The authors gratefully thank Colonel (IL) J.N. Pritzker, the Tawani Foundation, USA, the Austrian Federal Ministry of Science, Research and Economy (Sparkling Science SPA04_149 and SPA05_201), Alpine Forschungsstelle Obergurgl (AFO), Austrian Space Forum (&#214;WF), Roman Erler from the Hintertuxer Natur Eis Palast, the Austrian Federal Forestry and Base Manager Nick Cox from the Arctic Station in Ny Alesund (Svalbard). We are also indebted to Sabrina Obwegeser, Carina Rofner, and Fabian Drewes for their help during filming. Finally, we want to thank James Bradley for giving the voice for the concomitant video.

1. Calibration and validation
NOTE: For the pigment calibration, prepare dilution rows from stock solutions of chla and B-PE. The chla stock solution is diluted with acetone and B-PE is diluted with distilled sterile water. Later, 15 mL of each dilution step will be needed. Protect the pigments from light by wrapping them with aluminum foil. Store the chla in a freezer and the B-PE in a refrigerator until further use. A detailed protocol for the dilution row follows in sections 1.1 for chla and 1.2 for B-PE. Both the chla and the B-PE laboratory calibration for pigment detection and quantification with the L.I.F.E. instrument is described below. A previous calibration24 was made with the same pigments as in this study.
<ol>
	<li>Chlorophylla dilution row
	<ol>
		<li>Dissolve 1 mg of chla (purified from A. nidulans algae) with acetone in a 50 mL sample tube and dilute this chla stock solution with acetone to the following final concentrations: 1,000; 800; 640; 320; 160; 80; 40; 20; 10; 5; 1; and 0.5 ng/mL.</li>
		<li>Transfer 15 mL of each dilution into 50 mL sample tubes and cover them in aluminum foil due to light sensitivity. Store the tubes in a -20 &#176;C freezer until the calibration measurements are taken. 
		NOTE: The protocol can be interrupted here.</li>
		<li>Measure the chla absorption features from each dilution in a double-beam spectrophotometer as triplicates and calculate the chla content as described by Lorenzen25, which will be described in detail in section 2.2.2.</li>
	</ol>
	</li>
	<li>PE dilution row
	<ol>
		<li>Dilute a 4 mg/mL B-PE stock solution with sterile filtered water (pH = 7) to the following final concentrations: 1,000; 800; 640; 320; 160; 80; 40; 20; 10; and 5 ng/mL B-PE. Transfer 15 mL of each dilution in a 50 mL sample tube and cover it with aluminum foil. Store at 4 &#176;C until further use. 
		NOTE: The protocol can be interrupted here.</li>
	</ol>
	</li>
	<li>Setup for calibration
	<ol>
		<li>Build a rack as shown in Figure 5 to establish three measurement platforms, each 1.5 cm higher than the next. 
		NOTE: The rack and column height play an important role for the measurements because the surface of the liquids should remain in the focal point of the L.I.F.E. instrument as indicated in Figure 5.</li>
		<li>Add 5 mL of the highest concentrated dilution in a polyscintillation plastic vial and put it on the highest point of the rack. Measure the fluorescence intensity.</li>
		<li>Place the vial in the middle position of the rack and add another 5 mL (10 mL total volume). Measure the fluorescence intensity. Repeat the procedure at the lowest position on the rack with another 5 mL (15 mL total volume; total of 45 mm in column height). 
		NOTE: Adapt the exposure time for each dilution step to prevent detector saturation (gray values over 255 in 12-bit images) and for a sufficient signal-to-noise ratio of the weak fluorescence signals.</li>
		<li>Repeat steps 1.3.2 and 1.3.3 with all dilutions (steps 1.1.1 and 1.2.1) of chla and B-PE.</li>
		<li>Load the generated data files into the data reduction wizard to automatically count and sum up the gray values from each pixel line along the Y-axis (spatial distribution). 
		NOTE: Varying exposure times are compensated automatically by normalizing the fluorescence intensity to an integration time of 1 s.</li>
		<li>Calculate the pigment area density with a Poisson regression analysis using the known concentrations from the dilution series and the normalized fluorescence intensities that were calculated. Then normalize the fluorescence counts from the three different column heights to 1.5 cm (5 mL) by multiplying the counts from each column height with a factor (factors 1, 0.5, and 0.33, for the 5 mL, 10 mL, and 15 mL concentration solutions, respectively).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60447/60447fig05.jpg" /> 
Figure 5: Setup for the L.I.F.E. calibration with chla and B-PE under laboratory conditions. 
(A) Lens tube of the instrument. (B) Green laser for B-PE excitation. (C) Blue laser for chla excitation. (D) Scintillation vial. (E) Focal point of the L.I.F.E. instrument. (F) B-PE/water or chla/acetone solution with 5 mL, 10 mL, and 15 mL. (G) Spacers that keep the surface of each solution at the focal plane for three different volumes. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Sampling and sample processing
<ol>
	<li>Collection of snow and ice
	<ol>
		<li>Collect snow and supraglacial ice from a glacier into sterile polyethylene bags and store them frozen until further processing. 
		NOTE: For this study, the samples were collected at Midtre Lovenbreen (MLB), a polythermal glacier near the research village Ny-&#197;lesund in the high Arctic Archipelago of Svalbard (78&#176;53&#8217; N, 12&#176;03&#8217; E).</li>
		<li>Sample bacterial mats from the glacier forefield into sterile polyethylene bags and transport all samples to a laboratory for further processing.</li>
		<li>Melt frozen material slowly in the dark at 4 &#176;C. Filter liquid samples on GF/F filters (47 mm diameter) and note the filtered volume. Keep the filters frozen until further processing.</li>
	</ol>
	</li>
	<li>Chlorophylla measurements
	<ol>
		<li>Using the L.I.F.E. instrument, measure the filter in four random areas, each in triplicates using the green and blue laser. Calculate the overall pigment concentration by multiplying the area density with the filtered area and filtered volume. Normalize the pigment concentration (&#181;g/L) to a volume of 1 L.</li>
