Retinal and optical adaptations for nocturnal vision in the halictid bee Megalopta genalis
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- Greiner, B., Ribi, W.A. & Warrant, E.J. Cell Tissue Res (2004) 316: 377. doi:10.1007/s00441-004-0883-9
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The apposition compound eye of a nocturnal bee, the halictid Megalopta genalis, is described for the first time. Compared to the compound eye of the worker honeybee Apis mellifera and the diurnal halictid bee Lasioglossum leucozonium, the eye of M. genalis shows specific retinal and optical adaptations for vision in dim light. The major anatomical adaptations within the eye of the nocturnal bee are (1) nearly twofold larger ommatidial facets and (2) a 4–5 times wider rhabdom diameter than found in the diurnal bees studied. Optically, the apposition eye of M. genalis is 27 times more sensitive to light than the eyes of the diurnal bees. This increased optical sensitivity represents a clear optical adaptation to low light intensities. Although this unique nocturnal apposition eye has a greatly improved ability to catch light, a 27-fold increase in sensitivity alone cannot account for nocturnal vision at light intensities that are 8 log units dimmer than during daytime. New evidence suggests that additional neuronal spatial summation within the first optic ganglion, the lamina, is involved.
KeywordsVisual systemNocturnal visionApposition compound eyeRetina structureDim lightMegalopta genalis (Insecta)
Considering Megalopta’s remarkable nocturnal visual abilities, superposition eyes would be of great advantage to this bee. However, the more than 16,000 described bee species worldwide (Michener 2000) have formed a clearly established monophyletic lineage (Jander and Jander 2002) with the apposition eye as its dominant eye design. Even though Megalopta’s nocturnal visual abilities using apposition eyes are an extreme example, the African race of the honeybee Apis mellifera and the Asian apid A. dorsata can continue to forage on nights with a half-full moon present in the sky (Fletcher 1978; Dyer 1985). Theoretically, however, light levels at early dusk should render their insensitive apposition eyes blind (Warrant et al. 1996). Due to this clear contradiction, Megalopta’s eyes must have evolved distinct nocturnal adaptations. Several possibilities exist that may allow Megalopta to increase light capture at night. These include the development of (1) more sensitive optics, (2) larger and more sensitive rhabdoms, and/or (3) an eye design that changes from apposition during the day, to superposition at night, via morphological and optical circadian changes as found in the eyes of some tiger beetles (Brännström 1999). We explored these possible nocturnal adaptations by means of histology and optical measurements. Our study represents the first detailed description of the apposition compound eye of a nocturnal bee. The results are analysed and discussed in comparison with the well-studied worker honeybee Apis mellifera and females of the more closely related diurnal halictid bee Lasioglossum leucozonium.
Materials and methods
Females of Megalopta genalis (Meade-Waldo 1916) were collected in a 2-week period (25 March to 5 April 2003) on Barro Colorado Island (9°N, 79°W), Republic of Panama. Their distribution ranges from Mexico to southern Brazil, and they were common at our study site in Panama. Females live solitarily or in small groups (of up to ten bees), constructing nests and brood cells with their strong mandibles in small wooden branches. These can be primarily found above the ground and within the thicket of the rainforest. As these bees are only active at night during two activity periods shortly after dusk and before dawn, we used UV light tubes mounted over white linen sheets to attract females during these peak activity times. In this tropical semideciduous moist forest the dry season generally lasts from mid-December until mid-May (Leigh 1999). M. genalis is most abundant during the dry-to-wet season transition and during the first half, and at the end of, the wet season (Wolda and Roubik 1986).
Taxonomy of the bee species used in this study
Bees were immobilized by cooling them down at 4°C. Their head capsules were subsequently opened by cutting the mouthparts and the frontal head cuticule away. Optimal fixative penetration into the retina was achieved by cutting the most ventral rim of the eye away. In order to study the effect of dark adaptation vs. light adaptation in the retina in M. genalis, one group of bees was dissected under dim light conditions for dark adaptation and another group in bright light at 6:30 a.m., at the end of their activity period. The diurnal comparison species A. mellifera and L. leucozonium were dissected and fixed in bright light during daytime. We used a fixative consisting of a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (pH 7.2–7.5). The heads were fixed for 2–3 h at 4°C before being osmicated (2% OsO4 in distilled H2O) for 1 h. The heads were subsequently dehydrated in an ethanol series, transferred to propylene oxide and embedded in epoxy resin (Fluka).
