.05, *p* = 0.81, **Figure 3C**), although the average of 116 [46] ms is lower than the value of 200 [80] ms recorded for the much larger ring pattern, suggesting that the size and visual saliency of the landing surface is another factor that affects the timing of leg extension in *Megalopta*.

DISCUSSION

In this study, we investigate the effect of light intensity on flight control and landing in a nocturnal (*Megalopta*) and diurnal (*Bombus*) bee species. Overall, we find that flight control and landing precision in *Megalopta* is not strongly affected by light intensity, even over a five orders of magnitude decrease from twilight down to illumination levels approaching starlight. In contrast, ground speed and landing precision in *Bombus* decrease significantly over just a single order of magnitude decrease in light intensity, from illumination levels similar to an overcast day to those experienced just before twilight.

The finding that light intensity does not have a strong effect
on ground speed in *Megalopta* is consistent with previous
findings (Theobald et al., 2007), despite the large methodological
differences between analysing natural return flights in the earlier
study and analysing flights in an experimental tunnel in the
present study. Does this lack of dependence of ground speed
on light intensity come at the cost of flight performance in
*Megalopta*? Surprisingly, we found that flight accuracy, at least
in terms of positioning between the walls of a tunnel, does
not worsen even over a five orders of magnitude decrease in
light intensity. Despite the increased sensitivity afforded by
their optical specializations (Greiner et al., 2004a), the ability to
maintain the same level of flight control precision over such a
broad range of intensities strongly suggests that *Megalopta* rely

on neural summation strategies to improve visual reliability in
dim light. The lack of a change in ground speed in combination
with a negligible effect on precision makes it unlikely that
*Megalopta* rely heavily on temporal neural summation strategies
to control flight in dim light but that they more likely rely heavily
on spatial summation strategies to do this.

In contrast to *Megalopta*, we observe a strong effect of light intensity on flight control in *Bombus*, even over a single log unit change in light intensity. These findings are consistent with previous work (Reber et al., 2015) and suggest that, as light intensities fall, *Bombus* use neural temporal summation to improve visual reliability and that they compensate for the subsequent loss of temporal resolution by flying more slowly, as hornets (Spiewok and Schmolz, 2006) and honeybees (Menzel, 1981) also appear to do. This compensatory decrease in ground speed allows them to obtain enough visual information to continue to control their position accurately.

To date, all investigations into the effect of light intensity on
flight control in insects have focussed primarily on how light
intensity affects ground speed. However, insects must control
more than their speed to be able to fly safely in dim light.
One of the most challenging behaviors that flying insects must
perform is landing. To orchestrate a safe and efficient landing,
flying insects need to determine the moment when they will
contact the surface so that they can extend their legs in time.
One cue that stimulates this leg extension response in tethered
flies is the apparent rate of image expansion generated by the
surface, which is used to measure the relative distance to the
surface and the TC (Goodman, 1960; Wehrhahn et al., 1981;
Borst, 1986; Borst and Bahde, 1986)—once this apparent rate of
expansion reaches a certain threshold value, the leg extension
reflex is initiated. To investigate if changes in light intensity
affect landing precision in *Megalopta* and *Bombus*, we analyzed
the effect of light intensity on the timing of the leg extension
reflex.

As with the other parameters of flight control discussed above, the timing of the leg extension reflex is not affected by light intensity in *Megalopta*, while in *Bombus* precision is clearly lost and they extend their legs much later (i.e., closer to the nest) when light intensity decreases. One possible explanation for the lack of observeable effect of light intensity on TC in *Megalopta* is that the leg extension response is not mediated by visual cues. However, when we tested the effect of removing expansion cues from the landing surface, we find that *Megalopta* extend their legs later, suggesting that visual cues do indeed play an important role in the control of landing and that the neural summation mechanisms they employ do not affect their ability to measure the rate of expansion of optic flow cues generated by the landing surface, despite a four orders of magnitude decrease in light intensity (note that the bees in the tunnel experiments flew over five orders of magnitude difference in light intensity while in the landing experiments the flights were distributed over four orders of magnitude).

Although our results show that the timing of the leg extension
response in *Megalopta* is not affected by light intensity, the
patterns that we used in the experiment were not representative
of the natural landing surface of an unmodified nest stick, for
which the only strong contrast cues are provided by the edge of the stick and the dark entrance. Although the timing of the leg extension was reduced for landings at the nest stick in comparison to landings on the disk, suggesting that the size and visual saliency of the landing surface might be an important cue, we find no effect of light intensity in this situation either. This result further supports our finding that flight control and landing precision in *Megalopta* is extraordinarily robust to large changes in light intensity.

Considered together, the results of this study reveal that the visual control of flight and landing in *Megalopta* is not affected by large changes in light intensity, even at intensities similar to a moonless clear night sky (~10⁻³ lux, according to our own measurements). Under similar experimental conditions (but under much brighter limiting light levels), *Bombus* fly more slowly and the time between leg extension and landing decreases—elevating their risk of colliding with the landing surface (an event that was frequently observed at low light levels)—even for a decline in light intensity of just a single order of magnitude. These findings suggest that the neural summation strategies employed by these two species are fundamentally different. The reduction in ground speed and landing precision observed in *Bombus* as light levels fall strongly supports the hypothesis that they rely on neural temporal summation mechanisms to obtain enough visual information to see in dim light. In contrast, *Megalopta* do not fly more slowly and nor does their flight accuracy appear to suffer, even over a very large range of light intensities. This strongly implies that their temporal resolution does not vary with light intensity and that spatial summation is instead employed to ensure sufficient visual reliability to control flight at night. At first glance, these findings appear to contradict those of Theobald et al. (2007), who showed that flight trajectories become more tortuous as light intensity

decreases, suggesting a loss of precision. Our results suggest, however, that the apparent loss of accuracy is not due to a decrease in the accuracy of flight control *per se* but rather to a decrease in the ability of *Megalopta* to accurately locate the nest stick due to increased spatial summation (as evidenced by the shorter time between leg extension and landing at a natural nest, **Figure 2C**). Nonetheless, these bees could use coarser spatial landmarks in the rainforest to systematically home in on the general vicinity of their nest stick, thus eventually allowing them to locate it.

Here, we show that the neural summation strategies of *Megalopta* are adequate for the fine control of flight and landing while also enabling them to navigate over large distances back to their nest across a broad range of light intensities, which change rapidly and somewhat unpredictably in their equatorial habitat (Endler, 1993). An improved understanding of how *Megalopta* increase their visual sensitivity without sacrificing flight precision will not only be important for understanding how animals use neural adaptations to optimize sensory information when signal-to-noise ratios are low but also for the development of artificial visual guidance systems that are effective in dim light.

## FUNDING

Air Force Office of Scientific Research/European Office for Aerospace Research and Development (grant numbers FA8655-07-C-4011, FA8655-08-C-4004). The Swedish Foundation for Strategic Research (2014-4762).

## ACKNOWLEDGMENTS

We would like to thank the staff at the Smithsonian Tropical Research Institute for logistical support.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Baird, Fernandez. Wcislo and Warrant. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
<figure><img src="image_149.png" /></figure>

# Hunting in Bioluminescent Light: Vision in the Nocturnal Box Jellyfish *Copula sivickisi*

Anders Garm¹*, Jan Bielecki², Ronald Petie¹ and Dan-Eric Nilsson³

¹ Marine Biological Section, Department of Biology, University of Copenhagen, Copenhagen, Denmark, ² Department of Ecology evolution and Marin Biology, University of California, Santa Barbara, Santa Barbara, CA, USA, ³ Vision Group, Department of Biology, Lund University, Lund, Sweden

Cubomedusae all have a similar set of six eyes on each of their four rhopalia. Still, there is a great variation in activity patterns with some species being strictly day active while others are strictly night active. Here we have examined the visual ecology of the medusa of the night active *Copula sivickisi* from Okinawa using optics, morphology, electrophysiology, and behavioral experiments. We found the lenses of both the upper and the lower lens eyes to be image forming but under-focused, resulting in low spatial resolution in the order of 10–15°. The photoreceptor physiology is similar in the two lens eyes and they have a single opsin peaking around 460 nm and low temporal resolution with a flicker fusion frequency (fff) of 2.5 Hz indicating adaptions to vision in low light intensities. Further, the outer segments have fluid filled swellings, which may concentrate the light in the photoreceptor membrane by total internal reflections, and thus enhance the signal to noise ratio in the eyes. Finally our behavioral experiments confirmed that the animals use vision when hunting. When they are active at night they seek out high prey-concentration by visual attraction to areas with abundant bioluminescent flashes triggered by their prey.

**Keywords:** cubozoa, night active, eyes, spectral sensitivity, foraging

**OPEN ACCESS**

**Edited by:**
Robert Huber,
Bowling Green State University, USA

**Reviewed by:**
Netta Cohen,
University of Leeds, UK
Rhanor Gillette,
University of Illinois at
Urbana-Champaign, USA

*Correspondence:
Anders Garm
algarm@bio.ku.dk

**Specialty section:**
This article was submitted to *Invertebrate Physiology, a section of the journal Frontiers in Physiology*

**Received:** 17 October 2015

**Accepted:** 01 March 2016

**Published:** 30 March 2016

**Citation:**
Garm A, Bielecki J, Petie R and Nilsson D-E (2016) *Hunting in Bioluminescent Light: Vision in the Nocturnal Box Jellyfish Copula sivickisi*. *Front. Physiol.* 7:99.
doi: 10.3389/fphys.2016.00099

## INTRODUCTION

Within Cnidaria a small group, the cubozoans, have diverged to evolve an elaborate visual apparatus along with an according expansion of their nervous systems (Satterlie, 1979; Garm et al., 2007). The cubomedusae, or box jellyfish, all possess a similar set of 24 eyes distributed on four sensory structures called rhopalia. Each rhopalium holds eyes of four morphologically distinct types, the upper lens eye, the lower lens eye, the pit eyes, and the slit eyes, and offer a clear example of special purpose eyes (Yamasu and Yoshida, 1976; Pearse and Pearse, 1978; Matsumoto, 1995; Nilsson et al., 2005; Garm et al., 2008). The optics have been investigated only in two species, *Tripedalia cystophora* and *Chiropsella bronzie*. In these species, the lens eyes provide low spatial resolution in the order of 10° or worse (Nilsson et al., 2005; O'Connor et al., 2009). This does not allow visually guided hunting for prey. Instead they use vision to seek out habitats with high prey densities. This is best understood for the Caribbean species, *T. cystophora*, which feed on copepods accumulating in light shafts between the mangrove prop roots (Buskey, 2003; Garm et al., 2011). They avoid the dark roots but are attracted to the light shafts where the positively phototactic copepods gather. Once in the right habitat they hunt passively with extended and trailing tentacles and the actual prey capture is no different from the typical scypho- and hydromedusa which rely on the prey accidentally contacting a tentacle.
The small eyes (pupil diameter <100 µm) of *T. cystophora* agree with their diurnal activity pattern: they are only found hunting between the prop roots during the day and rest on the bottom at night (Garm et al., 2012). Still, in all other examined species it seems to be different. Several species are found actively swimming both day and night, but unfortunately most of the data originates from tank experiments which might induce artificial behaviors (Yatsu, 1917; Satterlie, 1979). In one case, the Australian *Chironex fleckeri*, individuals have been tagged in the wild, and the tracking revealed that this species has great variations between individuals but at least some were just as active during dark hours as during the day (Gordon and Seymour, 2009). Whether they hunt and capture prey during the night is still unknown though. Interestingly, one species, *Copula sivickisi*, from the Indo Pacific is strictly night active and sits inactive and attached to the substrate during the day (Garm et al., 2012). This species is predominantly associated with coral reefs where it hunts a variety of planktonic crustaceans in the surface waters at night. Like *T. cystophora* they have internal fertilization and mating happens in the dark only (Hartwick, 1991; Lewis and Long, 2005; Garm et al., 2012, 2015). Despite the strictly nocturnal behavior, they still have the same set of small eyes (Figure 1) as the day active *T. cystophora*. How they locate each other or prey items in the dark is unknown and could in principle be governed by random swimming and accidental encounters. But this would be rather inefficient, unless prey, and mate densities are very high. In a recent paper we suggested an alternative method (Garm et al., 2012). At least at Okinawa, Japan, *C. sivickisi* are co-localized with the bioluminescent dinoflagellate *Pyrocystis noctiluca* which is constantly triggered to emit light by encounters with a variety of planktonic crustaceans (Garm et al., 2012). We hypothesized that the medusa of *C. sivickisi* are attracted by the flashes of blue light and thereby aggregate in areas with high prey densities.

Here we test the hypothesis that *C. sivickisi* locate areas of high prey densities using the bioluminescent signals from *P. noctiluca*. We focused on the lens eyes since they are the only image forming eyes and examined their optics and used electroretinograms (ERGs) to investigate their receptor physiology, including spectral sensitivity and temporal resolution. Further, we conducted behavioral experiments to test if they are attracted by the flashes emitted by *P. noctiluca*. All the results clearly support the hypothesis.

# MATERIALS AND METHODS

## Experimental Organisms

Medusa of *C. sivickisi* were collected using light traps in the harbor at Akajima, Okinawa, Japan, and brought back to Akajima Marine Science Laboratory (AMSL) where they were kept in 40–80 l tanks with seawater at 29°C and 33 psu, and fed *Artemia* or copepods daily. The traps attracted medusae of both sexes and all sizes (1.5–8 mm in diameter). The electrophysiological experiments on the temporal resolution was carried out at AMSL using 10 adult animals of both sexes, but for spectral sensitivity measurements, 15 juvenile medusae were brought back to University of Copenhagen and raised to mature size

during 4–5 weeks. They were raised in a 50 l tank at 29°C and 33 psu and fed SELCO enriched *Artemia* daily. Circulation in the tank was created by aeration and half the water was exchanged every other week. The behavioral experiments were conducted at AMSL using adult males and females (bell diameter 8–10 mm) within 3 days of capture. The dinoflagellate, *P. noctiluca*, was caught at nighttime in the harbor using a 100 µm plankton net. After manually sorting them from the rest of the plankton samples they were kept in a 60 l tank under natural light conditions at AMSL.