		<li>Assess the chla contents of the GF/F filters with a laboratory standard according to the protocol by Lorenzen25. To do so, put each of the filters in a vial with 13 mL of acetone and store them in the dark at 4 &#176;C overnight. Next, take a vial and place it on ice before sonication for 2 min at 50% power in continuous mode. Squeeze and remove the filter from the vial.</li>
		<li>Attach tygon tubing to a syringe and remove the chla extraction-acetone mix from the vial. Replace the tygon tubing with a GF-5 filter holder. Transfer the solution into a quartz cuvette.</li>
		<li>After calibrating the absorbance spectrometer for acetone, place the sample in the cuvette into the spectrometer and measure the absorbance features between 400&#8211;750 nm. Next, remove the cuvette from the spectrometer and add 200 &#181;L of 2 M HCl to the sample. Then, repeat the absorbance measurement to measure the phaeophytin content in the sample.</li>
	</ol>
	</li>
	<li>Measurement of microbial activity via radiolabelled markers and impact of laser intensity and exposure time on productivity rates 
	CAUTION: Beware of the marker radioactivity (&#946;-radiation). Use a laboratory coat, gloves, and goggles, and work under a fume hood in a licensed isotope lab.
	<ol>
		<li>For bacterial production transfer five aliquots of bacterial mats into sterile polyethylene bags. Inactivate the controls with formaldehyde to a 4% final concentration. 
		NOTE: Three aliquots are used for the 3H-labeled leucine uptake and two aliquots are used as controls.</li>
		<li>Expose the mats with a blue laser (405 nm, 5 mW) and with a green laser (532 nm, 5 mW) for 10 s and 30 s each. Repeat the procedure with 50 mW lasers. Then, inactivate the samples that were not treated with formaldehyde. 
		NOTE: One mat is used for only one laser exposure. Use non-exposed microbial mats as controls.</li>
		<li>After the laser treatment, estimate the bacterial and primary production by incorporating 3H-leucine and NaH14CO3, respectively. For bacterial production measurements, use five aliquots per sample (20 mL) and add formaline (4% final concentration) to two of the parallels, which serve as controls for the abiotic incorporation of the marker. Add 3H-leucine (40 nM) to all samples of the various treatments and incubate them for 4 h under in situ conditions (0.1 &#176;C). Terminate the reaction by adding formaline to the remaining live samples.</li>
		<li>For bacterial production of bacterial mats, transfer labelled samples into cryovials. Extract cells with 5% trichloroacetic acid and centrifuge at 10,000 x g for 5 min according to the protocols by Kirchman26 and Bell27. Add scintillation liquid and put the cryovial into a polyscintillation vial. Analyze the samples with a liquid scintillation counter and calculate the uptake rates. 
		NOTE: Three aliquots are used for the 3H-labeled leucine uptake, two aliquots are used as controls.</li>
		<li>For primary production, prepare five replicates of various treatments (100 mL), wrap two of them into aluminum foil to mimic the dark samples, and add NaH14CO3 (1 &#181;Ci) to all. Incubate for 4 h in the ambient light and in situ temperature (0.1 &#176;C). Terminate the reaction by darkening the remaining three replicates and filter the sample onto GF/F filters (25 mm diameter).</li>
		<li>Add 100 &#181;L of 2 N HCl to the filters to remove all excess carbon and let it air out under the fume hood. Dry the samples on a heating plate at 80 &#176;C and place samples into polyscintillation vials.</li>
		<li>To measure radioactive disintegrations per minute (dpm) of all treatments of primary and bacterial production, place the cryovials into polyscintillation vials and add 5 mL of scintillation cocktail. Measure dpm with a liquid scintillation counter and calculate the uptake rates.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Calibration 
There was a linear correlation between pigment concentration and fluorescence intensity after normalizing photon counts to an exposure time of 1 s. Samples with low column height and low pigment concentrations led to an overestimation of the target pigments, compared to higher column heights with the same pigment concentration. Further, weak fluorescence signals required long exposure times for sufficient photon counts on the sensor. However, long integration times also increased the amount of stray light on the sensor, resulting in a decrease of the signal-to-noise ratio. In its current version, the software cannot distinguish between noise and signal during the data reduction process. Hence, low fluorescence intensity measurements led to a pigment overestimation because noise was counted as a signal that derived from the target pigments. Further, fluorescence intensities from more concentrated pigment solutions showed a greater variability than low concentration solutions. This effect could be explained by absorption processes within the pigment solutions that were used for the calibration curve.
Data validation for chlorophylla quantification 
After filtering ice and snow samples, the three-dimensional samples almost appeared as a two-dimensional specimen on the filter. This justified a direct comparison between L.I.F.E (area density) and spectrophotometric data (volumetric measurement).
The data set (Figure 8) indicated that high pigment concentration leads to an underestimation, whereas low pigment concentration leads to an overestimation of the actual value. This effect can be explained by the thickness of the filter cake and hence, the volumetric character of the sample. The laser penetration depth depended on the optical density and thickness of the specimen. High pigment contents were underestimated because the laser could not induce pigment fluorescence in deeper layers. However, in thin filter cakes, low fluorescence signals were captured due to low area densities of pigments. Apparently, the filter itself showed laser-induced signals after passing the 450 nm long-pass filter (Figure 12). This signal was misleadingly counted as fluorescence signal derived from chla. Thus, thin and too thick filter cakes are difficult to measure with the L.I.F.E. instrument.