Frontal, longitudinal and tangential serial sections, 1 μm thick, were cut on a Reichert Ultracut microtome using glass knives. Four tangential and six longitudinal section series were cut from the whole eye of light adapted M. genalis females, as well as two tangential and two longitudinal section series from the whole eye of dark adapted M. genalis. In A. mellifera and L. leucozonium two tangential and longitudinal sections were sectioned from the whole eye of both species. Moreover, we applied a re-embedding method (Ribi 1976, 1978) that enabled us to follow single ommatidia throughout their whole length in different regions of the eye of M. genalis. In order to do this, serial frontal sections, 25–50 μm thick, were cut on a Reichert sledge microtome, and embedded with epoxy between two acetate sheets. Sections containing complete and parallel ommatidia were selected and small areas containing six to ten ommatidia were cut out and re-embedded for tangential and frontal section series. Two tangential and four longitudinal section series of the whole length of the retina were cut from the dorsal and medial regions of the eye. The 1-μm-thick section series were placed on microscope slides and flattened on a 60°C hot-plate. These sections were then stained with toluidine blue and viewed under a Zeiss photo-microscope. Colour pictures were taken with Fuji AS100 film and the negatives scanned in digitally for further analysis.
The corneal micro-surface and facets were studied in the three bee species using a JEOL JSM-5600 scanning electron microscope. Furthermore, corneas of two M. genalis females and two L. leucozonium females were painted with a thin layer of nail polish to produce cornea replicas. After letting them dry for a few hours the cornea replicas could be carefully and completely removed by cutting along the corneal border of the eye with a microscalpel (Ribi 1978). Several incisions were necessary to flatten the retina on the microscope slide. The replicas were coated with coverslips, sealed with melted wax, and viewed under a Zeiss Axiophot microscope. Each replica was photographed with an Olympus DP 50 digital camera and analysed on a Compaq EvoW4000 PC using Adobe Photoshop 7.0. The largest possible circle was then fitted into the hexagonal facets across different eye regions to calculate the respective facet diameters (Praagh et al. 1980). Measurements of facet diameter were also made on M. genalis females mounted in a Leitz goniometer and viewed with a macroscope (for full details see Land and Eckert 1985; Rutowski and Warrant 2002).
The nodal point N was situated at a distance of one focal length f in front of the image and its position within the corneal lens was determined by measuring the distance from the image to the back of the lens and then subtracting this distance from the focal length.
Anatomical aspects of the retina in nocturnal bees
The nine retinula cells of each ommatidium give rise to retinula cell axons: six short visual fibres (svf) and three long visual fibres (lvf), ending in a straight projection to the lamina or the medulla, respectively. These retinula cell axons penetrate the basement membrane in a distinct axon bundle (Fig. 5h, i), forming pseudocartridges (Fig. 6d). On the way to the lamina, the first optic ganglion, these pseudocartridges pass through the fenestrated layer, a region below the basement membrane containing trachea and the cell bodies of lamina cells. After reaching the lamina, a network of tangential fibres keeps their hexagonal arrangement according to their assembly in the retina. Retinula cells originating in the overlying ommatidium target the same cartridge in the lamina.
Distal to the basement membrane a thick layer of pigments is present (Fig. 5i, k), which can be found within the secondary pigment cells as well as in the crystalline cone extensions that envelop the most proximal tip of the rhabdom (Fig. 5h). Consequently, no tapetum is visible in the retina of M. genalis females.
Anatomical aspects of the retina in diurnal bees
Anatomical and optical parameters in the eye of the nocturnal halictid M. genalis, the diurnal halictid L. leucozonium and the worker honeybee A. mellifera. All values are taken from the frontomedial region of the eye in both species
Number of facets per eye
Maximal corneal facet diameter
Crystalline cone length
Distal rhabdom diameter
Absorption coefficient of the rhabdom
Theoretical acceptance angle
Optical aspects of the nocturnal and diurnal bee retina
The optical sensitivity of a compound eye is greater if it views an extended scene with facets of greater area [(π/4)D2 μm2], with photoreceptors that view larger solid angles of visual space [(π/4)(d/f)2 sr, from the definition of solid angle] and with longer (l μm) and more absorptive (k μm−1) photoreceptors. Recent work (Stavenga 2003b) has shown that Eq. 3 has limitations in eyes operating according to the principles of waveguide optics, and in some eyes with F-numbers lower than 2. Therefore, some caution needs to be applied when interpreting sensitivity values obtained in the wave-guiding eyes of the diurnal bees. The eye of M. genalis, with 8-μm-wide rhabdoms, can clearly be approximated by geometric optics, and with F=2.7, Eq. 3 can be applied with safety.