## Anatomical Model

As a basis for our analyses of visual optics, a geometrically accurate model was made of the two lens eyes and their position in the rhopalium. The model was based on histological sections as well as fresh rhopalia photographed from the front, the side and from above. The shape of excised fresh lenses, together with histological sections, were used to determine the position and dimensions of all optically relevant structures. The model was based on five rhopalia from five fully-grown medusae.

## Focal-Length Measurements in the Lens Eyes

Fresh lenses from the lens eyes were excised from the eye by tearing the retinal cup with two needles, one on either side of the lens. Roughly 50% of the attempts delivered seemingly intact lenses. The isolated lenses were placed in seawater on a microscope slide and covered by a cover-slip, which was supported to form a 1 mm deep cavity. The lens was arranged such that the longest axis was perpendicular to the incoming light, as would be the case in an intact eye. The microscope condenser was removed and a pinhole of 0.5 mm was placed 10 cm below the preparation. Images were then taken through a high numeric aperture objective (x50), focusing first at the lens equator and then at every 10 µm to a distance well below the depth of best focus. Measurements were performed on five lenses from lower lens eyes and three lenses from upper lens eyes.

## Electrophysiology

For measurements of the dynamic range and spectral sensitivity, extracellular ERG recordings were obtained from seven lower lens eyes and seven upper lens eyes originating from 11 adult individuals of both sexes. A maximum of two rhopalia were used per animal and only one eye from each rhopalium. Rhopalia were dissected from the animals by cutting the rhopalial stalk and afterwards they were transfered to a petri dish in the electrophysiological setup containing seawater (29°C, salinity 33 psu). A custom made glass suction electrode was placed on the edge of either the upper or the lower lens eye and suction was applied until a slight migration of pigment into the electrode was observed. The diameter of the electrode tip was 1–3 µm resulting in an impedance of 2–5 MOhm. Recordings were amplified 1000 times and filtered (0.1 Hz high pass, 1000 Hz low pass, and 50 Hz notch filter) via a differential AC amplifier (1700, A-Msystems Inc., WA) and recorded using a custom made program for Labview (Labview 8.5, National Instruments, TX). The light stimulus was provided by an ultra-bright white LED (Luxeon III
<figure><img src="image_150.png" /><figcaption>**FIGURE 1** | **The visual system of Copula sivickisi.** (A) An adult male fishing with outstretched tentacles. Framed area indicates location of one of the rhopalia. (B) Close up of a rhopalium showing four of the six eyes. Note the large crystal (Cr) distally. (C) Resting medusae lying upside down but the rhopalia still orient with the upper lens eye pointing straight upwards due to the heavy crystal. (D) Close up of framed area in (C) showing upright rhopalium, arrowhead indicates upper lens eye. (E) Medusa laying on the side still having upright rhopalia. (F) Close up of framed area in (E) showing upright rhopalium, arrowhead indicates upper lens eye. (G) TEM micrograph of photoreceptors in the lower lens eye. The outer segments (OS) are modified cilia, and the photoreceptors also contain screening pigment (Pg). Note apparent holes in the outer segments. (H) Close up of OS showing the cilia projecting densely packed microvilli. LLE, lower lens eye; SE, slit eye; ULE, upper lens eye.</figcaption></figure>

star, Philips, San Jose, CA) placed in a Linos microbench system (Linos, Goettingen, Germany). The microbench was equipped with a series of neutral density filters and interference color filters (half width = 12 nm, CVI laser, Bensheim, Germany). The stimulus was presented to the eye using a 1 mm light guide close to the pupil to create a close to even illumination of the entire visual field.

The experimental protocol started with 15 min of dark adaptation. Then an intensity series was presented covering four log units in steps of 0.3 or 0.7 log units starting at the low intensity end (1.1 × 10¹W/sr/m²). This was followed by an equal quanta

(6 × 10¹⁸ photons/s/sr/m²) spectral series covering 410–680 nm in 20 steps and the protocol ended with a 2nd intensity series to ensure that the sensitivity had not changed during the experiment. Each stimulus lasted 25 ms and the stimuli were presented with 1 1/2 min in between. Only data from eyes lasting a full protocol, where the 2nd intensity series differed <15% from the 1st, were used for the analysis. This similarity between the two V-log I curves also shows that the initial dark adaptation, stimulus duration, and inter stimulus times were long enough to avoid a change in adaptational state during the experiments. The data were analyzed manually in the program Igor Pro 6.12A
(Wavemetrics, Lake Oswego, Oregon). The spectral data were transformed by the V-log I curve to obtain the relative sensitivity (see Coates et al., 2006 for details on this procedure).

The temporal resolution of the lens eyes was examined using flicker fusion frequency (fff) experiments. Five upper lens eyes and five lower lens eyes were presented with a sinusoidal stimulus for 10 s covering the frequency spectrum 0.5–20 Hz in 0.5 Hz steps while recording the ERG as described above. Initially, the eyes were adapted for 10 min to the mid intensity of the stimulus, which was followed by the sinusoidal stimuli starting at the low frequency end with 2 min at mid intensity between. A full protocol thus lasted 90 min. The recordings were analyzed using a fast forward fourier transformation on what equals five stimulus cycles. The returned value at the principle frequency was normalized and used to create an fff curve.

## Behavioral Experiments

Nine fully grown medusae were placed in a 20 l tank with seawater at 29°C and 33 psu and without circulation. The experiment was conducted within the natural activity period of the medusae at 10 p.m. and the medusae had last been fed the night before. The tank was kept in a fully darkened room and the animals were left for 30 min to adjust to the tank. A similar tank next to the medusa tank with the same conditions held ~300 *P. noctiluca* caught between 24 and 48 h prior to the experiments. The two tanks were separated by 0.5 cm to minimize possible transfer of vibrations. After the 30 min, aeration was started in the *P. noctiluca* tank and continued for 2 min. The bubbling had a frequency of 2–3 Hz and triggered the bioluminescence immediately. The behavioral response to the bioluminescence was recorded using a Sony handycam (Sony DCR-HC44) under infrared light (IR-65LED, Loligo Systems, Denmark; peak wavelength = 850 nm, intensity at surface = 27.5 W/m²/sr). The video was analyzed in a custom made program for Matlab 2013b (Mathwork, Inc., Natick, MA, USA) which tracked the position of the medusae from 2 min before the onset of the bioluminescence to 2 min after with a 2 s time resolution. In a further analysis of the video recording the tank was divided in four equally sized horizontal sectors (#1 closest to the bioluminescence, #4 the furthest away) and it was noted in which sector each of the medusae were positioned again with a time resolution of 2 s.

# RESULTS

## Morphology

In *C. sivickisi* the rhopalia carry the six eyes, as typical for cubozoans (**Figures 1A,B**). The rhopalia hang in the rhopalial niche suspended on a flexible stalk. Along with the heavy crystal in the distal end, this results in the rhopalium always keeping the same vertical orientation with the upper lens eye pointing straight upwards (**Figures 1C-F**). The sections of the lens eyes show that they have the same structure with a thin cornea, a slightly elliptic lens, a thin vitreous space, upright ciliary photoreceptors also holding the brown screening pigment, and retinal associated neurons (**Figure 1G**). The lens cells appear dead and devoid of organelles in the center while the peripheral cells facing the retina

have nuclei and other organelles. The photoreceptors of the two lens eyes are very similar and have outer segments of 40–70 µm depending on the area of the retina, with the central ones being the longest. The outer segments form dense layers of microvilli arising from a single cilium (**Figures 1G,H**). Interestingly, the outer segments in both lens eyes have large empty swellings along the central axis appearing like holes in the retina (**Figure 1G**).

## Optics

Isolated fresh lenses were slightly ellipsoidal with the longer axes in the pupil plane (**Figures 2A,B**). Using a compound microscope to project parallel light through the lens we determined the focusing properties of the lens by measuring the width of the beam as a function of distance behind the lens. Lenses from both the upper and lower eyes brought light to a focus at a surprisingly short distance—approximately 100 µm. At the plane of best focus, the beam was converged to a diameter of 15–20% of the lens diameter. To account for the variation in eye size and lens size (about ± 25%) we normalized all measurements to units of lens diameter and plotted the beam profile in an anatomical model of the eye (**Figures 2A–E**). This demonstrated that the plane of best focus is at the base of the retina in both the upper and lower lens eyes. Even though the f-number (focal ratio) is lower than that found in the related jellyfish *T. cystophora*, we estimate that the spatial resolution will be roughly the same, i.e., 10–20° in both upper and lower lens eyes (compare **Figures 2A,B,F–H**).

## Dynamic Range

The electrophysiologically recorded dynamic range was very similar in the upper and lower lens eyes, and flashes of light with varying intensity resulted in graded impulse responses typically biphasic (**Figure 3A**). The dynamic range covered at least four log units from 1.1 × 10 to 1.1 × 10⁵ W/m²/sr (**Figures 3A, 4A**). It might well be broader tough, since the V-Log I curves showed no sign of saturation in the high intensity end (**Figure 4A**).

## Spectral Sensitivity

The spectral sensitivity curves were also very similar in the two lens eyes. They had a single peak in the deep blue part of the visual spectrum around 460 nm (**Figure 5**). Using the least square of the mean method to fit the spectral sensitivity curve of the lens eyes to theoretical absorption curves of opsins (Govardovskii et al., 2000), returned the best match in both lens eyes to a single opsin peaking at 458 nm (**Figure 5**). In contrast to linear models where the R² is commonly used, the goodness of fit for non-linear models is best described by Akaike's Information Criterion (AIC). The AIC for the opsin fit was -53.7 and -35.9 for the upper and lower lens eye, respectively. Note that more negative values reflect a better model fit.

## Temporal Resolution

The temporal resolution of the two lens eyes was tested using two different methods, a direct and an indirect. In the indirect method the width at half height of the impulse response was measured, indicating a difference between the two eyes. The upper lens eye showed a decreasing half width with increasing
<figure><img src="image_151.png" /></figure>

**FIGURE 2** | Geometrical model and optics of the lens eyes. (A,B) Frontal view of anatomical models of the lower (A) and upper (B) lens eye of *C. sivickisi*. Red line indicates average beam width of a distant point light source, broken red lines indicate maximum and minimum beam width (N = 5 in A and N = 3 in B). (C,D) Examples of the appearance of beam cross-sections behind a lens of the upper lens eye photographed at the lens equator and at the focal plane. (E) The average beam profilers from (A,B) plotted in the sagittal plane of the rhopalium. The yellow field highlights beam width for a point source and thus the blur spot in the retina. (F) Optical model of the lower lens eye of *Chiropsella bronzie* for comparison (modified from O'Connor et al., 2009). (G,H) Optical models of the lower and upper lens eyes of *Tripedalia cystophora* respectively (modified from Nilsson et al., 2005).

light intensity of the 25 ms flashes, and at the highest intensity the
half width was 29 ± 0.5 ms (mean ± SEM; **Figure 4B**). The lower
lens eye was slower at all tested intensities and had a minimal
half width of 42 ± 4 ms (mean ± SEM; **Figure 4B**). The flicker
fusion frequency (fff) experiments provided stable responses
to the sinusoidal stimulus throughout the entire stimulation
period (**Figure 3B**). Interestingly, when measuring the temporal
resolution with this direct method the difference between the
upper and lower lens eyes disappeared. Both eyes were very
slow and showed a close to linear decline in the response to
a sinusoidal flicker going from 0.5 to 2.5 Hz (**Figures 3B, 4C**).
Above 2.5 Hz no response was seen and the fff is thus ~2.5 Hz
for both eyes. At low frequencies, <1.5 Hz the response peak
preceded the stimulus peak putatively due to build in temporal
filters. The same is seen for *T. cystophora* (O'Connor et al.,
2010).

**Response to Bioluminescence from**
**Pyrocystis noctiluca**

When the tank holding the nine medusae was kept in darkness,
the medusae displayed what we consider to be a natural foraging
behavior, swimming slowly with their tentacles fully or partly
extended near the surface. They did not distribute evenly but
preferred the ends of the tank over the middle part (Figures 6A,
7), which is a natural consequence of random exploratory
behavior. Importantly, they spent the same amount of time in the
two ends (two-sided unpaired student t-test, *p* = 0.88). There was
a marked change in behavior soon after the aeration was turned
on and the bioluminescence initiated (Figure 6B). Some medusae
swam directly to the end of the tank toward the bioluminescence
and stayed there throughout the 2 min. Others took 10-20 s
before swimming toward the light but all medusae spend most
of the 2 min in zone 1 close closest to the bioluminescence
<figure><img src="image_152.png" /></figure>

<figure><img src="image_153.png" /></figure>

**FIGURE 3** | Examples of ERGs. (A) Recordings from an upper lens eye showing the graded impulse response to 25 ms flashes of light (red line) covering four log units. Note also the longer time to peak with declining light intensity. (B) Recording from a lower lens eye showing the responses to 1, 2, and 3 Hz sinusoidal light stimuli (red traces). Note that the response peaks before max intensity at 1 Hz.

<figure><img src="image_154.png" /></figure>

<figure><img src="image_155.png" /></figure>

**FIGURE 4** | Physiology of the lens eyes. Upper lens eye (ULE) in blue and lower lens eye (LLE) in red. (A) V-log I curve showing a dynamic range of about four log units from ~10 to 1 × 10⁵ W/sr/m². (B) The half width of the impulse response decrease with increasing intensity. At max intensity the half width was 29 ms for ULE and 42 ms for LLE. (C) Both lens eyes are very slow and had flicker fusion frequencies (fff) of about 2.5 Hz. All curves are showing mean ± S.E.M. *N* = 7, except for (C) where *N* = 5.

flashes (Figure 6B). In the 2 min with bioluminescence the medusae spent significantly more time in zone 1 than any of the other zones of the tank (two-sided unpaired student *t*-test, *p* > 0.0001). They also spent significantly more time in zone 1 during activation of the bioluminescence than in darkness and less time in zone 4 (two-sided unpaired student *t*-test, *p* = 0.027 and 0.028, respectively). At the end of the 2 min, two medusae started mating (Figure 6B, parallel blue and green trace).