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/60447/60447fig12.jpg" /> 
Figure 12: Fluorescence signals from thick (A) and thin (B) filter cakes on a GF/F filter.&#160;(A) Self-shading prevents laser-induced fluorescence from deeper layers, which results in an underestimate of the actual pigment concentration. (B) Fluorescence emission from filter cake with overlay by filter reflections. (C) Raw data show filter reflection (gray). The spectral property of a laboratory derived chla fluorescence pattern is illustrated in red. Scale bar = 45 mm. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Limitations of the L.I.F.E. prototype 
During data reduction, the MATLAB coded software interpreted the raw images by summing up pixel lines within a given wavelength range. The current version of the software did not distinguish between organic and inorganic derived signals. The presence of multiple signals might lead to an overestimation of the actual pigment content. Long exposure times due to low fluorescence intensities led to a decrease of the signal-to-noise ratio, promoting the effect as described above (see Figure 8 and Figure 12).
A geode rock shown in Figure 13 exhibited red fluorescent light when exposed with green and blue light. Currently, it is not clear whether the fluorescence resulted from minerals or from porphyrin-based molecules. Hence, an overlay of biological and nonbiological signals may limit the application of this method and require the establishment of a fluorescence database specifically made for the L.I.F.E. prototype.
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/60447/60447fig13.jpg" /> 
Figure 13: Mineral fluorescence from a geode rock, found in Ny-&#197;lesund.&#160;The rock was excited with a 532 nm 50 mW laser (A) and a 405 nm 50 mW laser (B). Both pictures were captured with a polarization filter attached on the lens, which led to a falsification of the actual fluorescence colors. (C) True color image without the use of a polarization filter under daylight conditions. Scale bar = 40 mm. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Beutler and others29 concluded that characteristic emission spectra of cyanobacteria in marine ecosystems depend on environmental conditions. Also, the metabolic state has an impact on the fluorescence properties in phototrophic organisms30. The L.I.F.E instrument may distinguish between the algal and cyanobacterial fluorescence pattern by using bio-fingerprint libraries that contain spectral information of the specimen correlated with environmental conditions.
In dark-adapted chla molecules, all reaction centers are fully oxidized and available for photochemistry and no fluorescence yield is quenched31. Using the L.I.F.E. procedure, a specimen is first excited by a 532 nm laser (green) and then with a 405 nm laser (blue). During the second excitation by the blue laser, chla might show a decreased fluorescence response due to prior excitation by the green laser. Chla absorbs energy at 532 nm wavelength, despite its distance from its absorption maximum wavelength32. Before the actual chla measurement at 405 nm, the green laser may cause photochemical reactions, activating quenching mechanisms in the target pigments. Further, pre-illumination of marine phototrophic organisms did not lead to a change in spectral norm curves between 450 nm&#8211;600 nm while the standard deviation in fluorescence intensities increased by 25%29. Depending on the species, fluorescence intensities even increased in response to prior excitation. This topic requires further investigation.
Applicability 
We tested the L.I.F.E. instrument in various habitats with an emphasis on cryoconite holes. The laser was successfully applied in soil and biofilm habitats because of the absence of ambient light during the measurement. Cryoconite granules could be measured when sediment layers blocked light from beneath the hole (Figure 14A,C). Thin sediment cryoconite holes were permeable for stray light from beneath (Figure 14B). Stray light interferes with the measurement. Thus, pigment concentration in bare ice surfaces is not measurable under daylight conditions yet. Signal processing efforts are currently underway to enable operation of the system in high ambient light conditions.
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/60447/60447fig14.jpg" /> 
Figure 14: Cryoconite hole with liquid water on top.&#160;(A) Cryoconite on glacier with L.I.F.E. lens tube. Scale bar = 70 mm. (B) The sediment layer (red) is very thin. Stray light bleeds through the cryoconite layer. (C) The sediment layer is thick enough to block the stray light from beneath. This type of cryoconite hole is measurable with the L.I.F.E. instrument. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In conclusion, our L.I.F.E. instrument detected photoautotrophic organisms in terrestrial habitats such as soils, bacterial mats, biofilms, and in cryoconite holes on glacial surfaces. The target molecules were chla and B-PE. The spatial resolution was 30 &#181;m/px. The detection limit for chla was 250 pg/mL and 2 ng/mL for B-PE. After a laboratory calibration we were able to quantify pigment content in samples that were collected at our study site in the Arctic. We applied self-programmed software for an automated data reduction process. The effects of the presence of minerals and changing light conditions during the measurements require further investigation.
With climate warming, increasing temperatures lead to enhanced availability of liquid water, which results in higher biological activity on icy surfaces of autotrophic and heterotrophic nature. Vigorous efforts should be made to detect heterotrophic organisms in situ to give a full picture of active life in the cryosphere. This could be tested with other target pigments and appropriate laser excitation wavelengths. Hence, L.I.F.E. provides a suitable monitoring system providing high temporal and spatial resolution for supraglacial conditions in context with global change as well as possible astrobiological applications.

