Here, we fully characterize bioluminescence in H. brasiliana, an intertidal mari
ID: 225378 • Letter: H
Question
Here, we fully characterize bioluminescence in H. brasiliana, an intertidal marine snail in the Planaxidae, a family that contains approximately 20 species in six genera [43]. Reports of light production are available for three of the genera, namely Planaxis [41], Angiola [43] and the one studied here, Hinea [42]. We describe a unique mechanism of spatially amplifying a biolumines-cent signal using a hard, calcified shell for diffusion. Indeed, we demonstrate that the shell of the snail acts as a unique diffuser that propagates the specific wavelength of the bioluminescence, causing the light signal to appear enlarged to the receiver. MATERIAL AND METHODS Adult individuals (0.5-1.5 cm shell length) of H. brasiliana (Lamarck, 1822) were collected by hand at low tide under rocks from Merry Beach and Hastings Point, NSW, Australia. Photonic properties of the bioluminescence were measured directly from the body of a snail, after shell removal using a rotary circle cutter (400 series XPR Dremel, Wisconsin, USA). Bioluminescence was experimentally stimulated with potassium chloride (KC1, 200 mM final concentration), which is commonly used on bioluminescent invertebrates to depolarize tissues and trigger light production until exhaustion of their luminous constituents [44]. This treatment usually allows enough light to be produced for photonic characterization. The spectrum of the light production was recorded every second, following 1 s integration of emitted light, using a low-light SE200 Echelle Spectrograph (Catalina Scientific, Arizona, USA). The intensity of the light production was measured for several minutes (depending on the experimental treatment) every 0.2 s, using a Sirius luminometer (Berthold Inc., Germany) for all experiments. However, in order to describe the detailed kinetics of spontaneous and mechanically stimulated flashes, light measurements were also made every 0.01 s in an integrating light chamber, using a photon-counting Election Tubes model P10 232 photomultiplier fitted with a Uniblitz electronic shutter (Vincent Associates, New York, USA). The number of photons emitted was then expressed as photons per 10 milliseconds on the basis of radiometric calibration with a 310 multispectral source (Optronics Laboratories, Florida, USA), as used to characterize short flashing patterns [45, 46]. Bioluminescence was also assessed with an intact snail. However, the snail retracts deeply into its shell when manipulated and exposed to chemical stimulation. Such behaviour allows the snails to be hermetic to external stimulants, which failed to trigger light. In order to address this, two holes of about 1.5 mm were drilled (400 series XPR Dremel) through the ventral side of the second largest whorl of the shell (away from the side of light production), with the snail alive inside. This treatment rendered the snail 'permeable' to external chemical stimulants while keeping it deeply lodged in its shell. The snail was then placed in 200 mu l of artificial sea water to which KCl (as described above) or the neuro-mediator acetylcholine (Ach, 1 mM final concentration) was added to stimulate light production, which in this ease was recorded for 15min. The spectrum of light production was also measured from such snails in their shells, using KC1 as a stimulant (as described above). The calcified shell was characterized for various photonic attributes, using a whole shell from either live or methanol-preserved specimens. In all cases, the soft mollusc body was retracted far into the shell. To avoid potential disruption to the shell, no physical or chemical attempts were made to remove it. We first tested whether the shell equally transmits (or absorbs) all wavelengths of light. The spectrum of light transmitted through the shell was measured by using two 600 mu l-wide optic fibres, placed approximately 3 mm apart and in direct opposition (electronic supplementary material, figure S1); one emitted white light (tungsten-halogen calibrated, 300-1050 nm; Occan Optics, Florida, USA), while the other received the transmitted light, and was connected to the spectrograph for spectrum profile identification. Measurements were made with the shell placed mid-distance between the fibres, and without the shell for a control. The emitting fibre was placed into the aperture of the shell, positioned about 1 mm away from the internal side as guided using a micromanipulator (, Japan). For the transmission capacity (position a, electronic supplementary material, figure S1), the emitting/receiving fibre optics stayed aligned facing one another, and measurements from the different samples placed in between the two fibres were recorded under the same acquisition parameters (exposure time and gain). For the diffusion capacity (gradual lateral move from position a to b, electronic supplementary material, figure Si), the acquisition parameters from each sample were such that the light measured by the receiving fibre when facing the emitting fibre in position a was about 75 per cent of the optimal intensity range for the instrument, and the receiving fibre optic laterally and gradually (by millimetre steps) moved to position b (electronic supplementary material, figure Si) using the micro-manipulator for light measurements of the diffused light, without changing any acquisition parameters. (a) Light transmission We then analysed the shell for its light transmission capacity and compared it with standard Zenith diffuser material of thicknesses 100, 250 and 500 mu m (SphereOptics, New Hampshire, USA). For comparison, the shell thickness was measured using an ocular micrometer on a Nikon SMZ1500 stercomicroscope. Light transmittance was quantified by using the same setting as above, with the two optic fibres placed in direct opposition. However, in this case, one emitted a set beam of blue-green light (522 + 40 nm; Ocean Optics), which was observed to be the colour range of bioluminescence in this species [41], while the other received the transmitted light, and was connected to the spectrograph for quantification. We measured transmittance with no material placed between the fibres (100% control transmittance) and determined the exposure time that provided intensity values within the optimal resolution of the spectrograph. Keeping the settings constant, we repeated measurements with samples placed mid-distance between the fibres, as described above. Transmittance of the samples was then expressed relative to the initial full control transmittance with no sample. (b) Light diffusion The shell was also analysed for its diffusion capacity and compared with the same three thicknesses of the standard Zenith diffuser material. We used the same settings as described above with the exception that the receiving opticExplanation / Answer
Answer:
Bioluminescence can also be done by using labeled florescence vector, such as either GREEN/FITC or RED/TEXAS RED.
The injection of florescence vector in animals has been studied extensively and it is doable.
To answer the second question, Full paper has to be understood. here in only Materials & Methos. Hence please understand the aim and objectives o the artcle and then it is possible to look if there is any problem in methods.