DISCUSSION

Our results show that the lenses of both lens eyes of *C. sivickisi* form under-focused images on the retinae, generating poor spatial resolution with large blur spots in the range of 10–20° depending on retinal location. Further, the eyes are color-blind, having a single opsin with peak sensitivity in the blue part of the spectrum close to 460 nm. Both lens eyes have very low temporal
<figure><img src="image_156.png" /></figure>

<figure><img src="image_157.png" /><figcaption>**FIGURE 5** | Spectral sensitivity of the lens eye. (A) The spectral sensitivity curve of the upper lens eye (blue line) has a single peak in the deep blue part of the spectrum and has a good match with the absorption curve of a 458 nm opsin (red line, AIC = -53.7). (B) Similar to the upper lens eye the spectral sensitivity of the lower lens eye (blue line) peaks in the deep blue part of the spectrum and had a best match with a single opsin peaking a 458 nm (red line, AIC = -35.9). The curves are showing mean ± S.E.M., N = 7.</figcaption></figure>

resolution with fff's around 2.5 Hz. We also show that under dark conditions the medusae are attracted to bioluminescent flashes produced by the dinoflagellate *P. noctiluca*, which is occurring in high densities in their natural habitat. In conclusion all data support our hypothesis, that the medusae use vision to find parts of the habitat where hunting will be most successful. They are unlikely, however, to use vision to directly spot or pursue individual prey items due to their low spatial resolution.

## Low Intensity Vision for Spotting Bioluminescence

So far *C. sivickisi* is the only cubomedusa known to be strictly night active (Garm et al., 2012). Still, they have a visual system similar to all other examined cubomedusae. Our morphological data show that the lens eyes of *C. sivickisi* are structurally similar to other box jellyfish eyes (Claus, 1878; Berger, 1900; Yamasu and Yoshida, 1976; Pearse and Pearse, 1978; Nilsson et al., 2005). Fully grown, the upper and lower lens eyes have a diameter of about 150 and 200 µm, respectively. In general, small eyes with pupils in the order of 100 µm provide only low spatial resolution, especially at the low light intensities present at night. But our results also point to several features, both in the morphology and in the physiology, which will enhance the photon capture. The outer segments are relatively long and have very dense membrane stacking when compared to other box jellyfish (Laska and Hündgen, 1982; Martin, 2004; O'Connor et al., 2010). Assuming that this implies more photopigment, it will enhance the photon capture, and can thus be interpreted as adaptation for a nocturnal life-style. If there is more opsin per photoreceptor cell it will also result in additional dark noise, which will set a limit to the dimmest stimulus that can be discriminated from noise (Barlow, 1956).

The photon capture is further optimized by a long integration time shown by the very low temporal resolution with flicker fusion frequencies of about 2.5 Hz. Such long integration time is typically found in night active or deep sea animals and are adaptations for vision in low light intensities (Warrant, 2004; Warrant and Locket, 2004). The long integration times has the obvious disadvantage of causing motion blur of objects

moving across the visual field. The large acceptance angles of the photoreceptors, estimated to be 10–20°, will also enhance photon capture but reduce the ability to spot individual bioluminescent flashes. This supports the notion that the lens eye of *C. sivickisi* are tuned for finding the direction toward the densest population of bioluminescent organisms rather than guiding behavior toward single bioluminescent flashes. Detection of bioluminescence is further supported by the spectral sensitivity of the lens eyes peaking close to 460 nm which is a fairly good match with the peak emission of 473–478 nm from *P. noctiluca* (Hastings and Morin, 1991). Further, the flashes typically have a duration between 100 and 200 ms which is slightly faster than the temporal resolution we find for both lens eye and this ensures a maximum photon capture from each flash (Hastings and Morin, 1991).

The slight mismatch between the spectral sensitivity of the photoreceptors and the *P. noctiluca* emission is probably hinting at another important visual task for the medusae. At dawn the medusae come to a rest and anchor themselves to the underside of stony corals (Garm et al., 2012). This means that they have to seek out the coral in the morning light and having a spectral sensitivity in the deep blue part of the spectrum will optimize the contrast between the reef structures and the surrounding ocean water. Clear ocean water peaks at about 450 nm (McFarland and Munz, 1975) whereas coral reef structures, though varying, typically reflect very little blue light and much more in the green and red part of the spectrum (Schalles et al., 2000; Hochberg et al., 2004). The peak sensitivity at about 460 nm could thus be seen as a compromise allowing both habitat recognition and prey detection.

## Retinal Cavities for Noise Reduction

As mentioned above, dark noise is a major problem for vision at low light intensities. Interestingly, the retinal structure with a large fraction (about 50%) of empty spaces found in *C. sivickisi* might be a unique way to minimize this problem. In the closely related diurnal species *T. cystophora* the lens eyes have very similar shapes and sizes, but there is much less empty space in the retina (<10–15%; Nilsson et al., 2005). There is a possibility that the retinal cavities are part of the adaptation to a nocturnal
<figure><img src="image_158.png" /></figure>

<figure><img src="image_159.png" /><figcaption>**FIGURE 6** | Swim trajectories from behavioral experiments. (A) In darkness the medusae swim in most of the tank but had a preference for the corners of the tank. (B) When the dinoflagellate *Pyrocystis noctiluca* is emitting bioluminescent light to the left of the tank the medusae spend most of the time in the left side of the tank. Each trace represents 2 min with 2 s time resolution and each color represents a single medusa (N = 9). It is the same nine medusae in (A,B) and timewise the trajectories from (A) continues in (B). See also **Figure 7**. Note the parallel run of the blue and green trance in the bottom left where a mating started. * indicates start of trajectory, • end.</figcaption></figure>

<figure><img src="image_160.png" /><figcaption>**FIGURE 7** | Distribution of the medusae in darkness (black bars) and with bioluminescence (light blue bars). The tank was divided into four zones with zone 1 closest to the tank with *P. noctiluca* and zone 4 the furthest away. In darkness the medusae spend most time at the ends of the tank but with no significant difference between zone 1 and zone 4. When the bioluminescence of the algae is activated the medusae spend most of the time in zone 1. Asterisks indicate significant difference at the 0.05 level. Bars are means ± S.E.M., N = 9.</figcaption></figure>

life-style. The longitudinal structure of the empty spaces will concentrate light to the microvillar sections between the spaces, because these have a higher refractive index and will trap some light by total internal reflections. This will boost absorption in the photopigment. The spaces will also reduce the microvillar volume of the retina, and if this means fewer rhodopsin molecules it will lead to less thermal noise. We thus hypothesize that the pronounced cavities in the *C. sivickisi* retina is yet another adaptation for a nocturnal life style, providing both a better signal and less noise. However, without modeling the ray path in the retina and measuring both density and thermal instability of the rhodopsin, it is not possible to assess to which degree the retinal spaces will improve the signal to noise ratio.

## Visually Guided Hunting in *C. sivickisi*

Like many other cnidarian medusae, the medusa of *C. sivickisi* is a predator feeding for a large part on pelagic crustaceans (Larson, 1976; Mackie, 1980; Buskey, 2003; Colin et al., 2003; Garm et al., 2012). While most medusae behave as plankton organisms

following the currents, cubomedusae are agile swimmers actively choosing their location. Even though a higher number of test animals than used here would be needed to understand the full details of the hunting behavior of *C. sivickisi*, the statistical significance of our results show that they use vision to place themselves in areas with maximum prey density. This they do using the bioluminescence emitted when their crustacean prey contact the dinoflagellate *P. noctiluca* which can be present in high densities in the habitat. The observed mating during the experiments indicates that the results could be influenced by a group effect (tendency to aggregate). Since the medusae in general displayed natural behavior (including mating) in the tank and since they are also found in close vicinity to conspecifics when hunting in the natural habitat we trust that even though a group effect might have been present it has not resulted in unnatural behavior during the experiments.

The hunting behavior of *C. sivickisi* is similar to the diurnal box jellyfish *T. cystophora*, which is visually attracted to light shafts produced by gaps in the mangrove canopy. These light shafts also attract their crustacean prey, and when these occur in large numbers, their light scattering effect makes the light shafts much more visible. The role of vision in *C. sivickisi* and *T. cystophora* is not to spot individual prey animals, which is typically implied by the term “visually guided predation.” But both species are clearly engaged in visually guided foraging, and it may be the passive mode of prey capture in medusae that have prevented the evolution of high resolution vision in box jellyfish. In principle terms, the foraging mode of *C. sivickisi* and *T. cystophora* is an important example of a visual task that may drive evolution from low resolution to high resolution vision. It can be seen, therefore, as an important intermediate between simple low resolution vision for habitat selection and the high resolution vision which putatively drove the evolution of large eyes and brains in vertebrates, cephalopods and arthropods (Nilsson, 2009, 2013).
## AUTHOR CONTRIBUTIONS

AG designed the experiments, conducted some of the experiments, wrote the initial draft of the MS incl the figures, and financed most of the work. JB conducted some of the experiments. RP conducted some of the experiments. DN conducted some of the experiments and financed part of the work. All authors helped finalize the MS.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Garm, Bielecki, Petie and Nilsson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
# Heat Perception and Aversive Learning in Honey Bees: Putative Involvement of the Thermal/Chemical Sensor AmHsTRPA

Pierre Junca and Jean-Christophe Sandoz *

Evolution, Genomes, Behavior and Ecology, CNRS, Univ. Paris-Sud, IRD, Université Paris-Saclay, Gif-sur-Yvette, France

The recent development of the olfactory conditioning of the sting extension response (SER) has provided new insights into the mechanisms of aversive learning in honeybees. Until now, very little information has been gained concerning US detection and perception. In the initial version of SER conditioning, bees learned to associate an odor CS with an electric shock US. Recently, we proposed a modified version of SER conditioning, in which thermal stimulation with a heated probe is used as US. This procedure has the advantage of allowing topical US applications virtually everywhere on the honeybee body. In this study, we made use of this possibility and mapped thermal responsiveness on the honeybee body, by measuring workers' SER after applying heat on 41 different structures. We then show that bees can learn the CS-US association even when the heat US is applied on body structures that are not prominent sensory organs, here the vertex (back of the head) and the ventral abdomen. Next, we used a neuropharmalogical approach to evaluate the potential role of a recently described Transient Receptor Potential (TRP) channel, HsTRPA, on peripheral heat detection by bees. First, we applied HsTRPA activators to assess if such activation is sufficient for triggering SER. Second, we injected HsTRPA inhibitors to ask whether interfering with this TRP channel affects SER triggered by heat. These experiments suggest that HsTRPA may be involved in heat detection by bees, and represent a potential peripheral detection system in thermal SER conditioning.

**Keywords:** insects, thermoreception, nociception, aversive learning, AmHsTRPA

## INTRODUCTION

In associative learning, animals associate sensory stimuli or their own behavioral responses with particular outcomes, possessing a positive or negative hedonic value for the animal. In classical (or Pavlovian) learning, an initially neutral stimulus such as an odor, sound or color (conditioned stimulus-CS) is associated with a salient appetitive or aversive outcome, like the presence of food or of a noxious stimulus (unconditioned stimulus—US; Pavlov, 1927). Learning success critically depends on the salience of the involved stimuli for the animal, especially on the subjective intensity of the US (Rescorla, 1988; Hammer, 1993; Scheiner et al., 2005). Understanding Pavlovian conditioning therefore implies a careful analysis of how a particular US is detected at the sensory

**OPEN ACCESS**

**Edited by:**
Sylvia Anton,
Institut National de la Recherche
Agronomique, France

**Reviewed by:**
André Fiala,
Georg-August-Universität Göttingen,
Germany
Tatsuhiko Kadowaki,
Nagoya University, Japan

**\*Correspondence:**
Jean-Christophe Sandoz
sandoz@egce.cnrs-gif.fr

**Specialty section:**
This article was submitted to
Invertebrate Physiology,
a section of the journal
Frontiers in Physiology

**Received:** 17 July 2015
**Accepted:** 20 October 2015
**Published:** 25 November 2015

**Citation:**
Junca P and Sandoz JC (2015) Heat Perception and Aversive Learning in Honey Bees: Putative Involvement of the Thermal/Chemical Sensor AmHsTRPA. Front. Physiol. 6:316.
doi: 10.3389/fphys.2015.00316
level and how its information is processed within the animal
brain.

In honeybees, both appetitive and aversive conditioning can
be studied in laboratory conditions thanks to two dedicated
protocols (Gurfa and Sandoz, 2012; Tedjakumala and Gurfa,
2013). The conditioning of the proboscis extension response
(PER), in which bees associate an odor CS with a sucrose US,
is a well-established assay that mimics the final part of bees'
foraging behavior, when they experience a floral aroma together
with nectar. It has been used for decades for unraveling the
neural mechanisms of appetitive learning (Bitterman et al., 1983;
Menzel, 1999; Gurfa and Sandoz, 2012). In this paradigm, data
are already available about how the sucrose US is detected and
processed in the bee brain. Sucrose is detected by dedicated
sugar receptors (AmGr1) on gustatory neurons within specific
sensilla on the bees' antennae, mouthparts and tarsi (de Brito
Sanchez, 2011; Jung et al., 2015). These neurons project to the
subesophageal ganglion, where they are thought to directly or
indirectly contact a single octopaminergic neuron, VUM-mx1
(ventral unpaired median neuron 1 of the maxillary neuromere),
which represents the appetitive reinforcement in the bee brain
(Hammer, 1993). It converges at multiple sites with the olfactory
pathway, allowing the formation of the odor-sucrose association
(Menzel, 1999, 2012).

By contrast, very little information is yet available concerning
US detection and perception in aversive conditioning. The most
influential aversive learning paradigm is based on the bees' sting
extension response (SER). This response represents the final
stage of bees' aggressive response to the presence of a potential
intruder in front of the hive (Breed et al., 2004), classically
elicited by many sensory stimuli (dark colors, moving objects,
etc., Free, 1961) and by honeybees' alarm pheromones (Free,
1987). In fixed bee in the laboratory, SER is triggered by noxious
stimuli, such as an electric shock (Núñez et al., 1983) or a
strong tactile contact (Zhang and Nieh, 2015). In the initial
version of the aversive conditioning, bees learned to associate
an odor CS with an electric shock US (Vergoz et al., 2007;
Roussel et al., 2009). As the electric shock is an unnatural
stimulus for bees, a recent study proposed a modified version
of SER conditioning, in which the electric shock is replaced
by a thermal stimulation with a heated probe as US (Junca
et al., 2014). Heat is a natural stimulus for bees and temperature
variations play an important role in the life of honeybees. At
the colony level, bees strictly regulate the hives' temperature, as
deviations from normal brood temperature results in increased
mortality as well as in morphological and behavioral defects
(Himmer, 1927; Koeniger, 1978; Tautz et al., 2003; Groh et al.,
2004; Jones et al., 2005). High temperatures are critical, and in
summer, when temperatures rise above the thermal optimum of
the hive (~34°C), workers stand at the hive entrance and fan their
wings to decrease in-hive temperature. Foragers also bring water
inside the hive, thereby cooling air temperature (Lindauer, 1954).
At the individual level, bees strictly avoid temperatures above
44°C and respond with a sting extension to heat stimulations
(Junca et al., 2014). They thus perceive a high temperature as an
aversive stimulus, and can associate an odorant with such a heat
stimulus.