The cryosphere harbors sea ice, glaciers, high mountain lakes, snow areas, lake ice, melt water streams, and permafrost. These areas cover approximately 11% of the earth&#8217;s landmasses1,2 and are overarched by the atmosphere as a recognized cryospheric environment. Recent studies show that massive areas of the cryosphere are quickly retreating3,4. The Antarctic5,6, the Alps7, the Arctic8, and other regions show negative ice mass balances. The retreat of ice caps and glaciers leads to the depletion of our largest freshwater reservoir on Earth. In some areas, glacier retreat is unstoppable5.
For a long time, ice ecosystems were considered sterile environments. However, despite harsh conditions, the presence of active life in the earth&#8217;s cryosphere is evident9,10,11,12,13,14,15. Due to the trend towards massive losses of ice by melting, the cryosphere is going through a shift in biological activity, affecting adjacent habitats. To understand those partially irreversible changes we require methods to investigate biological activity in ice under in situ conditions with high spatial and temporal resolution.
In supraglacial environments, life can be found in cryoconite holes, snow covers, melted water, streams, and on bare ice surfaces. However, the most obvious supraglacial habitats are cryoconite holes. They appear globally in glaciated environments and were first described by the Swedish explorer Adolf Erik Nordenskjold during an expedition to Greenland in the 1870&#8217;s16,17. The name originates from the Greek words &#8220;kryos&#8221; (cold) and &#8220;konia&#8221; (dust). Aeolian-derived dark organic and inorganic debris attach on the ice surface and reduce the albedo locally. Solar radiation promotes melting of the debris into deeper ice layers, forming cylindrical basins with sediment (cryoconite) in the bottom9. Cryoconite holes cover 0.1&#8211;10% of glacial ablation zones11.
Cryoconite communities consist of viruses, fungi, bacteria, cyanobacteria, microalgae, and protozoa. Depending on the region, metazoan organisms such as rotifers, nematodes, copepods, tardigrades, and insect larvae can also be found. Edwards and others18 describe cryoconite holes as &#8220;ice-cold hotspots&#8221;. They also traced functional genes in cryoconite holes that are responsible for N, Fe, S, and P cycling. The mini lake ecosystems respire and photosynthesize at rates found in much warmer and more nutrient-rich habitats11. These findings emphasize the important role of microbial sequestration in supraglacial environments. Beside living communities in cryoconite holes, bare ice surfaces are inhabited by ice algae. Their physiology is well studied19 but their spatial distribution has not been assessed20. Their presence in supraglacial environments decreases the albedo and hence promotes melting that leads to a nutrient outwash and nutrient input into downstream habitats9. Increasing temperatures and hence, a higher availability of liquid water, affects the net ecosystem productivity in these icy ecosystems.
In supraglacial environments, photosynthetically active organisms transform inorganic carbon and nitrogen into organic, available sources for the microbial food web21,22. Until now there are few studies that estimate supraglacial carbon fluxes11,20,23. The discrepancy in proposed rates of carbon flux results from a low spatial and temporal data resolution. Further, the spatial distribution of supraglacial communities outside cryoconite holes is barely assessed. Cook and others20 predicted in their models that supraglacial algal communities fix up to 11x more carbon than contemporary cryoconite holes due to their large surface coverage. The detection of supraglacial algal communities securing sample integrity is still impeded due to missing tools for in situ detection and quantification.
In response to difficulties in logistics, ice ecosystems are less frequently studied than habitats in temperate areas. Data resolution depends on the number of samples assessed and depend on the accessibility of the study sites. Standard sampling methods such as sawing, coring, and subsequent melting involve manipulation of the microbial community. For example, chlorophylla (chla) assessment in solid ice samples is impossible with standard methods without substantial interference. Hence, melting-induced temperature changes within the investigated microbial communities are unavoidable. In response to the thermolability of the photosystem II and other cellular structures in psychrophiles22, laboratory analyses of melted ice samples will always lead to a falsification of in situ conditions.
Non-destructive in situ measurements are the only reasonable way to obtain reliable data. This goal can be achieved using fluorescence-based methods. Due to their light harvesting function, chla and B-phycoerythrin (B-PE) are present in organisms that contribute to the carbon cycle in supraglacial environments, as has been proved by Anesio and others11. Hence, these fluorescent molecules are suitable biomarkers for the quantification of microbial mediated carbon fluxes in ice ecosystems.
In this study, we present the development, calibration, and applicability of a novel non-invasive tool for in situ quantification of chla and B-PE molecules in terrestrial and ice ecosystems. The prototype device is based on laser-induced fluorescent emission, also known as L.I.F.E. The optical instrument (Figure 1) captures fluorescent biomarker signatures after laser-induced fluorescence excitation. The procedure is nondestructive and can be performed at the study site or in a laboratory.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60447/60447fig01.jpg" /> 
Figure 1: The L.I.F.E. prototype.&#160;Left: Photo of the instrument without a protective lid. Right: Schematic illustration of the instrument. Total mass = 5.4 kg (laser and optics = 4.025 kg, laptop = 1.37 kg). Aluminum frame = 32.5 cm x 20.3 cm x 6.5 cm. Optical tube: 18.4 cm x 4 cm (diameter). CCD: bluefox mv220g sensor; F: servo-steered long-pass filters (450 nm and 550 nm); L: optical lenses; M1: mirrors; M2: dichroic mirror; MC: microcontroller; P: prism; PBS: polarizing beam splitter; S: slit aperture made of adjustable razor blades. Scale bar = 70 mm. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The portable dual-wavelength kit weighs 4.5 kg and is used on a tripod in combination with an external computer. The field setup is quick and easy. The instrument is attached to the tripod, and the lens tube is attached to the device along with a USB cable and the camera cable. The external computer is connected to the instrument using a USB cable. The tripod legs are adjusted in such a way that the lens tube is directed towards and covers the specimen. Then, a 5 mW green laser hits the sample after passing a polarizing beam splitter that redirects polarized light towards the optical axis of the spectrometer. The specimen exhibits a fluorescent light, illustrated in red in Figure 1. Half of the collimated light passes the polarizing beam splitter and is focused through a servo-steered long-pass filter that removes the laser signals. Next, the signal hits an aperture slit that consists of two adjustable razor blades. A prism spectrally separates the fine line of light orthogonal to the slit aperture before the signal is captured by the sensor. The procedure is repeated with a blue laser. The raw data are transferred automatically to a portable computer that is also used for the software operation.
