Michio Sugahara

On the three visual pigments in the retina of the firefly squid, Watasenia scintillans

SummaryThe deep-sea bioluminescent squid, Watasenia scintillans, has three visual pigments: The major one (A1 pigment) is based on retinal and has λmax = 484 nm, the second one (A2 pigment) is based on 3-dehydroretinal and has λmax = 500 nm, and the third one (A4 pigment) is based on 4-hydroxyretinal and has λmax = 470 nm. The distribution of these 3 visual pigments in the retina was studied by HPLC analysis of the retinals in retina slices obtained by microdissection. It was found that A1 pigment was not located in the specific region of the ventral retina receiving the down-welling light which contains very long photoreceptor cells, forming two strata. A2 and A4 pigment were found exclusively in the proximal pinkish stratum and in the distal yellowish stratum. The role of these pigments in the retina is hypothesized to involve spectral discrimination. The extraction and analysis of retinoids to determine the origin of 3-dehydroretinal and 4-hydroxyretinal in the mature squid showed only a trace amount of 4-hydroxyretinol in the eggs. Similar analysis of other cephalopods collected near Japan showed the absence of A2 or A4 pigment in their eyes.

Studies on cephalopod rhodopsin. Conformational changes in chromophore and protein during the photoregeneration process

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (lamda >520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.

Differences in Heat Sensitivity between Japanese Honeybees and Hornets Under High Carbon Dioxide and Humidity Conditions Inside Bee Balls

Upon capture in a bee ball (i.e., a dense cluster of Japanese honeybees forms in response to a predatory attack), an Asian giant hornet causes a rapid increase in temperature, carbon dioxide (CO2), and humidity. Within five min after capture, the temperature reaches 46°C, and the CO2 concentration reaches 4%. Relative humidity gradually rises to 90% or above in 3 to 4 min. The hornet dies within 10 min of its capture in the bee ball. To investigate the effect of temperature, CO2, and humidity on hornet mortality, we determined the lethal temperature of hornets exposed for 10 min to different humidity and CO2/O2 (oxygen) levels. In expiratory air (3.7% CO2), the lethal temperature was ≥ 2° lower than that in normal air. The four hornet species used in this experiment died at 44–46°C under these conditions. Hornet death at low temperatures results from an increase in CO2 level in bee balls. Japanese honeybees generate heat by intense respiration, as an overwintering strategy, which produces a high CO2 and humidity environment and maintains a tighter bee ball. European honeybees are usually killed in the habitat of hornets. In contrast, Japanese honeybees kill hornets without sacrificing themselves by using heat and respiration by-products and forming tight bee balls.

An intermediate in the photoregeneration of squid rhodopsin.

Abstract Squid rhodopsin made alkaline with borate-NaOH buffer is bleached by irradiation with yellow light. After irradiation, rhodopsin is regenerated from a substance with an absorption maximum of 380 nm (P 380 ). This regeneration of rhodopsin is scarcely observed in a solution made alkaline with sodium carbonate instead of borate buffer. The reaction velocity of the conversion of P 380 to rhodopsin is dependent not only on temperature and pH but also on the concentrations of borate buffer and digitonin. P 380 is produced from acid metarhodopsin during irradiation with yellow light and its chromophore must be 11- cis -retinal. This substance with λ max of 380 nm is an intermediate in the photoregeneration process of squid rhodopsin.

Oriental Orchid (Cymbidium floribundum) Attracts the Japanese Honeybee (Apis cerana japonica) with a Mixture of 3-Hydroxyoctanoic Acid and 10-Hydroxy- (E)-2-Decenoic Acid

The flower of the oriental orchid Cymbidium floribundum is known to attract the Japanese honeybee Apis cerana japonica. This effect is observed not only in workers but also drones and queens; that is, it attracts even swarming and absconding bees. A mixture of 3-hydroxyoctanoic acid (3-HOAA) and 10-hydroxy-(E)-2-decenoic acid (10-HDA) was identified as the active principles from the orchid flower, whereas these compounds individually have no such activity. Both compounds are also mandibular gland components of worker honeybees with related compounds. This strongly supports the idea that orchid flowers mimic bee secretions, although the ecological consequences of this relationship remain unknown. Because the flower is used to capture swarms, the present identification may contribute to the development of new techniques in traditional beekeeping for Japanese bees as well as A. cerana in Southeast Asia.

Apis Cerana Japonica Discriminates between Floral Color Phases of the Oriental Orchid, Cymbidium floribundum

Foragers of the Japanese honeybee (Apis cerana japonica) were attracted by flowers of an oriental orchid (Cymbidium floribundum) and were observed to carry the pollinia on their scutella. After the removal of pollinia from the flowers, their labial color changed from white to reddish brown. Both artificial removal of pollinia and ethrel treatment of the flowers also induced this labial color change. Labia in color-changed flowers showed a decreased reflectance of wavelengths less than 670 nm compared to control intact flower. Both reflectance irradiance spectra and ultraviolet photographs showed that only the nectar guide in white (unchanged) flowers reflected ultraviolet light, and that this reflectance decreased with labial color change. Dual choice experiments showed that the honeybee foragers preferentially visited flowers having white labia rather than reddish brown. We suggest that Japanese honeybees discriminate between the floral phases of C. floribundum using color vision.