Changing the nature of the aversive reinforcement has opened
new possibilities for studying US detection and processing.
Contrary to the electric shock, which requires using EEG gel and
does not easily allow topical applications, the heated probe can be
used for precisely stimulating particular parts of the bees' body.
In the appetitive modality, US perception varies according to
which structure is stimulated with sucrose: mouthparts, antennae
and foreleg tarsi (Marshall, 1935; Scheiner et al., 2004; de
Brito Sanchez et al., 2008). Several studies have dissected the
differential contributions of these potential USs in appetitive
olfactory learning (Bitterman et al., 1983; Sandoz et al., 2002;
Scheiner et al., 2005; Wright et al., 2007; de Brito Sanchez et al.,
2008). First, these studies showed that all three locations support
some level of conditioning, although sucrose solution applied to
the proboscis leads to higher acquisition success compared to
antennal or tarsal USs. This effect is thought to be related to the
mouthparts' higher sensitivity to sucrose compared for instance
to the tarsi (de Brito Sanchez et al., 2008). In addition, the location
of the sucrose US can have an effect on the duration of memory
retention and the types of memories produced (Wright et al.,
2007). PER conditioning with an antenna-only US supports,
shorter memory retention (<24 h) than when bees receive the US
on the mouthparts (>96 h; Wright et al., 2007). Thus, different
US locations may support different learning and/or retention
performances. Sucrose detection is limited to a few structures
on the bee body, which have evolved to arbor gustatory sensory
organs involved in appetitive behaviors. In aversive learning, by
contrast, bees learn to associate an odor with a noxious stimulus,
potentially leading to an injury. Contrary to the detection of
food stimuli, animals must be able to avoid injuries on their
whole body. Until now, we showed that thermal stimulation of
the antennae, mouthparts and foreleg tarsi all trigger SER and
can act as aversive US, yielding a similar learning success (Junca
et al., 2014). In the present study, we asked if in bees, the aversive
thermal US must be detected by dedicated sensory organs to act
as US (as in appetitive conditioning) or if thermal detection is a
more general sensory ability and heat applied anywhere on their
body may act as US.

The use of heat as US may also allow searching for the involved
peripheral receptors. In the animal kingdom, a wide range of
receptors belonging to very different families have been shown to
be responsible for temperature detection, from cold to extreme
heat (Clapham et al., 2001). Among them, Transient Receptor
Potential (TRP) channels seem to be especially important
(Montell et al., 1985; Clapham, 2003; Voets et al., 2005). In
invertebrates, Drosophila possesses several types of TRP channels
involved in high temperature detection. Among them, members
of the TRPA subfamily are essential for responding to heat,
like Painless and dTRPA1 (Tracey et al., 2003; Hamada et al.,
2008; Kwon et al., 2010; Neely et al., 2011). Unfortunately,
no TRPA1 receptor is known in honeybees and AmPain is
poorly described (Matsuura et al., 2009). However, honey bees
express HsTRPA, a Hymenoptera-specific non-selective cationic
channel belonging to the TRPA subfamily and activated by
temperatures above 34°C (honeybee gene: AmHsTRPA, Kohno
et al., 2010). When expressed in a heterologous system, this
channel's current response increases rather monotonically with
increasing temperature without showing any maximum at least until 42°C (it was not tested for higher temperatures). Such response is reminiscent of the SER probability increase observed from room temperature until 65°C in worker bees (Junca et al., 2014). To this day, *HsTRPA* thus represents the best candidate for thermal detection involved in aversive thermal conditioning. This TRP channel is a joint thermal and chemical sensor, being also triggered by exogenous activators like AITC (allyl isothiocyanate), CA (cinnamaldehyde) and camphor (*Kohno* et al., 2010). Two exogenous inhibitors, Ruthenium Red (RuR) and menthol have also been isolated (*Kohno* et al., 2010). The existence of both activators and inhibitors for this receptor provides us with the opportunity to test whether *HsTRPA* is necessary and/or sufficient for thermal detection assessed through SER.

In this study, we first mapped thermal responsiveness all over the honeybee body, by measuring workers' SER after applying heat on 41 different structures. We, then, assessed the aversive olfactory conditioning performances of bees when applying the thermal US on body structures that are not prominent sensory interfaces, the vertex (back of the head) and the ventral abdomen. We next used a neuropharmalogical approach to evaluate the role of HsTRPA for heat detection. First, we performed topical applications of HsTRPA activators on the bee to assess if it is sufficient for triggering SER. Second, we injected HsTRPA inhibitors to ask whether interfering with this TRP channel affects SER triggered by heat.

# MATERIALS AND METHODS

## Animals

Experiments were performed on honey bees caught on the landing platform of several hives on the CNRS campus of Gif-sur-Yvette, France. After chilling on ice, bees were harnessed in individual holders so that both sting- and proboscis extension could be clearly monitored in the same harnessed position. Bees were fed with 5 µl of sucrose solution (50% w/w) every morning to standardize satiety levels and were conserved in a dark and humid box between experiments.

## Stimulations

Thermal stimulations were provided for 1 s by means of a pointed copper cylinder (widest diameter: 6 mm; length: 13 mm), mounted onto the end of a minute soldering iron running at low voltage (HQ-Power, PS1503S). Temperature at the end of the cylinder was controlled using a contact thermometer (Voltcraft, Dot-150). Sucrose stimulations were provided for 1 sec with a soaked toothpick to the bees' antennae.

## Thermal Sensitivity Map of the Bee Body

We first aimed at determining whether noxious thermal stimulation of the bees' different body parts triggers a SER and if thermal sensitivity varies among them. Thermal stimulations (65°C for 1 s) were applied on 41 different areas of the bees' body (see **Figure 1A**). Although, bees' encounters with such a high temperature would be very rare in natural conditions,

this stimulation was chosen in order to study bees' thermal nociceptive system. Recent studies in Drosophila have shown that insects possess a nociceptive system which quickly and strongly responds to potentially deadly temperatures and allows them to avoid such stimuli (Tracey et al., 2003; Neely et al., 2011). Our previous work already showed that a short (1 s) stimulation at this temperature triggers clear SER responses when applied on the antennae, the mouthparts or the forelegs of the bees, without inducing any long-lasting effect on bees (Junca et al., 2014). Eleven median unpaired structures were tested: labrum, clypeus, back of the head, mesoscotum, mesosternum, 1-2, 3-4 sternites, 5-6 sternites, 1-2 tergites, 3-4 tergites, 5-6 tergites. Fifteen paired body parts were also tested on the left or right side independently: antenna flagellum, antenna scape, compound eye, mandible, proximal forewing, distal forewing, protarsus, protibia, profemur, mesotarsus, mesotibia, mesofemur, metatarsus, metatibia, metafemur. To avoid any fatigue of the bees, only four structures were tested per bee. In addition to thermal stimulations, tactile controls were applied on the same structures to verify that sting extension was a consequence of thermal stimulation. Tactile stimulations were performed with a duplicate copper probe which remained at ambient temperature. For each bee, the order of stimulation of the different structures, as well as whether each stimulation was performed with the heated or with the control probe, were determined randomly prior to starting the experiment. The eight stimulations were performed at 10 min intervals. In this experiment, two groups of 20 bees were tested each day.

## SER Conditioning with a Thermal US on the Vertex and the Ventral Abdomen

To assess whether or not bees are able to perform aversive olfactory conditioning with a thermal US on body parts that do not correspond to sensory organs, SER conditioning experiments were carried out with a thermal stimulus (65°C) on 3-4 sternites or on the back of the head as reinforcement. In a differential aversive conditioning procedure, one odorant (the CS+) was associated with a thermal reinforcement (the US), while another odorant was presented without reinforcement (the CS-). The odor CSs were 2-octanone and nonanal (Sigma Aldrich, Deisenhofen, Germany). Five microliters of pure odorant were applied onto a 1 cm² piece of filter paper which was transferred into a 20 ml syringe (Terumo, Guyancourt, France) allowing odorant delivery to the antennae. Half of the honeybees received thermal reinforcement when 2-octanone (odor A) was presented and no reinforcement when nonanal (odor B) was presented, while the reversed contingency was used for the other half of the bees. Both groups were conditioned along 16 trials (8 reinforced and 8 non-reinforced) in which odorants were presented in a pseudo-random sequence (e.g., ABBABAAB) starting with odorant A or B in a balanced way across animals. The inter-trial interval (ITI) was 10 min. Each conditioning trial lasted 36 s. The bee was placed in the stimulation site in front of the air extractor, and left for 18 s before being exposed to the odorant paired with the US. Each odorant (CS+ or CS−) was delivered manually for 4 s. The thermal stimulus started 3 s after odorant onset and finished with the odorant (1 s temperature
A

<figure><img src="image_161.png" /></figure>

Temperature

Tactile

B

<figure><img src="image_162.png" /></figure>

**FIGURE 1** | Impact of thermal stimulation of 41 structures of the honeybee body on sting extension responses (SER). (A) Map of the bee body showing the names of the tested structures. Gray areas were not accessible in our holding setups and were thus not tested. (B) Percentage of SER observed for thermal stimulations on the 41 different body parts using a heated copper probe (n = 555, four structures tested per bee). As control, tactile stimulations with an unheated probe were given. Prox, proximal; dist, distal; L, Left; R, Right.
stimulation). The bee was then left in the setup for 14 s and was then removed. The temperature of 65°C was chosen for the US because this stimulation induced a high rate of SER in the previous experiments. One group of 16 bees was tested daily.

## HsTRPA Involvement in Thermal Sting Extension Response

We investigated the putative involvement of the thermal/chemical sensor HsTRPA in heat sensitivity as measured by sting extension. To this end, we evaluated the effects of known HsTRPA activators and inhibitors. We focused on the SER triggered by thermal stimulation on the mouthparts, as this is the US commonly used for aversive thermal conditioning (Junca et al., 2014; Cholé et al., 2015).

In a first experiment, we asked if topical application of a chemical HsTRPA activator on the mouthparts directly triggers SER, as a thermal stimulation does. Kohno et al. (2010) isolated three exogenous molecules able to activate this channel: allyl isothiocyanate (AITC), cynnalmaldehyde (CA), and camphor (Sigma Aldrich, Deisenhofen, Germany). These compounds were applied with a soaked toothpick at two concentrations per drug in distilled water: AITC (1 mM and 100 mM), CA (1 mM and 100 mM), camphor (3 and 300 mM). As controls, thermal stimulation (65°C) as above and a toothpick soaked with distilled water (vehicle) were applied to the mouthparts. Activator solutions and controls were provided in a randomized order with a 10 min interval. Two groups of 18 bees divided in three subgroups for each activator were tested each day.

We also evaluated the effect of injections of HsTRPA inhibitors on SER triggered by heat. A small hole was pricked into the cornea of the median ocellus to allow the insertion of a 1 µl microsyringe (Hamilton company, Reno, Nevada, USA). Different groups of bees were injected with 1 µl Ringer solution, menthol in Ringer, or ruthenium red (RuR) in Ringer (Sigma Aldrich, Deisenhofen, Germany). Two concentrations were tested for each drug: menthol (0.5 and 5 mM), RuR (0.1 and 1 mM). One hour after the injections (Kohno et al., 2010), bees received a thermal stimulation (65°C) and a tactile control on the mouthparts, in a randomized order for each bee. Stimulation were performed at 10 min intervals. In a further experiment, bees were injected with the highest inhibitor concentrations (RuR 5 mM or menthol 1 mM) or Ringer and were then subjected to a thermal responsiveness experiment (Junca et al., 2014). One hour after inhibitor injection, bees received a succession of six stimulations of increasing temperature (from ambient temperature ~25 to 75°C), in steps of 10°C. Thermal stimulations alternated with tactile controls, provided as above with an identical unheated probe. Stimulation were applied during 1 s and the bees' SER was noted.

We also verified that the application of HsTRPA inhibitors did not have any non-specific deleterious effects on bees' behavioral responsiveness. We thus chose to assess their potential effect on bees' PER responses to sucrose. After injections with the inhibitors (RuR 5 mM, menthol 1 mM) or Ringer, we performed a typical sucrose responsiveness protocol as described in Scheiner et al. (2004). Bees were presented sucrose solutions of increasing concentration, following an exponential progression (0, 0.1, 0.3,

1, 3, 10, 30% w/w). Sucrose stimulations were alternated with water controls. Sucrose and water stimulations were provided with a soaked toothpick to the bees' two antennae simultaneously, and the PER (extension or not of the proboscis) was noted.

Each trial lasted 38 s. One bee at a time was placed in the setup, and left for 20 s before stimulus application started. The stimulation lasted for 1 s. The bee was then left in the setup for 17 s before being removed. For a given bee, all stimulations were performed at 10 min intervals.

## Statistical Analysis

All recorded data were dichotomous, with a sting or proboscis extension being recorded as 1 and a non-extension as 0. When comparing the responses of the same bees to thermal and tactile stimulations on the different structures composing the heat sensory map, pairwise McNemar comparisons were used. Differences in thermal or in tactile responses among body structures were assessed using a Chi² test. When comparing responses to thermal or tactile stimuli across wider areas (lateralization, core/periphery, body parts), Chi² tests were used. For pairwise comparisons, as body parts were composed of three structures (head, thorax, abdomen), each structure was involved in two comparisons. A Bonferroni correction for multiple comparisons was thus applied, and the significance threshold was $\alpha_{corr} = 0.05/2 = 0.025$. When analyzing within group the effect of topical applications of HsTRPA activators, McNemar tests were used to compare drug application to water control. To compare between groups the responses of bees injected with HsTRPA inhibitors or vehicle, Fisher's exact test were used. As three groups were involved, the significance threshold was corrected for multiple comparisons as $\alpha_{corr} = 0.025$. To analyze thermal and sucrose responsiveness curves or aversive conditioning curves, we used repeated measure ANOVAs with stimulus (thermal vs. tactile, sucrose vs. water or CS+ vs. CS−) and trial as repeated factors. For aversive conditioning, following standard procedures, only bees which responded to the US at least three times in the course of acquisition were kept for analysis (vertex: 2%; 3-4 sternites: 29%). To test the effect of inhibitors on thermal and sucrose responsiveness, thermal, or sucrose response curves were compared using repeated measure ANOVAs with drug as a between-group factor. Monte Carlo studies have shown that it is permissible to use ANOVA on dichotomous data only under controlled conditions, which are met in these experiments (Lunney, 1970). Statistical tests were performed with STATISTICA 5.5 (Statsoft, Tulsa, USA).