The instrument is controlled by an external computer using a LabVIEW environment that synchronizes the picture-taking with the CCD camera, switching on/off lasers, and rotating the long-pass filter wheel. The graphical user interface (GUI) is divided into three main sections. The exposure adjustment is done manually. Although the correction between exposure time and signal intensity is linear (Figure 2B), the maximum exposure time is limited to 10 s because longer integration times lead to a significant decrease in the signal-to-noise-ratio. The comment field is used for the description of the sample (Figure 2A). In the right section, raw images are displayed as soon as the measurements are finished. This feature is crucial for immediate data evaluation in the field (Figure 2C&#8211;E). Red areas indicate overexposed pixels, which can be avoided by reducing the exposure time.
The subsequent raw data reduction process is decoupled from the image acquisition procedure and can be done at any time after image acquisition.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60447/60447fig02.jpg" /> 
Figure 2: L.I.F.E. graphical user interface for the data acquisition and raw data evaluation.&#160;(A) The software enables manual text input for sample descriptions. (B) The exposure time can be adjusted before the measurement. (C&#8722;E) The raw images are displayed on the right side of the interface. (E) Red colors indicate a saturation of the sensor. (F) The activation of the RUN MEASUREMENT button triggers the data acquisition process. In the array (G), all commands executed automatically during data acquisition are displayed. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60447/60447fig03.jpg" /> 
Figure 3: Example of a raw image.&#160;Left: Raw data of chla standard in acetone solution, recorded with the L.I.F.E instrument. Due to the optical properties of the device, the signal is displayed as a warped line. Right: Interpretation of the raw image per pixel (px). The spectral axis (5 nm/px resolution) is plotted against the spatial axis (30 &#181;m/px resolution). <a href="https://www.jove.com/files/ftp_upload/60447/60447fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The 12-bit gray-scale raw images show a spatial component due to the one-dimensional aperture slit and a spectral component due to the prism in front of the CCD (Figure 3). In response to optical constraints the raw images are distorted. Therefore, they need to be cropped and dewarped by applying a code that recognizes the degree of distortion. This is done with a software wizard (Figure 4). Next, the wavelength calibration is done with the 532 nm laser. The green light is produced by frequency doubling of a 1,064 nm infrared laser. Both wavelengths can be detected by the CCD and therefore, the spectral position of each pixel can be calculated in dewarped images automatically (Figure 4).
The picture is then cropped down to a given wavelength range (550&#8211;1,000 nm for green laser measurements and 400&#8211;1,000 for blue laser measurements). Gray values from each pixel in a selected pixel line are counted and summed up. A gray value can range from 0&#8211;255. After that, every pixel line accounts for one number. Further on-screen software instructions lead to the generation of a plot showing the gray value counts from each pixel line plotted against the spatial coordinates. This allows for a quantitative spatial discrimination of chla and B-PE simultaneously in the sample. Additionally, the spectral properties of a sample can be plotted from selected pixel lines automatically.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60447/60447fig04.jpg" /> 
Figure 4: Dewarping raw images.&#160;Left: Raw image captured with a green laser. No filter was used. Signals are displayed at 532 nm and 1,064 nm. Exposure time = 0.015 s. Center: The cropped 532 nm signal is used as a reference line to dewarp a set of images. Right: The dewarped image from the raw image source. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>aceton</td>
			<td>Merck</td>
			<td>67-64-1</td>
			<td></td>
		</tr>
		<tr>
			<td>B-Phycoerythrin</td>
			<td>Invirtrogen</td>
			<td>P6305</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorophyll a standard</td>
			<td>Sigma-Aldrich</td>
			<td>C6144-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>formaline</td>
			<td>Merck</td>
			<td>HT501128</td>
			<td>36%</td>
		</tr>
		<tr>
			<td>GF/C filters</td>
			<td>Whatman</td>
			<td>WHA1822025</td>
			<td>25mm diameter</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Merck</td>
			<td>H1758</td>
			<td>36,5-38%</td>
		</tr>
		<tr>
			<td>L.I.F.E. Prototype</td>
			<td>University of Innsbruck</td>
			<td></td>
			<td>built on demand</td>
		</tr>
		<tr>
			<td>LabView</td>
			<td>National Instruments</td>
			<td></td>
			<td>Software, Laboratory Virtual Instrumentation Engineering Workbench</td>
		</tr>
		<tr>
			<td>Leucine, L-[4,5-3H], 1 mCi</td>
			<td>Perkin Elmer</td>
			<td>NET1166001MC</td>
			<td>radioactive</td>
		</tr>
		<tr>
			<td>Liquid scintillation cocktail Beckman Ready Use</td>
			<td>Beckman</td>
			<td>not more available, can be compensated by Ultra Gold, Packard</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid scintillation counter</td>
			<td>Beckman</td>
			<td>out of stock</td>
			<td>LSC 6000 IC</td>
		</tr>
		<tr>
			<td>NaH14CO3 (4 &#181;Ci/ml)</td>
			<td>DHI Denmark</td>
			<td>4 &#956;Ci/ml, 1 ml</td>
			<td>radioactive</td>
		</tr>
		<tr>
			<td>Osmonics polycarbonate filters</td>
			<td>DHI Denmark</td>
			<td>PCTE</td>
			<td>25mm diameter, 0,2&#181;m pore size</td>
		</tr>
		<tr>
			<td>Polyscintillation vials</td>
			<td>Perkin Elmer</td>
			<td>WHA1825047</td>
			<td>20ml</td>
		</tr>
		<tr>
			<td>sample tubes</td>
			<td>Sigma Aldrich</td>
			<td>T2318-500EA</td>
			<td>Greiner centrifuge tubes, 50ml</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Hitachi</td>
			<td>NA</td>
			<td>Model U2001, any photometer for absorption spectroscopy measuring at 664nm and 750nm would be appropriate</td>
		</tr>
		<tr>
			<td>trichloric acetic acid (TCA)</td>
			<td>Merck</td>
			<td>T6399</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>ultrasonic probe</td>
			<td>nano lab</td>
			<td>QS1T-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser-induced fluorescence emission (l.i.f.e.) as novel non-invasive tool for in-situ measurements of biomarkers in cryospheric habitats