After conversion from radians to degrees, M. genalis females have a theoretical acceptance angle Δρ of 4.7°, which is nearly 3 times wider than found in worker honeybees (Δρ=1.7°) and the diurnal halictid bee (Δρ=1.6°).
The retina of the nocturnal bee Megalopta genalis has the same general anatomical organisation as the typical diurnal bee apposition eye of the worker honeybee Apis mellifera and the diurnal halictid bee Lasioglossum leucozonium. However, there are a number of specific differences that have most likely evolved for Megalopta’s purely nocturnal activity. The most distinct nocturnal adaptations are (1) the 1.8 times larger ommatidial facets and (2) the 4–5 times wider rhabdom (Fig. 8). A nocturnal transition to a superposition eye design, as found in some tiger beetles, was not found. Furthermore, having determined the focal lengths, we can, with limitations, discuss Megalopta’s nocturnal optical adaptations in terms of sensitivity and spatial resolution.
The advantages of being nocturnal
A nocturnal lifestyle has two major advantages: lower competition for scarce food resources and fewer predators (Roubik 1989; Warrant et al. 1996). With many flowers opening at night, the purely nocturnal bee M. genalis has the great advantage of being able to exploit abundant food sources when diurnal bees are inactive. Pseudobombax septanatum (Bombaceae), a large canopy tree common on Barro Colorado Island in Panama, has been reported as being a possible pollen host of M. genalis (Roulston 1997). These plants produce large white flowers that open at sunset and are highly attractive to diurnal bees in the mornings (Roulston 1997). A similar beneficial effect of lower competition has also been reported for matinal, as well as crepuscular, bees (Kerfoot 1967; Linsley and Cazier 1970; Shelly et al. 1993). As usual predators of large bees, one can consider birds, toads, dragonflies, spiders, robber flies, lizards and mantids. Many of these predators are active during the day and most likely contribute little to the mortality of bees active at night (Wolda and Roubik 1986). Furthermore, nest parasites are known to mainly use visual cues and only enter the nests when the host is absent (Wolda and Roubik 1986).
An apposition eye with nocturnal adaptations
According to Kerfoot (1967) and Engel (2002), the particular features that distinguish bees with nocturnal activity are enlarged eyes and ocelli, a large body size, and pale body pigmentations. Jander and Jander (2002) have shown that the surface area of the eyes is 1.8 times larger in nocturnal bees (including Megalopta) than in diurnal bees. These results fit well with our 1.8-fold increase in facet diameter found in the nocturnal bee compared to the diurnal bees (Fig. 8c, d). A larger eye with larger facet diameters will, under the same light conditions, accept more light than a smaller eye with smaller facets, leading to greater sensitivity (Warrant and McIntyre 1993). We will return to these larger facets later.
As mentioned in the “Results”, the outer corneal curvature of the eye is flat and thus has an infinite radius of curvature (r1≈∞), whereas the inner corneal surface is convex, with a curvature of r2=−22 μm. Assuming realistic refractive indices for the cornea (n1=1.45) and the crystalline cone (n2=1.34) (Stavenga 2003b), defining n0=1 as the refractive index of the outer medium (air), noting that the axial separation of the corneal curvature centres (s) is 48 μm and using the radii of curvature of the corneal lens given above, we can calculate a focal length of 200 μm. This theoretical value is twice as large as the experimentally measured focal length, indicating that an inhomogeneous lens might be present in order to focus light onto the tip of the rhabdom. To clarify this, exact refractive index measurements of the corneal lens cylinder are needed, and these will be the subject of further investigations.
The corneal outer surface of M. genalis features distinctly noticeable corneal protuberances or nipple-array (Fig. 8a). In comparison, worker honeybee facets are smooth (Fig. 8b), and protuberances are only formed at the facet borders; a clear separation line towards the smooth facet surface can be distinguished (Fig. 8b). Regarding the function of this corneal nipple-array, it has been shown that for moths in particular these are likely to function as an anti-reflection coating when they reach a height of 50 nm or greater (Miller 1981). Corneal nipples not only decrease reflections from the cornea, effectively camouflaging the animal, they also increase the intensity of a focused image by as much as 4%. Corneal protuberances of less than 50 nm height show a tendency towards coalescence, as seen on the corneas of flies (Miller 1981). A similar coalescence could be observed on the facets of L. leucozonium. In this case, the protuberances are not thought to have an anti-reflection effect. Although the exact height of the corneal nipples of M. genalis still needs to be determined, we present the first indications that the corneal protuberances in M. genalis could be involved in increasing light flux into the eye, an important factor for a photon starved animal.