## RESULTS

### Thermosensory Map of the Bee Body Assessed by Sting Extension

We first aimed to map the heat sensitivity of the different parts of the honeybee body, by applying a heated probe and measuring sting extension responses (SER). Heat was applied for 1 s, and heat stimulations were alternated with tactile controls in a pseudo-randomized order. In total, 41 different structures were tested (Figure 1A, four structures tested per bee, n = 555 bees).
<figure><img src="image_163.png" /><figcaption>**FIGURE 2** | Tactile and heat sensitivity maps obtained by measuring sting extension responses. The maps represent the percentage of SER observed after tactile (A) or thermal (B) stimulation of each structure of the bee body, using a color scale from light red (0–10% of SER) to dark red (>50% of SER). Gray areas were not accessible in our holding setups and were thus not tested (NT). Sting extensions are mainly due to thermal input as seen from the comparison of both maps.</figcaption></figure>

**Figure 1B** presents the percentage of responses obtained for each structure to heat and to the tactile control. The proportion of SER to heat stimulation varied among tested structures ($\text{Chi}^2$ test: $\text{Chi}^2 = 235.7, P < 0.001$, 40 df, from 13.9% SER for the left distal wing to 92.5% SER for the dorsal part of the head (vertex)). Likewise, responses to tactile control stimulations varied according to the tested structure ($\text{Chi}^2$ test: $\text{Chi}^2 = 104.8, P < 0.001$), from 0% SER (right mandible and right distal wing) to 32% SER (vertex). Overall, 38 out of the 41 tested structures exhibited significantly higher responses to heat than to the tactile control (McNemar test: $\text{Chi}^2 > 4.17, p < 0.05$; exceptions: left distal wing, 5.6 sternites, 5.6 tergites: $\text{Chi}^2 < 1.78, NS$).

**Figure 2** presents the same data on a schematic individual, using a color scale from light red (0–10% of SER) to dark red (>50% of SER). This map shows strong variations in the responses of the different body parts to heat stimulations, more so than for tactile stimulations. To evaluate this observation statistically, we next analyzed the responses of different body parts according to their localization (**Figure 3**). First, we asked whether bees' tactile and heat sensitivities are lateralized (**Figure 3A**). We found that responses to tactile and to heat stimuli were identical between the bees' left and right appendages (tactile: $\text{Chi}^2 = 0.10, 1$ df, NS; temperature: $\text{Chi}^2 = 0.04, 1$ df, NS). Second, we asked if a difference in sensitivity exists between the honeybees' body and its different appendages (**Figure 3B**). We found that SER were significantly more frequent when stimulating the body than when stimulating the appendages, both for thermal stimulation ($\text{Chi}^2 = 10.1, 1$ df, $p < 0.01$) and for tactile stimulation ($\text{Chi}^2 = 35.4, 1$ df, $p < 0.001$). Lastly, we examined tactile and heat sensitivity according to the bees' antero-posterior axis (**Figure 3C**). A significant heterogeneity appeared among body parts (head, thorax, abdomen) in the bees' responses to thermal stimuli ($\text{Chi}^2 = 14.4, 2$ df, $p < 0.001$) but not to tactile stimuli ($\text{Chi}^2 = 5.40, 2$ df, NS). Thermal responses

were highest for the head (56.8% SER) and lowest for the abdomen (40.4% SER), and all body parts differed from the others (head/thorax: $\text{Chi}^2 = 5.99, p < \alpha_{\text{corr}} = 0.025$; head/abdomen: $\text{Chi}^2 = 15.9, p < \alpha_{\text{corr}} = 0.025$; thorax/abdomen: $\text{Chi}^2 = 6.39, p < \alpha_{\text{corr}} = 0.025$). We thus conclude that although the whole honeybee body is sensitive to thermal stimuli, differences in thermal sensitivity appear among body parts.

## Thermal Aversive Reinforcement on Main Body Structures

If honey bees are able to detect heat on their whole body and to respond with a SER, one may then wonder whether such stimulations may also act as an aversive reinforcement in a conditioning procedure. Our previous work showed that heat application on the antennae, the mouthparts or the front legs may operate as aversive reinforcement in olfactory SER conditioning (Junca et al., 2014). These structures are however all known sensory organs, acting as interfaces between the animal and its environment. Here, we chose two structures, the rear part of the head (vertex) and the ventral abdomen (3-4 sternites), which are not dedicated sensory structures, and asked whether 65°C stimulations of these structures can act as reinforcement in a differential olfactory conditioning procedure. In this protocol, bees had to differentiate between an odor associated with the thermal stimulation (CS+) and an explicitly non-reinforced odor (CS-).

Bees learned the task efficiently in both situations (**Figure 4**). When the vertex was stimulated (**Figure 4A**, *n* = 37), bees' SER to the CS+ increased significantly (from 6 to 54%, ANOVA for repeated measurements—RM-ANOVA, $F_{(7, 238)} = 4.13, p < 0.001$), while their responses to the CS- remained low and stable ($F_{(7,238)} = 0.27, NS$). Consequently, bees' responses to the CS+ and CS- developed differently in the course of training (stimulus × trial interaction: $F_{(7, 238)} = 3.89, p < 0.001$). When the 3-4 sternites were stimulated (**Figure 4B**, *n* = 57), bees' SER to the CS+ increased along trials (from 9 to 49%, $F_{(7, 392)} = 5.99, p < 0.001$) while responses to the CS- did not change throughout the experiment ($F_{(7, 392)} = 1.81, NS$). Accordingly, bees' responses to the CS+ and CS- developed differently in the course of training (stimulus × trial interaction: $F_{(7, 392)} = 7.66, p < 0.001$). These results, obtained on the vertex and the ventral abdomen, suggest a general ability of bees to associate odorants (CS) with thermal stimulations on their body (US).

## Impact on SER of Topical Applications of HsTRPA Activators

The previous experiments showed that bees perceive a heat stimulus on their whole body and can use this information in the context of aversive conditioning. But how does heat detection take place at the peripheral level? We focused on HsTRPA, so far the only well-described thermal receptor in the honey bee. As a previous study isolated chemical activators of this receptor *in vitro* (Kohno et al., 2010), we first wondered if topical application of these chemicals is sufficient for triggering a SER. We thus evaluated the effect caused by the application on the bees' mouthparts of a toothpick soaked with AITC (allyl
<figure><img src="image_164.png" /></figure>

**FIGURE 3** | Tactile and heat sensitivity according to the location of the structures. (A) Bilateral symmetry: responses of left (green) or right (magenta) structures were pooled and compared. Stimulations on both sides induced similar SER rates. (B) Body/appendages: data were pooled for all appendages (green: antennae, mouthparts, legs, wings) and for main body parts (magenta). Body structures responded significantly more than appendages to both tactile and heat stimulations. (C) Heat sensitivity according to the antero-posterior axis: data were pooled separately for head (blue), thorax (green), and abdomen (magenta). A gradient of thermal response intensity was found from head to abdomen. Different letters indicate significant differences in $Chi^2$ tests.

isothiocyanate), CA (cinnamaldehyde) or camphor, in three groups of animals. We focused here on the mouthparts because thermal stimulation of this structure is routinely used in our aversive conditioning experiments (Junca et al., 2014; Junca et al. in preparation). As controls, identical stimulations with a water-soaked toothpick (solvent control) and a heated copper probe (65°C, positive control) were applied. Stimulations were given at 10 min intervals in a randomized order. Two concentrations of each drug were tested.

At the lower concentrations (Figure 5A; 1 mM AITC, n = 39; 1 mM CA, n = 39; 3 mM camphor, n = 41), no effect of the drugs was observed. As expected, honey bees exhibited high SER to the heated probe and low responses to the water control stimulation, with a clear difference between both stimulations (Mc Nemar test, $Chi^2 > 24.04, p < \alpha_{corr} = 0.025$). However, drugs generally induced low response rates, which were not statistically higher than the water control (Mc Nemar test, $Chi^2 < 3.20$, NS). At the 100 times higher concentrations (Figure 5B; 100 mM AITC, n = 37; 100 mM CA, n = 36; 300 mM camphor, n = 36), one of the three drugs was effective in triggering SER. As above, in all groups, thermal stimulation led to strong responses but the water control did not (Mc Nemar test, $Chi^2 > 24.04, p < 0.025$). While CA and camphor application did not elicit any clear response

(Mc Nemar test, $Chi^2 < 1.50$, NS), AITC induced 32% SER, which was significantly higher than the water control (Mc Nemar test, $Chi^2 = 8.10, p < 0.025$). We thus conclude that only one HsTRPA activator was effective when applied topically on the bees' mouthparts, and only at a very high concentration.

## Impact of HsTRPA Inhibitors on Heat Sensitivity

We then asked whether HsTRPA is necessary for bees to detect heat and respond with a sting extension. We focused here on SER triggered by thermal stimulation on the mouthparts, the US commonly used for aversive thermal conditioning (Junca et al., 2014; Cholé et al., 2015). Two chemical inhibitors of HsTRPA have been identified *in vitro* (Kohno et al., 2010), menthol and ruthenium red (RuR). If drug injections provoke a decrease in SER triggered by heat, it would position HsTRPA as a good candidate for high temperature detection. To test this hypothesis, three groups of bees received an injection of 1 µl menthol, RuR, or Ringer (vehicle) as a control, in the median ocellus. After 1 h, bees were then subjected to a thermal stimulation (65°C) to the mouthparts and a tactile control at 10 min intervals in a randomized order. Two concentrations of each drug were tested.
<figure><img src="image_165.png" /></figure>

**FIGURE 4** | Thermal aversive conditioning with US application on the head and the abdomen. Differential olfactory SER conditioning with a US consisting in thermal stimulation of (A) the rear of the head (vertex) or (B) the ventral abdomen (3-4 sternites). In both cases, honey bees managed to differentiate between the CS+ (red dots) and the CS− (white dots) along the 8 trials (\*\*p < 0.001).

<figure><img src="image_166.png" /></figure>

**FIGURE 5** | Effect of topical application of HsTRPA activators on sting extension responses. The bees' mouthparts were stimulated with AITC (ally isothiocyanate), CA (cinnamaldehyde) or camphor at two concentrations, (A) 1–3 mM or (B) 100–300 mM (all drugs in green). A thermal stimulation (red) or a water control (white) were used as controls. Only 100 mM AITC led to significant SER compared to the water control (*p* < αcorr = 0.025).

When the lower concentrations of inhibitors were tested (Figure 6A; 0.5 mM menthol, *n* = 40; 0.1 mM RuR, *n* = 39; Ringer *n* = 43), no effect was observed. In all three groups, honey bees exhibited high SER to the heated probe and low responses to the tactile control, with a clear difference between these stimulations (Mc Nemar test, $\text{Chi}^2 > 20.0, p < 0.001$). No difference was observed among groups in SER to the thermal stimulation ($\text{Chi}^2 = 1.13, \text{2 df, NS}$) or to the tactile control ($\text{Chi}^2 = 1.86, \text{2 df, NS}$). At the 10 times higher concentration (Figure 6B; 5 mM menthol, *n* = 64; 1 mM RuR, *n* = 61; Ringer *n* = 62), both drugs were effective in blocking SER. Although, in all three groups responses induced by thermal stimuli were still significantly higher than responses to tactile controls (Mc Nemar test, $\text{Chi}^2 > 26.0, p < 0.001$), SER to the heat stimulus was different among groups ($\text{Chi}^2 = 17.4, \text{2 df, p < 0.001}$). In particular, responses to heat were lower in both drug-injected groups compared to the Ringer control group (Fisher's exact test, RuR: $\text{Chi}^2 = 8.95, p < \alpha_{corr} = 0.025$; menthol: $\text{Chi}^2 = 17.3, p < 0.025$). RuR- and menthol-injected groups displayed comparable rates of SER to the thermal stimulus (Fisher's exact test, $\text{Chi}^2 = 1.5, \text{NS}$). No difference appeared among groups in SER to the tactile stimulus ($\text{Chi}^2 = 0.14, \text{2 df, NS}$).

Thus, HsTRPA inhibitors appear to inhibit SER to heat. We next aimed to confirm and expand this result by characterizing

the impact of HsTRPA inhibitors on thermal sensitivity along an increasing temperature gradient, as usually tested for measuring bees' aversive responsiveness (Junca et al., 2014; Junca et al. in preparation). Bees were thus injected with the higher dose of each inhibitor or with Ringer, as above, but were then subjected to a series of thermal stimulations at increasing temperatures on the mouthparts alternated with tactile controls (Figures 7A–C). All stimulations were applied at 10 min intervals.

Bees' SER increased significantly with increasing temperature in all three groups (RM-ANOVA, trial effect: Ringer: *n* = 40, $F_{(5, 195)} = 21.6, p < 0.001$; RuR: *n* = 38, $F_{(5, 185)} = 10.8, p < 0.001$; menthol: *n* = 40, $F_{(5, 195)} = 9.84, p < 0.001$). By contrast, responses to alternated tactile stimuli did not increase, and even decreased in the Ringer group, throughout the experiment (RM-ANOVA: ringer: $F_{(5, 195)} = 2.46, p < 0.05$; RuR: $F_{(5, 185)} = 1.22, \text{NS}$; menthol: $F_{(5, 195)} = 1.05, \text{NS}$). Accordingly, in all three groups, responses to the temperature stimulus evolved differently from those triggered by tactile controls (RM-ANOVA, stimulus × trial interaction: Ringer: $F_{(5, 195)} = 24.6, p < 0.001$; RuR: $F_{(5, 185)} = 10.2, p < 0.001$; menthol: $F_{(5, 195)} = 9.17, p < 0.001$). However, responses to heat were significantly different in the three groups (Figure 7D, RM-ANOVA, stimulus effect: $F_{(2, 115)} = 5.47, p < 0.01$; stimulus × trial interaction: $F_{(10, 575)} =$
<figure><img src="image_167.png" /></figure>

**FIGURE 6** | Impact of HsTRPA inhibitors on SER to thermal stimulations. Bees were injected in the median ocellus with menthol, ruthenium red (RuR) or Ringer as control. Sting extensions were recorded in response to 1 sec thermal stimulation (65°C; red) and tactile stimulation (white). (A) At low concentration (0.5 mM menthol and 0.1 mM RuR), no effect of the inhibitors appeared. (B) At 10 times higher concentrations (5 mM menthol and 1 mM RuR) both drugs significantly inhibited SER responses to heat. Different letters indicate significant differences among groups (*p* < αcorr = 0.025).