Global warming affects microbial communities in a variety of ecosystems, especially cryospheric habitats. However, little is known about microbial-mediated carbon fluxes in extreme environments. Hence, the methodology of sample acquisition described in the very few studies available implies two major problems: A) high resolution data require a large number of samples, which is difficult to obtain in remote areas; B) unavoidable sample manipulation such as cutting, sawing, and melting of ice cores that leads to a misunderstanding of in situ conditions. In this study, a prototype device that requires neither sample preparation nor sample destruction is presented. The device can be used for in situ measurements with a high spectral and spatial resolution in terrestrial and ice ecosystems and is based on the Laser-Induced Fluorescence Emission (L.I.F.E.) technique. Photoautotrophic supraglacial communities can be identified by the detection of L.I.F.E. signatures in photopigments. The L.I.F.E. instrument calibration for the porphyrin derivates chlorophylla (chla) (405 nm laser excitation) and B-phycoerythrin (B-PE) (532 nm laser excitation) is demonstrated. For the validation of this methodology, L.I.F.E. data were ratified by a conventional method for chla quantification that involved pigment extraction and subsequent absorption spectroscopy. The prototype applicability in the field was proven in extreme polar environments. Further testing on terrestrial habitats took place during Mars analog simulations in the Moroccan dessert and on an Austrian rock glacier. The L.I.F.E. instrument enables high resolution scans of large areas with acceptable operation logistics and contributes to a better understanding of the ecological potential of supraglacial communities in the context of global change.