The organisation of the nine retinula cells in M. genalis is very similar to the retinal structure in the diurnal bees. No anatomical differences based on taxonomy were found. As L. leucozonium is of much smaller body size, rhabdom size and volume are only compared between the equally sized A. mellifera and M. genalis. In M. genalis the distal tip of the fused rhabdom, measured in the frontomedial region of the eye, is a remarkable 4 times wider than in the worker honeybee (Fig. 8e–h). This wider and also longer rhabdom (for values see Table 2) leads to a 17.5-fold greater photoreceptive volume in M. genalis, representing a significant improvement in sensitivity. Apart from the effect of an increased rhabdom width, a wide pupil during dark adaptation also leads to a greater sensitivity.
During dark adaptation, pigment migration in the nocturnal bee was only seen inside the retinula cells (Fig. 7b). No retraction of the rhabdoms and secondary pigments—necessary for the formation of a clear zone—was visible. This disproves our hypothesis that Megalopta might possess a functional superposition eye at night. The retinula cell pigment migration (Fig. 7b) represents a so-called “longitudinal pupil” and this type of pupil is also found in day-active insects such as flies, butterflies (Stavenga 1979) and the honeybee (Kolb and Autrum 1972, 1974; Stavenga and Kuiper 1977; Autrum 1981). During light adaptation, the retinula cell pigments tightly surround the rhabdom, absorbing much of the propagating light and leading to higher spatial acuity (Fig. 7a). During dark adaptation the pigments migrate away from the rhabdom, resulting in higher sensitivity, but at the cost of spatial resolution (Fig. 7b) (Land and Osorio 1990). Furthermore, in the light-adapted state one can observe large cisternae, also known as palisades, surrounding the rhabdom. In locusts, where they are prominent in the dark-adapted retina, these palisades are thought to improve the light-guiding properties of the rhabdoms (Horridge and Barnard 1965). This is probably not the case in M. genalis, as the palisades almost disappear during dark adaptation.
An adaptation to dim light that is particularly common in deep sea and nocturnal animals is a mirror-like structure called the tapetum. Tapeta are mostly developed at the proximal base of the retina and reflect the light back into the rhabdoms, giving the retina a second chance to absorb light (Land and Nilsson 2002). In many insects, tapeta are constructed of modified tracheal structures ensheathing the proximal end of the rhabdom and are capable of producing a characteristic eye glow (Ribi 1980). Due to its nocturnal lifestyle, one might expect M. genalis to have a tapetum at the proximal base of the retina. Indeed, by viewing the eye from the direction of illumination we were able to observe a bright eye glow that could easily arise from a tapetum. However, the pigmented enlargements of the crystalline cone extensions optically isolate the most proximal end of the rhabdom (Fig. 5h) and secondary pigment cells construct a thick layer of pigments above the basement membrane absorbing all stray light (Fig. 5i, k). These features can also be observed in the diurnal bees studied, which have neither a tapetum nor an eye glow. From this we conclude that no tapetum exists in the retina of M. genalis females. However, the cause of the bright eye glow is still unclear and is currently being investigated.