<figure><img src="image_168.png" /></figure>

**FIGURE 7** | Effect of HsTRPA inhibitors on thermal responsiveness. Different groups of bees were injected with Ringer as control (A) or with an HsTRPA inhibitor, either menthol (5 mM; B) or ruthenium red (RuR, 1 mM; C) SER was measured in response to increasing temperatures (red) alternated with tactile controls (white). (D) Comparison of thermal response curves among the three groups (Ringer: gray circles; menthol: light blue triangles; RuR: orange squares). Both inhibitors decreased heat responsiveness (\*p* < 0.05; \*\*p* < 0.001).

2.03, *p* < 0.05). In particular, weaker responses were observed in the RuR- and menthol-injected groups compared to the Ringer control (RM-ANOVA, stimulus × trial interaction, Ringer/RuR: *F*_(5, 380)_ = 2.59, *p* < 0.05; Ringer/menthol: *F*_(5, 390)_ = 2.78, *p* < 0.05). No difference appeared between the groups injected with HsTRPA inhibitors (RuR/menthol: *F*_(5, 380)_ = 0.73, NS). Lastly, no difference appeared among groups in the responses to the tactile controls (RM-ANOVA, stimulus effect: *F*_(2, 115)_ = 1.29, NS; stimulus × trial interaction: *F*_(10, 575)_ = 0.74, NS).

The previous experiment confirmed that HsTRPA inhibitors affect thermal responsiveness measured by means of SER. Most probably, this result is due to the effect of the inhibitors on HsTRPA receptors. However, theoretically, it could also be due to a non-specific detrimental effect of the drugs on the bees' physiological state, even though no such effect was apparent by simple observation. In the next experiment, we thus checked the possible effect of HsTRPA inhibitors in another context and another hedonic modality—the appetitive
modality. To this end, we measured bees' PER in a typical sucrose responsiveness protocol (Scheiner et al., 2004). After Ringer or HsTRPA inhibitor injections as above, bees were thus subjected to a series of stimulations on the antennae with sucrose solutions at increasing concentrations alternated with water controls (Figures 8A-C). All stimulations were applied at 10 min intervals.

Bees' PER increased significantly with increasing sucrose concentrations in all three groups (RM-ANOVA, trial effect: Ringer: *n* = 39, *F*_(6, 228) = 21.9, *p* < 0.001; RuR: *n* = 38, *F*_(6, 234) = 24.1, *p* < 0.001; menthol: *n* = 40, *F*_(6, 222) = 21.9, *p* < 0.001). Responses to the control water stimulations remained stable for Ringer and menthol but slightly increased for RuR (ringer: *F*_(6, 228) = 1.63, NS; RuR: *F*_(6, 234) = 2.20, *p* < 0.05; menthol: *F*_(6, 222) = 1.45, NS). In all groups, sucrose responses evolved differently from responses to water controls (RM-ANOVA, stimulus × trial interaction: Ringer: *F*_(6, 228) = 8.03, *p* < 0.001; RuR: *F*_(6, 234) = 6.50, *p* < 0.001; menthol: *F*_(6, 222) = 10.0, *p* < 0.001). However, responses evolved similarly in the three groups both for sucrose stimulations (Figure 8D; RM-ANOVA, stimulus effect: *F*_(2, 114) = 1.44, NS; stimulus × trial interaction: *F*_(12, 684) = 0.68, NS) and for the water controls (stimulus effect: *F*_(2, 114) = 0.85, NS; stimulus × trial interaction, *F*_(12, 684) = 0.68, NS). We conclude that HsTRPA inhibitors have no effect on bees' PER responses to sucrose, suggesting that their effect on heat-evoked SER is not due to a general behavioral impairment.

## DISCUSSION

Our study provides the first heat sensitivity map of the honeybee, measured using heat-induced SER. This map reveals that responses are symmetrical between body sides, that body structures are more sensitive than the appendages and it shows a gradual decrease in thermal sensitivity from the head to the abdomen. We then demonstrated that heat application does not need to be located on specific structures (mouthparts, antennae or protarsi) to serve as an aversive US in SER conditioning. Indeed, bees learned successfully when the US was provided on the vertex or on the ventral abdomen (3-4 sternites). Lastly, we observed that HsTRPA activators (AITC, CA, camphor) applied topically on the bees' mouthparts did not easily induce SER (only AITC at the higher dose) whereas inhibitor injections (RuR, menthol) significantly decreased SER to heat. This impact of HsTRPA inhibitors was specific of SER to heat, since no effect was observed on PER responses to sucrose.

## Thermal Body Map

We observed that bees' heat sensitivity, as measured by the induced SER, varied among body structures. Control tactile stimulations also led to variations in responses among body structures but on a much smaller scale compared to heat-triggered responses. Thus, most of the observed SER were due to heat application. The map showed clearly that heat detection is a general phenomenon and is not restricted to a few dedicated sensory structures, like the antennae, mouthparts

or tarsi (Junca et al., 2014). A possible explanation for this observation may originate from the high temperature (65°C) used for thermal stimulation, which may have induced activation of nociceptive pathways responsible for preserving the animals' physical integrity. Such system should be differentiated from fine-tuned thermosensory pathways which detect temperatures in the physiological range and employ dedicated thermosensitive sensilla (coelocapitular sensilla) on the bee antenna (Lacher, 1964; Yokohari et al., 1982; Yokohari, 1983). The existence of nociceptive pathways in insects has been recently demonstrated in Drosophila larvae, in which the detection and avoidance of noxious heat, bright light, or strong mechanical stimuli is operated by class IV multidendritic neurons that express a range of nocisensor proteins (Im and Galko, 2012). These neurons extend their dendrites within the derma and are widely distributed along the body surface (Hwang et al., 2007). Although, strongly remodeled, they survive through metamorphosis and may play a similar role in adults (Kuo et al., 2005; Shimono et al., 2009). The wide field heat sensitivity we have found in this study would fit with the existence of an analogous neuron family in honeybees. To this day, however, they have not yet been described. Alternately, thermosensation may also involve some of the many sensory hairs present on the bee body. Only a few structures of the bee body did not elicit more SER when they were thermally stimulated than with the tactile control: the tip of the abdomen and the distal part of the forewings. A possible lack of nociceptive neurons in the wings may explain this observation. At the tip of the abdomen, it would seem rather unlikely that nocisensor neurons are utterly absent. Rather, the proximity between the heat stimulus and the sting chamber might have prevented any sting extension, the animal attempting to avoid any internal injuries.

Responses to heat were compared among body parts. First, we did not find any lateralization bias on the paired appendages. The opposite would have been surprising. Indeed, organisms expressing such an asymmetrical perception would suffer from obvious disadvantages (Corballis, 1998). The physical world is indifferent to left and right, and any lateralized deficit might leave an animal vulnerable to attacks on one side or unable to attack prey or competitors appearing on one side (Vallortigara and Rogers, 2005). Second, peripheral structures appeared less sensitive than body structures. This difference was mostly due to a lower sensitivity of appendages to tactile stimuli, which could be related to the fact that appendages are more likely to come in contact with mechanical substrates than the body. Lastly, we observed a gradient of decreasing thermal responsiveness from the head to the abdomen. The brain located in the head capsule contains neuropils essential for processing and integrating information from many sensory modalities (gustatory, olfactory, visual, tactile, etc) as well as for motor control, navigation, learning, and memory processes among others (Menzel, 1999, 2012). Therefore, physical integrity of the head is crucial for bees to be able to assess their environment and exhibit adapted behaviors, and noxious stimulations located close to the head should trigger stronger responses.
<figure><img src="image_169.png" /></figure>

<figure><img src="image_170.png" /></figure>

<figure><img src="image_171.png" /></figure>

<figure><img src="image_172.png" /></figure>

**FIGURE 8** | Effect of HsTRPA inhibitors on sucrose responsiveness. Different groups of bees were injected with Ringer as control (A) or with a HsTRPA inhibitor, either (B) menthol (5 mM) or (C) Ruthenium red (RuR, 1 mM). Proboscis extension responses (PER) were measured in response to sucrose solutions at increasing concentrations (blue) alternated with water controls (white). (D) Comparison of sucrose response curves among the three groups (Ringer: gray circles; menthol: light blue triangles; RuR: orange squares). Inhibitor injections did not impact sucrose responsiveness (NS: Non Significant; \*\*\*p < 0.001).

## SER Learning on the Vertex and the Ventral Abdomen

In a previous study, we demonstrated that thermal SER conditioning is successful with a heat US on the mouthparts, the antennae and the tarsi of the forelegs (Junca et al., 2014). Such structures are well known sensory organs (Hammer, 1993; de Brito Sanchez et al., 2008; Giurfa and Sandoz, 2012; Jung et al., 2015). We show here that heat stimulation on body structures that are not dedicated sensory organs (vertex, ventral abdomen) can also act as US in SER conditioning. This observation supports our current putative neural model of thermal aversive conditioning in honeybees (Figure 9). Associative learning relies on the convergence of CS and US information at one or several locations in the brain. The olfactory (CS) pathway is well known in bees (Menzel, 1999; Giurfa, 2007; Sandoz, 2011): axons of olfactory receptor neurons (ORN) located on each antenna project to the antennal lobes (AL) where they synapse with approximately 4000 local interneurons (not shown) and 800 projection neurons (PN). Projection neurons then convey processed information to higher-order brain structures, the mushroom bodies (MB) and the lateral horn (not shown). For aversive learning, the US pathway is mostly unknown, but our results may provide some new clues. Except for the case in which an antenna heat US is used (Junca et al., 2014), and for which thermo-sensory neurons from the antenna are thought to project to the antennal lobe (Yokohari, 1983; Nishino et al., 2009), all other heat stimulations probably rely on thermal detection by the above-mentioned putative multidendritic neurons. It is unlikely that this information also projects to the antennal lobe.

Rather, it can be expected from neuroanatomical work in other insects (for instance on the mechanosensory system, Pflüger et al., 1988; Newland and Burrows, 1997) that such putative thermo-sensitive/nociceptive neurons would first project to the respective ganglia of the ventral nerve cord, i.e., to subesophageal, thoracic or abdominal ganglia depending on the location of the stimulation (SEG, TG, and AG in Figure 9). From there, information could be conveyed by ascending interneurons toward the brain, possibly to a thermal/nociceptive integration center (TNC in Figure 9), as suggested by several observations. In the Asian bee *Apis cerana*, immediate early gene (Acks) expression mapping showed that exposure to a high temperature (46°C) induces neural activity in several brain regions: within the mushroom body, intrinsic neurons (class I and II Kenyon cells), and in a region of the protocerebrum located between the dorsal and the optic lobe (Ugajin et al., 2012). Thus, stimulation with a high temperature presumably induces activity in one thermo-sensitive center and in the mushroom bodies, a well-known multimodal integration and association center of the bee brain. Our working hypothesis is that neurons from the putative thermo-sensory center could then activate aversive reinforcement circuits, which would converge with the olfactory pathway and induce learning-associated plasticity, in particular in the mushroom bodies. Previous work on SER conditioning indicated that dopaminergic neurons (dopN in Figure 9) are involved in aversive reinforcement, because pharmacological blockade of dopamine receptors disrupts aversive learning (Vergoz et al., 2007). Dopamine neurotransmission is also necessary for aversive learning in other insects (Drosophila,
<figure><img src="image_173.png" /></figure>

**FIGURE 9** | Working model of aversive olfactory conditioning of SER using a thermal US. Putative pathways involved in (A) the expression of SER after thermal stimulation, (B) the acquired SER after learning a CS-US association, are shown. (A) At the periphery, stimulation of the different structures with a high temperature is thought to activate thermosensitive neurons (possibly class IV multidendritic neurons), which would first project to the respective relays on the ventral nerve cord, the subesophageal ganglion (SEG), thoracic ganglia (TG), or abdominal ganglia (AG). As a second step, interneurons would project to a thermal/nociceptive center (TNC) in the brain. Antennal thermal stimulation induces activity in the antennal lobe (AL) but possibly also activates the TNC. Activation of this center would stimulate premotor descending neurons (DN) which would in turn trigger stinging motor patterns in the terminal abdominal ganglion (TAG), producing SER (Ogawa et al., 1995). (B) Olfactory learning: odorants are detected on the antenna by olfactory receptor neurons (ORNs) projecting to the AL. Then information is prominently conveyed to the mushroom bodies (MB) by projection neurons (PN). Activation of dopaminergic neurons (dopN) by the TNC would inform the olfactory pathway of the aversive thermal reinforcement. Associative plasticity at the level of MB extrinsic neurons (EN) feeding onto the sting premotor descending neurons would allow the CS to elicit SER after learning.

Schwarzel et al., 2003; Schroll et al., 2006; crickets, Unoki et al., 2005). The bee brain contains a complex arrangement of many dopamine-immunoreactive neurons (Schäfer and Rehder, 1989; Schürmann et al., 1989). Among dopamine neurons, three clusters are especially interesting as they contain processes that project to the mushroom body calyces and lobes (especially the α-lobe), and may thus provide aversive reinforcement information (Tedjakumala and Giurfa, 2013). Co-activation of CS and US pathways could modify the strength of synapses between the specific Kenyon cells representing the learned odorant and mushroom body extrinsic neurons (EN in **Figure 9**) feeding onto the sting extension premotor system. After learning, presentation of the odor CS alone would trigger SER thanks

to this modification. Further work is needed to confirm the different putative elements of this working model. The present study started this task by evaluating potential receptors detecting temperature at the periphery (see below).