Laboratory calibration for B-PE 
The response signals of the B-PE dilution row were measured with the L.I.F.E. instrument in a dark room at 20 &#176;C (Figure 6). The count rate depended on both the concentration and the column height of the measured sample. Low concentration and low column height B-PE specimen fluoresced stronger compared to samples of the same concentration and higher column height.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60447/60447fig06.jpg" /> 
Figure 6: B-PE laboratory calibration.&#160;B-PE content and column density calibration is shown. Normalized count rates were calculated for a column height of 1.5 cm. Reprint with permission28. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A Poisson regression was used for the final calibration line fit. There was a linear correlation between the area densities and pixel gray value counts. The function of the curve was y = 81.04x (Figure 7), which means that a gray value count rate of 8,104 in a 1 s exposed sample equaled an area density of 100 ng/cm2 B-PE. The chla calibration is set up in an analogue manner. The function was y = 8.94x.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60447/60447fig07.jpg" /> 
Figure 7: Final calibration curve for B-PE.&#160;The gray value counts were normalized to an exposure time of 1 s and plotted against the area density. Reprint with permission28. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Application on cryoconite samples from Svalbard and laboratory validation of data 
The mean values of the L.I.F.E. measurements and the single measurements of the same samples derived from conventional extraction using acetone and subsequent analysis with a spectrophotometer are illustrated in Figure 8.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60447/60447fig08.jpg" /> 
Figure 8: Data validation with natural samples.&#160;The samples (MLB) are ranked by chla content, based on the results of a laboratory spectrophotometer (single values) and compared with the chla fluorescence data measured on four random areas per filter. The error bars represent the standard deviation of the L.I.F.E. measurements. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Chla contents ranging from 48 &#181;g/L&#8211;67 &#181;g/L were underestimated, and lower chla contents ranging from 0.7 &#181;g/L&#8211;7 &#181;g/L were overestimated by the L.I.F.E. prototype. The standard deviations from the L.I.F.E. measurements were low.
Comparison of spectral data from in situ measurements with laboratory standards 
Chla spectra were comparable between cryoconite samples and those from purified from A. nidulans algae. The fluorescence peaks in all samples were located at 700 nm&#8211;710 nm. However, spectra derived from cryoconite samples showed higher noise signals between 400 nm&#8211;650 nm and from 800 nm&#8211;1,000 nm compared to spectra of the chla pigment standard (Figure 9).
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/60447/60447fig09.jpg" /> 
Figure 9: Spectral data interpretation.&#160;Measurements of four cryoconite granules (blue) and a chla standard pigment solution (red) after excitation with 405 nm lasers. The spectra were recorded 1 year after sample collection. The samples were kept frozen and were not exposed to light prior to the measurement. In response to wavelength calibration issues, the fluorescence peak is located at 700 nm&#8211;710 nm instead of 680 nm. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Automated cryoconite grain analysis 
In an example of an automated analysis of a cryoconite hole (Figure 10), the highest pigment area densities were observed at pixel line 50. The sample spectrum after excitation with a 532 nm laser showed a peak with a cut off at a gray value of 255 in response to oversaturation of the sensor. This peak derived from the green laser and not from the fluorescent signal.
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/60447/60447fig10.jpg" /> 
Figure 10: Automated data analysis of a single cryoconite granule with a diameter of 1 mm.&#160;The sample was collected at Vestre Br&#248;ggerbreen (VBB) and measured within 4 h after sampling in a dark laboratory room at the Arctic Station (GB) facility in Ny-&#197;lesund. The left column shows B-PE measurements and the right column represents chla data. The raw images are displayed on top. Laser-induced fluorescence responses are displayed in gray. Red areas indicate the response from standard pigments. The middle section illustrates the spatial distribution of the target pigments. The spectral properties of the fluorescence signal are displayed in the lower images. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Impact of laser excitation on productivity in bacterial mats 
Neither primary nor bacterial productivity were affected when increasing the power of the laser and/or the exposure time (Figure 11). No significant differences were detected under laser treatments with increased power.
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/60447/60447fig11.jpg" /> 
Figure 11: Productivity measurements of samples from Svalbard.&#160;Bacterial mats were exposed with green and blue lasers of varying laser intensities and exposure times. The data are colored according to the laser wavelength source (green and blue). <a href="https://www.jove.com/files/ftp_upload/60447/60447fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Laser-Induced Fluorescence Emission L.I.F.E. as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats at JoVE.com