Optical adaptations to dim light
Insects living in dim habitats generally possess compound eyes of lower F-number, indicating greater sensitivity to extended sources of light (Warrant and McIntyre 1991). The F-numbers of nocturnal insects with superposition eyes usually range between 0.5 and 1.2 (Warrant and McIntyre 1993). Despite its nocturnal activity, the high F-number of M. genalis indicates that its eye is more typical of diurnal apposition eyes, which generally have F-numbers higher than 2.1 (Warrant and McIntyre 1993). There are, however, apposition eyes with exceptionally low F-number, such as those of the deep sea isopod Cirolana borealis with an F-number of 1.0 (Nilsson and Nilsson 1981), implying that dim habitats can lead to specialised apposition eyes with high sensitivity. Megalopta’s high F-number suggests that its eye is not only adapted to high sensitivity, but also maintains a construction that supports reasonable spatial resolution, no doubt necessary for its difficult orientation tasks at night. An F-number of 2 or greater also ensures that in eyes with wide rhabdoms all of the light that reaches the rhabdom remains trapped within it (Stavenga 2003b). With an F-number of 2, light strikes the rhabdom with an angle of incidence of 10°, the maximum angle that ensures total internal reflection within the rhabdom (Warrant and McIntyre 1993; Stavenga 2003b). In eyes with low F-numbers (i.e. with a large aperture relative to the focal length), a large amount of light can escape and spread to neighbouring rhabdoms, leading to a degradation of image quality and a loss in spatial resolving power. The nocturnal mosquito Anopheles gambiae, with thin near-hemispherical lenses and an F-number of 0.78, avoids this problem by possessing unique conical rhabdoms that are able to trap light from much wider angles (Land et al. 1997, 1999). Consequently, with an F-number of 2.7, M. genalis can capture all of the incident light within its wide rhabdoms.
Despite the fact that a higher F-number indicates the importance of spatial resolution for M. genalis, its apposition eyes must of course be sensitive enough to see at night. This is reflected in the optical sensitivity S of the eye, a quantity that takes account of other factors when considering sensitivity (Eq. 3). In M. genalis, S is considerably higher than in the diurnal bees. In the nocturnal bee, the photoreceptors absorb 27 times more light when viewing an extended light source, a finding confirmed in another study that relies on electrophysiological measurements (Warrant et al., in preparation). With an optical sensitivity of 2.69 μm2 sr, the nocturnal apposition eye of M. genalis is nevertheless considerably less sensitive than that of the nocturnal superposition eye of the elephant hawkmoth Deilephila elphenor, which has an optical sensitivity of 69 μm2 sr (Warrant, unpublished data). Theoretical approximations of the acceptance angle Δρ resulted in a value that is nearly 3 times larger in M. genalis (Δρ=4.7°) than in the diurnal bees (for values see Table 2). This greater acceptance angle in M. genalis indicates higher sensitivity at the cost of poorer resolution. This approximate value is relatively close to the value measured experimentally in the frontal part of Megalopta’s eye (Δρ=5.6°; Warrant et al., in preparation).
Considering the fact that M. genalis is constrained by its unsuitable apposition eyes, it performs astonishing orientation and homing tasks at night. One might imagine that they have exploited all possible retinal and optical adaptations to find the ideal trade-off between maximised sensitivity and spatial resolution. In fact, the adaptations we have described for this nocturnal bee are extreme and unique for an insect apposition eye, as one might expect for an animal that must cope with up to a hundred million times less light than a diurnal bee. Although a 27 times higher sensitivity to light in M. genalis is a remarkable improvement, this alone cannot account for their ability to see at light levels 8 log units dimmer. Therefore, we must assume that higher neuronal mechanisms, increasing sensitivity via summation of photons in space and time, are involved (Warrant 1999). Temporal summation, which takes place in the retina and in the brain, involves the summation of photons over a longer time period, similar to a camera with a long exposure time. However, its cost in making vision slower could be detrimental for fast moving animals like bees. For spatial summation, the eyes could sum signals from large groups of ommatidia, in order to improve their photon catch (Warrant et al. 1996; Warrant 1999). However, this only occurs at the cost of courser spatial resolution. Our current idea is that spatial summation takes place very early in visual processing, and therefore most probably resides in the lamina, the first optic ganglion in the optic lobe of the brain. Recent findings of wide laterally branching first-order interneurons in Megalopta’s lamina (Greiner et al., in preparation) indicate that spatial summation may play a major role in the impressive visual abilities of this nocturnal bee.
We would like to thank Almut Kelber, Doekele Stavenga, Mark Holdstock and the two anonymous referees for critically reading the manuscript, Ladina Ribi for the taxonomic drawing of Megalopta, Rita Wallén and Carin Rasmussen for histological and technical support, Rikard Frederiksen for help with the corneal replicas, Mikael Sörenson and Jan Tengö for taxonomical help, as well as Victor Gonzales and Sara Juhl for help with fieldwork. We thank William T. Wcislo and the staff of the Smithsonian Tropical Research Institute, Panama City, for their help and the Autoridad Nacional del Ambiente of the Republic of Panama for permission to export bees. The histological work was partly done at the Center for Visual Sciences, Research School of Biological Science, Australian National University, Canberra.