## Putative Involvement of HsTRPA in Heat Perception

We assessed the possible involvement of HsTRPA in heat-triggered SER using topical applications of activators and injections of inhibitors. We observed that topical application of HsTRPA activators is not sufficient for triggering SER, except when a very high concentration (100 mM) of AITC was used as stimulus. This result might appear surprising since all three tested drugs were potent activators of the channel *in vitro* (Kohno et al., 2010). However, if thermosensation is carried out by a similar class of class IV multidendritic neurons as in Drosophila (Im and Galko, 2012), it is likely that the thermal channels are located in the epidermis, i.e., below the cuticle, so that direct contact of the activators with the channel is not possible, or at least difficult. Heat could diffuse through the cuticle to activate the channel, but chemical activators would not. In our view, therefore, this result does not invalidate a potential role of HsTRPA in thermal sensitivity and nociception in bees. Concerning the SER increase observed with AITC stimulation, we cannot be sure at this stage that it is not related to a possible aversive gustatory effect of this compound when presented to the mouthparts, because AITC was found to inhibit PER responses when added to sucrose solution (Kohno et al., 2010). However, in the same study, the effect of AITC was reversed by RuR, suggesting a possible involvement of HsTRPA. Until now no SER in response to bitter or repellent gustatory stimuli has been reported. It will be necessary to test the effect on SER of AITC application on other locations of the bee body, while also checking if known aversive gustatory stimuli (salt or bitter compounds) can trigger SER when applied on the mouthparts. This will be addressed in more details in the future.

Injections of HsTRPA inhibitors produced significant blocking of SER in response to heat. This effect is similar to the reversal of the suppression of PER by heat in previous work (Kohno et al., 2010). In this study, heating a sucrose solution to 70°C was found to decrease bees' PER to sucrose, compared to an unheated solution. Both RuR and menthol restored normal PER responses in the presence of the heated sucrose solution, presumably by blocking HsTRPA activity (Kohno et al., 2010). The effective inhibitor concentrations in our study were about 10 times higher than the concentrations that significantly modified bees' warmth (36.5°C) avoidance in a thermal gradient (0.1 mM RuR and 0.5 mM menthol, Kohno et al., 2010). It is possible that inhibition of the highly-sensitive stinging response requires higher inhibitor concentrations (i.e., more general blocking of HsTRPA channels) than a fine-tuned behavior like warmth avoidance. Alternately, the mode of injection performed in the two studies (ocellar injection in the present study, injection between the antennae in Kohno et al., 2010) might be involved. Performing both experiments in the same conditions may clarify this question. As a control for the effect of the drugs on thermally-induced SER, we tested the effective concentrations on bees' PER to sucrose and found that neither RuR nor menthol
had any effect. If indeed both compounds act on HsTRPA, as we suppose, such a result could have been expected since responses to sucrose are mediated by dedicated gustatory receptors, mostly AmGr1 (Jung et al., 2015). This confirms, however, that RuR and menthol did not reduce SER to heat through a non-specific effect on bees' general responsiveness to stimuli, but rather specifically inhibited their responses to heat.

For the moment, we need to remain cautious about the involvement of HsTRPA in bees' heat sensitivity, as a neuropharmalogical approach alone is not sufficient for demonstrating the role of this TRP channel *per se*. Indeed, the chemical activators and inhibitors we have used are also known to be inhibitors/activators of other members of the TRP family in other species. For instance, in mammals, menthol is able to activate TRPM8 (cold, Behrendt et al., 2004), while RuR is a non-specific inhibitor of TRPM8 (Story et al., 2003) and all four TRPV channels (fine temperature deviation to extreme heat, Clapham et al., 2001; Clapham, 2003). It would thus be especially important in the future to use a technique for blocking HsTRPA more specifically, for instance using RNA interference (Farooqui et al., 2003; Louis et al., 2012), especially because bees express other TRP channels. In invertebrates, channels belonging to the TRPA subfamily are more specifically involved in thermal detection (Matsuura et al., 2009). Most prominently, TRPA1 and Painless have been well described in Drosophila and were shown to be crucial for thermal nociception (Tracey et al., 2003; Hamada et al., 2008; Kohno et al., 2010; Neely et al., 2011). In addition, Pyrexia, another TRP channel, plays a significant part in heat detection and tolerance in this species (Lee et al., 2005). The honeybee genome, as that of other Hymenoptera, does not contain any TRPA1 channel. It is

thought that *HsTRPA*, which has evolved from the duplication of an ancestral hygrosensor (*Wtrw*), has gained thermoresponsive properties, which may have resulted in the loss of *TRPA1* in Hymenoptera (Matsuura et al., 2009). Consequently, HsTRPA is considered as a prominent thermosensor in bees and our results suggest it is involved in heat sensitivity leading to SER. However, homologs of the Drosophila genes painless and pyrexia have been described in the honey bee genome, and named *AmPain* and *AmPyr* respectively (Matsuura et al., 2009). It would thus be important to evaluate next the possible involvement of these two channels in heat sensitivity and thermal aversive conditioning. Thanks to the thermal sensitivity map we have established, future studies will be able to compare the relative sensitivity of the different body parts with the expression patterns of *AmHsTRPA*, *AmPain* and *AmPyr* in the bee body. In addition, SER triggered by heat stimulation, coupled to the use of RNA interference will allow testing the involvement of each channel.

In conclusion, this study constitutes a first step for understanding heat perception and aversive SER conditioning in honey bees. Our current results suggest that a RuR- and menthol-sensitive thermal receptor, possibly HsTRPA, is involved in heat sensitivity leading to sting extension and may represent the peripheral US detector in our aversive conditioning protocol.

## ACKNOWLEDGMENTS

We are thankful to Hanna Cholé for helpful comments on this manuscript, and to all members of the Evolbee team at CNRS Gif-sur-Yvette for insightful discussions. PJ thanks the French Research Ministry and JCS the CNRS for funding.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Junca and Sandoz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
<figure><img src="image_174.png" /></figure>

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# Concept of an Active Amplification Mechanism in the Infrared Organ of Pyrophilous *Melanophila* Beetles

Erik S. Schneider<fnref n="1" />†, Anke Schmitz²† and Helmut Schmitz²*†

Jewel beetles of the genus *Melanophila* possess a pair of metathoracic infrared (IR) organs. These organs are used for forest fire detection because *Melanophila* larvae can only develop in fire killed trees. Several reports in the literature and a modeling of a historic oil tank fire suggest that beetles may be able to detect large fires by means of their IR organs from distances of more than 100 km. In contrast, the highest sensitivity of the IR organs, so far determined by behavioral and physiological experiments, allows a detection of large fires from distances up to 12 km only. Sensitivity thresholds, however, have always been determined in non-flying beetles. Therefore, the complete micromechanical environment of the IR organs in flying beetles has not been taken into consideration. Because the so-called photomechanic sensilla housed in the IR organs respond bimodally to mechanical as well as to IR stimuli, it is proposed that flying beetles make use of muscular energy coupled out of the flight motor to considerably increase the sensitivity of their IR sensilla during intermittent search flight sequences. In a search flight the beetle performs signal scanning with wing beat frequency while the inputs of the IR organs on both body sides are compared. By this procedure the detection of weak IR signals could be possible even if the signals are hidden in the thermal noise. If this proposed mechanism really exists in *Melanophila* beetles, their IR organs could even compete with cooled IR quantum detectors. The theoretical concept of an active amplification mechanism in a photon receptor innervated by highly sensitive mechanoreceptors is presented in this article.

**Keywords:** *Melanophila*, pyrophilous insect, infrared receptor, photomechanic receptor, fire detection, active amplification

## INTRODUCTION

With 13 recent species jewel beetles of the genus *Melanophila* mainly can be found in the boreal and temperate forests of the holarctic zone (Bellamy, 2008). According to the current state of knowledge, males and females approach forest fires because their larvae can only develop in wood of freshly burnt trees (Linsley, 1943; Apel, 1988, 1989, 1991). As an adaptation to the pyrophilous way of life the 1 cm long black beetles (Figure 1A) are equipped with special antennal smoke receptors (Schütz et al., 1999) and one pair of metathoracic IR organs (Evans, 1964; Vondran et al., 1995; Schmitz et al., 1997). An IR organ consists of a little array of dome-shaped sensilla which is situated at the bottom of a little pit (Figures 1B, 2A,B). The inner spherule of each sensillum is innervated by a ciliary mechanosensitive cell (Figure 2C; Vondran et al., 1995; Schmitz et al., 2007). Thus, the

**OPEN ACCESS**

**Edited by:**

Sylvia Anton,
Institut National de la Recherche
Agronomique, France

**Reviewed by:**

Maria Hellwig,
University of Vienna, Austria
Daniel Robert,
University of Bristol, UK

*Correspondence:

Helmut Schmitz
h.schmitz@uni-bonn.de

**Specialty section:**

This article was submitted to
Invertebrate Physiology,
a section of the journal
Frontiers in Physiology

Received: 09 October 2015

Accepted: 30 November 2015

Published: 21 December 2015

**Citation:**

Schneider ES, Schmitz A and
Schmitz H (2015) Concept of an
Active Amplification Mechanism in the
Infrared Organ of Pyrophilous
Melanophila Beetles.
Front. Physiol. 6:391.
doi: 10.3389/fphys.2015.00391

<footnote n="1">¹Institute of Zoology, University of Graz, Graz, Austria, ²Institute of Zoology, University of Bonn, Bonn, Germany</footnote>

<footnote>†These authors have contributed
equally to this work.</footnote>
IR sensilla do not only respond to IR radiation but also to mechanical stimuli. A bimodality has already been demonstrated by electrophysiological experiments: single sensilla respond to weak vibratory stimuli with distinct action potentials (see Figure 3C in Schmitz and Bleckmann, 1998).

There are several hints in the literature that beetles are not only able to detect forest fires but also fires and heat sources of anthropogenic origin from distances of 30 km, 60 km, and even more than 100 km (Van Dyke, 1926; Linsley, 1943; Linsley and

Hurd, 1957). Currently it seems not very likely that beetles use the smell of smoke to detect fires from greater distances. It could not be demonstrated that *Melanophila* beetles could be lured by the smell of smoke (Evans, 1964) or that beetles resting at temperatures of 25°C could be aroused by smoke (unpublished data). A recent study, however, shows that beetles can be attracted by certain volatiles emitted by burning or smoldering wood (Paczkowski et al., 2013). In the study crawling beetles were tested in a two arm olfactometer at a temperature of 30°C. No information about the sex and mating state of the beetles is provided in this study. So these data are more likely suited to show that beetles (e.g., mated females?), once having landed on a burnt tree, can detect a suitable spot for oviposition by olfactory cues. Evaluations of satellite images very often yielded the result that the large smoke plume from a forest fire initially is driven away from the fire by the wind in a narrow angle over distances of many kilometers and finally gradually ascends to higher altitudes. So only beetles inside the smoke plume have a chance to become aware of the fire by olfactory cues. In contrast, beetles that are already close to the fire but outside the smoke plume most probably can see the plume but are not able to smell the smoke. Also the light of the flames—generally only visible at night—may not play an important role, because *Melanophila* beetles, as nearly all jewel beetles, are diurnal (Evans et al., 2007).

All threshold sensitivities published so far were measured in non-flying beetles (highest sensitivity 60 µW/cm², see **Table 1**).

<figure><img src="image_175.png" /><figcaption>**FIGURE 1** | (A) *Melanophila cuspidata* dorsally glued to a needle flying in front of a wind tunnel. Red box encircles area shown in (B). (B) Boundary between meso- and metathorax. Cx, coxa; EPI, episternite; VE, ventrite with IR organ (IR) close to the boundary to the mesothorax.</figcaption></figure>

<figure><img src="image_176.png" /><figcaption>**FIGURE 2** | (A) IR organ of *Melanophila cuspidata*. About 40 sensilla are inside the pit organ. Sensilla belong to a more basic type because wax glands (WG) are still intergrated into the domes of the IR sensilla (inset shows single sensillum). On the left a not fully differentiated sensillum can be seen which still has a bristle. (B) IR organ of *Melanophila acuminata*. The pit organ contains more than 80 sensilla of a more specialized type (wax glands clearly separated from the domes, see lower part of the image). Image modified after Schmitz et al. (2007). (C) General scheme of a *Melanophila* sensillum. d, dendrite; St, stalk. Image modified after Schmitz et al. (2007). (D) Air dried sensillum opened up in the center with a focused ion beam (FIB). WG, wax gland; St, stalk.</figcaption></figure>
<table><caption>TABLE 1 | Sensitivity thresholds of biological and technical IR sensors.</caption><thead><tr><th></th><th>Threshold</th><th>Source/Explanation</th></tr></thead><tbody><tr><td>Melanophila Behavioral experiments</td><td>60 μW/cm²</td><td>Evans, Ecology (1966); Determined by behavioral experiments with non-flying beetles</td></tr><tr><td>Pyroelectric sensor (room temperature) (DIAS 256 LTI)</td><td>8.4 μW/cm²</td><td>Sensorarray DIAS Infrared, Dresden</td></tr><tr><td>Rattlesnake Crotalus atrox</td><td>3.3 μW/cm²</td><td>Ebert and Westhoff, JCP A (2006)</td></tr><tr><td>Microbolometer (room temperature)</td><td>2.3 μW/cm²</td><td>Sensorarray Information provided by Dr. N. Hess, DIAS Infrared GmbH, Dresden. Compiled after: annual SPIE-Conferences "Infrared Technology," Orlando, USA, 1999-2011</td></tr><tr><td>Cooled quantum detector (AIM HgCdTe)</td><td>79 nW/cm²</td><td>Sensorarray AIM Infrarotmodule GmbH, Heilbronn AIM Data Sheet 2nd-3rd-Generation</td></tr><tr><td>Melanophila Coalinga-Simulation</td><td>40 nW/cm²</td><td>Schmitz and Bousack, PLoS ONE (2012)</td></tr><tr><td>Cryogenic cooled quantum detector (Hamamatsu P5274 Serie)</td><td>250 pW/cm²</td><td>High sensitivity single element quantum detector Hamamatsu MCT photoconductive detectors Data sheet P2748/P5274 series</td></tr></tbody></table>

Theoretical calculations, however, show that these sensitivities only allow a detection of a large fire from a distance of about 12 km (Evans, 1966; Schmitz and Bleckmann, 1998).