Laser-Induced Fluorescence Emission L.I.F.E. as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats

Carbon fluxes in the cryosphere are hardly assessed yet but are crucial regarding climate change. Here we show a novel prototype device that captures the phototrophic potential in supraglacial environments based on laser-induced fluorescence emission (L.I.F.E.) technology offering high spectral and spatial resolution data under in situ conditions.

Laser-Induced Fluorescence Emission (L.I.F.E.) as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats

Global warming affects microbial communities in a variety of ecosystems, especially cryospheric habitats. However, little is known about ...

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7.8K Views.  University of Innsbruck. Carbon fluxes in the cryosphere are hardly assessed yet but are crucial regarding climate change. Here we show a novel prototype device that captures the phototrophic potential in supraglacial environments based on laser-induced fluorescence emission (L.I.F.E.) technology offering high spectral and spatial resolution data under in situ conditions.

Video: Laser-Induced Fluorescence Emission L.I.F.E. as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

Using Deuterium Oxide as a Non-Invasive, Non-Lethal Tool for Assessing Body Composition and Water Consumption in Mammals

Body condition scoring systems and body condition indices are common techniques used for assessing the health status or fitness of a species. Body condition scoring systems are evaluator dependent and have the potential to be highly subjective. Body condition indices can be confounded by foraging, the effects of body weight, as well as statistical and inferential problems. An alternative to body condition scoring systems and body condition indices is using a stable isotope such as deuterium oxide to determine body composition. The deuterium oxide dilution method is a repeatable, quantitative technique used to estimate body composition in humans, wildlife, and domestic species. Additionally, the deuterium oxide dilution technique can be used to determine the water consumption of an individual animal. Here, we describe the adaption of the deuterium oxide dilution technique for assessing body composition in big brown bats (Eptesicus fuscus) and for assessing water consumption in cats (Felis catis).

This article describes the deuterium oxide dilution technique in two mammals, an insectivore and carnivore, to determine total body water, lean body mass, body fat mass, and water consumption.

Body condition scoring systems and body condition indices are common techniques used for assessing the health status or fitness of a species. Body ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

Mistletoe Eradicator - A Novel Tool for Simultaneous Mechanical and Chemical Control of Mistletoe

Some species of mistletoes, an aerial plant hemiparasites, play a Janus-like role in ecosystems by being rich biodiversity hotspots due to their trophic and topic associations with various organisms and at the same time acting as biological pirates, exhausting the host trees by stealing water and nutrients via the haustorium. Many crop and timber trees are attacked by mistletoes, for example, dwarf mistletoes parasitize conifers, representatives of genus Tapinanthus target guava, and Phoradendron mistletoe target pecans. Recently, an increasing infestation of the European mistletoe (Viscum album subsp. album L.) in monoculture stands of common walnuts (Juglans regia L.)&#160;was recorded for the first time during 2018 in different regions of Kashmir Valley, India. The common chemical approach based on the spraying of mistletoe exophytes with the growth regulators, ethephon and glyphosate, was not efficient since V. album resprouted in the next vegetation season. Hand pruning of mistletoe was time-consuming, and a polyethylene covering resulted in the localized rotting of host tissues. The most efficient approach to eradicate mistletoe was the use of a novel tool called Mistletoe Eradicator, which allows to use mechanical and chemical means of mistletoe control simultaneously. It consists of a manipulating pole with the terminal pruning saw having a container filled with the growth regulator, from which the liquid slowly drops onto the saw blade through the nozzle. It allows working both from the ground and in the tree canopy. After the targeted application of 5%-10% ethephon or glyphosate to the base of the just pruned V. album stems (stubs), exophyte resprouting was significantly reduced 9 months after the pruning as compared to the only pruned or sprayed exophytes. An alternative variant of Mistletoe Eradicator was constructed in Ukraine filled just with a food color additive to track the liquid dropping and was successfully tested.

The protocol describes an easy-to-handle tool called Mistletoe Eradicator which combines mechanical and chemical approaches and is efficient in the control of mistletoe infestation on the host trees. The manuscript uses the European mistletoe (Viscum album subsp. album L.) as an example.

Some species of mistletoes, an aerial plant hemiparasites, play a Janus-like role in ecosystems by being rich biodiversity hotspots due to their trophic ...