The results of a simulation of a huge man-made fire provide further evidence that *Melanophila* beetles might be able to detect IR radiation emitted by remote fires from much larger distances (Schmitz and Bousack, 2012). In this study a big oil-tank fire was modeled that burnt in 1925 for 3 days in Coalinga (California) and attracted “untold numbers” of *Melanophila consputa*. This event has also been documented in the entomological literature (Van Dyke, 1926). The site of the fire in the woodless Central Valley of California suggests that most beetles detected the fire by IR radiation from distances of 130 km. This would imply a threshold sensitivity of only 40 nW/cm² (Table 1), corresponding to an energy at a single sensillum of $1.3 \times 10^{-17}$ J. If the threshold of the *Melanophila* IR organ should really be in this range, the biological IR receptor would be two orders of magnitude more sensitive than all current uncooled technical IR sensors and would be able to compete with much more expensive cooled quantum detectors (cf. Table 1).

However, a sensitivity three orders of magnitude higher than the highest sensitivity ever published (Table 1) is only explainable by active amplification mechanisms. Until now, active amplification of very weak input signals has only been reported in the context of hearing: in the cochlear amplifier in the inner ear of vertebrates (Hudspeth, 1989, 1997; Gillespie and Müller, 2009), in antennal ears of mosquitoes and the fly *Drosophila* (Göpfert and Robert, 2001, 2003; Göpfert et al., 2005, 2006; Mhatre, 2015) and recently discovered also in the tympanal ears of a tree cricket (Mhatre and Robert, 2013). An amplification of 1000-fold can be achieved by electromotive outer hair cells in the mammalian cochlea (Robles and Ruggero, 2001; Ashmore et al., 2010). Like hair cells and scolopidia in

vertebrate and insect ears, respectively, the mechanosensitive sensory cells that innervate the IR sensilla in *Melanophila* are ciliary mechanoreceptors. Therefore, the search for active amplification mechanisms appears highly challenging.

## MORPHOLOGICAL PREREQUISITES

Starting point for the development of the concept was the consideration that the IR organs are situated on the metathorax just below the wing hinges (fulcra) of the alae (Figures 1A, 4A,B). Thus, the IR organs are located at a site strongly subjected to vibrations during flight. Additionally, a detail so far not understood was considered: the sphere of a sensillum is attached by a little stalk to the outer cuticular dome (Figures 2C,D). These stalks are missing in the photomechanic IR sensilla of pyrophilous *Aradus* bugs which are quite similar to the *Melanophila* IR sensilla but are not enclosed in a pit organ (Schmitz et al., 2010). By this constellation, vibrations of the spheres in flying *Melanophila* beetles can be proposed to affect the receptor potentials of the mechanosensory cells.

<figure><img src="image_177.png" /><figcaption>FIGURE 3 | (A) Morphology of the pleural region of the metathorax in beetles shown here for the rhinoceros beetle *Oryctes rhinoceros* (after Darwin and Pringle, 1959). The episternite with the fulcrum and the apodem of the basalar muscle (ApoB) are connected by a posterior hinge (red arrow) to the ventrite. In the region of the sternopleural suture (SPS, green) a hinge membrane allows movement of the episternite against the ventrite (Pringle, 1957). (B) Dorsoventrally oriented section through episternite and ventrite in the anterior region (indicated by red line in A) in the buprestid beetle *Chrysobothris solieri*. (C) Corresponding section about 150 µm in posterior direction.</figcaption></figure>
To realize genuine amplification, however, a mechanism has to be implemented permitting a precise regulation of the depolarization amplitude. The morphological prerequisites for such a regulation mechanism were found in the two hitherto investigated species *Melanophila acuminata* and *M. cuspidata*. The structural preadaptation for the evolution of an adjustable beat mechanism most probably was a special feature of the metathoracic pleural region in beetles: the sternopleural suture (SPS, **Figures 3A-C**). Episternite and ventrite are tightly connected only by a posterior hinge and thus can be moved against each other—especially at their anterior edges (Pringle, 1957). Amongst other purposes this mechanism mainly serves for lifting the fulcrum during flight. When the basalar muscle contracts, the episternite is pulled against the ventrite and the leading edge of the wing is pronated during the downstroke (Darwin and Pringle, 1959). The opposing edges of the episternite and the ventrite are formed in a manner so that the episternite can glide over the ventrite (as in the non IR-sensitive jewel beetle *Chrysobothris solieri*, closely related to the *Melanophila*-species, **Figures 3B,C**).

It turned out that in IR-sensitive *Melanophila*-species especially the morphology of the opposing anterior edges of episternite and ventrite has been modified. The slim ventral edge of the episternite can be beaten in a trench on the dorsal edge of

the ventrite, thus a kind of impact edge is realized (Figures 4D, 5A-C). According to the current conception beating of the two sclerites against one another is accomplished by contractions of the basalar muscle, which extends from the dorsal apodeme (ApoB; shown in Figure 4B) posteriorly to the basal region of the ventrite. By regulating the power of the muscle contractions during a search flight sequence (see below), the vibrations of the spheres caused by the proposed mechanism can be controlled. Therefore, also the magnitude of receptor depolarizations could be regulated.

In this context it is of great importance that the IR sensilla are innervated by ciliary mechanoreceptors. Specialized arthropod mechanoreceptors innervated by ciliary mechanosensory cells are the most sensitive receptors known. This could be shown for trichobothria in spiders, where energies of $1.5 \times 10^{-19}$ J to $2.5 \times 10^{-20}$ J are still sufficient for a suprathreshold stimulation of the receptors (Humphrey et al., 2003; Barth, 2004) and also for filiform hairs in insects (Thurm, 1982). Filiform hairs in crickets serving for detection of faint airflows can already generate an action potential if energies are still in the range of $k_B T$ ($k_B$: Boltzmann constant; T: temperature), i.e., about $4 \times 10^{-21}$ J (Shimozawa et al., 2003).

At the threshold, these ultrasensitive mechanoreceptors already work within the range of thermal noise of Brownian

<figure><img src="image_178.png" /><figcaption>**FIGURE 4** | (A) Outer and (B) inner view of the pleural metathorax of *Melanophila cuspidata*. The ventral anterior region of the episternite is developed as impact edge which can be beaten in a furrow of the ventrite. In (B) the apodeme of the basalar muscle (ApoB) is shown which is connected to the fulcrum (tissue removed). Red arrow points to posterior hinge shown in (C). (C) Section through hinge in dorso-ventral direction, outside on the left). (D) Dorso-ventral section through the pleural metathorax (indicated in A by red line). By contraction of the basalar muscle the edge of the episternite is beaten in the dorsal furrow of the ventrite (red arrow). Vibrations caused by the beats most probably are conducted to the IR sensilla. AS, air sac below IR organ; SPS, sternopleural suture.</figcaption></figure>
molecular motion and therewith close to the physical limit (Barth, 2004). It can be concluded that for a subthreshold depolarization of the mechanosensitive sensory cell innervating the IR sensilla most probably vibration amplitudes of the spheres of less than one nanometer are already sufficient.

In the two *Melanophila* species investigated so far, *M. acuminata* and *M. cuspidata*, morphological differences were found. It appears that the IR detection system (i.e., IR-organ plus the structures used for the proposed beat mechanism) in *M. cuspidata* is more ancient and relatively simple: IR organs contain less sensilla into which the wax glands are fully integrated (**Figure 2A**). So an unrestrained vibration of the sphere most probably may be somewhat hampered and less precise. An explanation could be that *M. cuspidata* is distributed in the Mediterranean region where fires are more frequent than in northern Europe (San-Miguel and Camia, 2009). Thus, the necessity to detect fires also from very large distances seems not to be predominant. In contrast, *M. acuminata* is distributed in the boreal forests of the northern hemisphere (in Europe northern distribution up to Fennoscandia; Horion, 1955), where forest fires are less frequent. Accordingly, a higher evolutionary pressure with respect to the sensitivity of the IR organs can be proposed. In *M. acuminata*, the IR organs contain significantly more IR sensilla from which wax glands

are clearly separated (**Figures 2B-D**). It can be concluded that unobstructed vibrations of the spheres are possibles. Furthermore, an additional damping system obviously has developed. This system may allow a precise "fine tuning" of the depolarizations of the IR sensilla caused by the beat mechanism. By a system of at least two muscles of hitherto unknown origin a damping cushion of about 300 µm lengths can be brought down into the inner trench of the ventrite (**Figures 5A-D**). Thus, at a given contraction power of the basalar muscle a very precise adjustment of the beat intensity and consequently of the evoked pre-depolarizations could be adjusted. Despite intense search in two further specimens such a damping cushion could not be found in the Mediterranean *M. cuspidata* (cf. **Figure 4**).

HOW IT COULD WORK

According to the present idea how *Melanophila* beetles may be able to become aware of a fire from large distances, beetles use a combination of visual cues (view of a big cloud against the horizon) and IR radiation. To make sure that a smoke plume and not a cloud bank is approached over distances of many kilometers a zone of IR emission has to exist at the base of the cloud above tree top level. IR sensitive *Melanophila* beetles, therefore, will conduct search flights for fire

<figure><img src="image_179.png" /><figcaption>**FIGURE 5** | (A) Top view (SEM micrograph) onto the furrow at the dorsal edge of the ventrite in *Melanophila acuminata* (all tissue removed). Light-blue area indicates region in which the proposed damping cushion (pad) can be brought into contact with the ventrite. (B) Section through region marked with red line in (A) showing the beating edge of the episternite and the narrow furrow in the ventrite. Same orientation as in **Figure 4A**. (C) Section through region marked in (A) with red line: here the damping pad (P) with its musculature is shown. (D) Section through the pad about 150 µm posteriorly. Pad becomes smaller toward its posterior end. After about 300 µm the pad is not longer present.</figcaption></figure>
detection. While doing so, beetles especially examine potential smoke plumes in view of additional IR emission. By the beat mechanism a cyclic depolarization of the IR sensilla with wing beat frequency appears possible (Figures 6A,B). In beetles, the basalar muscle serves as a well-developed direct wing depressor, which, together with the indirect dorso-longitudinal muscles (main muscle for propulsion), is used for wing downstroke. In principle the basalar muscle can be classified as a steering muscle (Nachtigall, 2003). By adjusting the contraction power of the basalar muscle, the wing inclination angle during the downstroke of the wing (pronation) and in this way propulsion

and buoyancy is adjusted. During a supposed search flight sequence, which may last for a few seconds only, the beetles could tune the contractions of the basalar muscles exactly so that the peak amplitudes of the oscillatory receptor potential almost reach the spike-triggering threshold. At the same time a slight reduction in propulsion and buoyancy would not be disadvantageous. To tune the IR organs to maximal sensitivity in anticipation of arriving IR radiation, the beetles should be able to adjust the intensity of the beat mechanism and therewith the probability of impulse generation by sensory feedback. As a result only a certain, most probably very low, percentage of sensilla in both organs generate action potentials.

With respect to symmetry the inputs of both IR organs could be permanently compared by central comparator neurons. Such central units enable acoustically communicating insects to approach, e.g., a sound source by paired hearing organs (von Helversen and von Helversen, 1995; Stumpner and von Helversen, 2001). A mechanical prestimulation of only a few sensilla in the *Melanophila* IR organ could be explainable by the fact that the sensilla show minute differences in their dimensions (cf. Figures 2A,B). In case of an oscillatory mechanical stimulation with constant intensity some sensilla will already generate first action potentials whereas most others will remain just below the threshold. At the peak of a given subthreshold depolarization additional IR radiation will slightly increase the height of the amplitude (Figure 6B). This will result in a few more spiking sensilla in the organ exposed to IR radiation. The asymmetry in the inputs of both organs immediately could be detected by comparator neurons. Therewith the beetle gets information about the spatial direction from which IR radiation arrives. This information could be combined with visual cues (e.g., a smoke plume) and the beetle should be able to directly approach a fire.

Based on theoretical considerations it seems essential that the vibrations of the spheres caused by the proposed beat mechanism have to be strongly damped. By appropriate damping an uncontrolled soaring up of the system can be suppressed and it can be ensured that the spheres all are in a defined initial state before the next impact impulse arrives. Ideally a creeping case (i.e., damping so strong that no oscillation can arise) or at least an aperiodic limit case (i.e., strong damping ensures oscillation of the sphere with only one zero crossing) has to be proposed. In this way beat impulses of constant intensity will always cause monotonic depolarization amplitudes. Most probably damping is realized by a slender margin of fluid surrounding each sphere (Figure 2C). This margin with a thickness of about 0.3 µm consists of the apical extensions of the two outer enveloping cells (Vondran et al., 1995). By this specific feature a fluidic damping system is build. Subthreshold depolarizations of the receptors most probably are already evoked by sub-nanometer vibrations of the spheres. The necessary small scale dislocation of water is allowed by compensatory air sacs below the IR organ (Figure 4D, AS).

Provided that the cuticular apparatus (i.e., mainly the spheres) is able to convert the energies of absorbed IR photons effectively into mechanical energy (so-called photomechanic mechanism

<figure><img src="image_180.png" /></figure>

<figure><img src="image_181.png" /><figcaption>**FIGURE 6** | Schematic depiction of the proposed beating mechanism. (A) Coupling in muscular energy (blue double arrows) from the flight motor results in vibration of the cuticular spheres. This in turn causes the stimulation of the mechanoreceptive sensory cells by which the spheres are innervated. By sensory feedback the sensory cells can be systematically depolarized with wing beat frequency. In contrast, energy for active amplification in hearing organs is generated by molecular motors of the sensory cells itself (green double arrows at the sensory cells). (B) In non-flying beetles the receptor potential of the IR sensilla is set at about -70 mV. Slight fluctuations are caused by thermal and mechanical noise. When the beetles take off, IR sensilla become depolarized by the beating mechanism: (1) by increasing beat intensity, the beetle is able to release action potentials in the absence of IR radiation. (2) by tuning the beat intensity and—in *Melanophila acuminate*—by additional fine tuning of the damping system, depolarization can be adjusted in a way that the generator potentials of most sensilla remain just below the threshold. (3) in turn IR radiation superimposed to the thermal noise at the peaks of the receptor potentials causes the generation of (additional) action potentials in exposed sensilla.</figcaption></figure>