In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters

Reporter gene technology is widely used to measure activity of hormone analogs, and bioluminescent in vitro assays have allowed rapid screening of numerous chemicals either to identify new agonists or antagonists of hormones or to detect the presence of endocrine disrupters in the environment. Stable bioluminescent cell lines have been established and they provide reproducible dose–response curves and accurate determination of in vitro efficiencies of various chemicals. In vivo, however, these molecules can be metabolized, bound by proteins, or stored in fats and thus could display efficiencies different from those observed in vitro. In vivo assays, such as the uterotrophic bioassay, require numerous sacrificed animals, and responses not only are dependent on an estrogenic action but also imply other factors. For a faster assay and to avoid the use of numerous animals, we developed an in vivo biosensor constituted of stable bioluminescent cells implanted in nude mice. MCF-7 bioluminescent cell lines were chosen since their proliferation is low in the absence of estrogen and the xenograft size can thus be stable for several weeks. Luciferase gene expression was monitored noninvasively with a cooled charge-coupled device camera. Quantitative analysis allowed us to compare in vitro and in vivo actions of different estrogenic compounds (estradiol, estrone) and endocrine disruptors (ethynylestradiol, genistein, octylphenol, and 2,4′-dichlorodiphenyldichloroethylene) in the same cell lines and to follow hormone action on a living animal as a function of time. Different administration protocols have been used and good correlation was observed for most products. However, we found that ethynylestradiol was the most efficient chemical when orally administered.     

Video rate bioluminescence imaging of secretory proteins in living cells: localization, secretory frequency, and quantification

We have developed a method of video rate bioluminescence imaging to investigate protein secretion from a single mammalian cell and analyzed the localization, secretory frequency, and quantification of secreted protein. By detecting the luminescence signals from the Gaussia luciferase (GLase) reaction using a high-speed electron-multiplying charge-coupled device (EM-CCD) camera, video rate imaging was performed with a time resolution within 500 ms/image over 30 min in living cells. As a model study, we applied the method to visualize the glucose-stimulated insulin secretion from clustered pancreatic MIN6 β cells using the fused protein of GLase with preproinsulin. High-quality video images clearly showed that the glucose-stimulated insulin secretion from the clustered MIN6 β cells oscillated within a period of a few minutes over 10 min. In addition, the glibenclamide-induced insulin secretion from the clustered MIN6 β cells was visualized, suggesting that bioluminescence video rate imaging is a useful method for validating drug action in living cells.     

A simple and sensitive high-throughput GFP screening in woody and herbaceous plants

Green fluorescent protein (GFP) has been used widely as a powerful bioluminescent reporter, but its visualization by existing methods in tissues or whole plants and its utilization for high-throughput screening remains challenging in many species. Here, we report a fluorescence image analyzer-based method for GFP detection and its utility for high-throughput screening of transformed plants. Of three detection methods tested, the Typhoon fluorescence scanner was able to detect GFP fluorescence in all Arabidopsis thaliana tissues and apple leaves, while regular fluorescence microscopy detected it only in Arabidopsis flowers and siliques but barely in the leaves of either Arabidopsis or apple. The hand-held UV illumination method failed in all tissues of both species. Additionally, the Typhoon imager was able to detect GFP fluorescence in both green and non-green tissues of Arabidopsis seedlings as well as in imbibed seeds, qualifying it as a high-throughput screening tool, which was further demonstrated by screening the seedlings of primary transformed T₀ seeds. Of the 30,000 germinating Arabidopsis seedlings screened, at least 69 GFP-positive lines were identified, accounting for an approximately 0.23% transformation efficiency. About 14,000 seedlings grown in 16 Petri plates could be screened within an hour, making the screening process significantly more efficient and robust than any other existing high-throughput screening method for transgenic plants.     

Detection of gene loops by 3C in yeast

“Chromosome conformation capture” (3C) is a powerful method to detect physical interaction between any two genomic loci. 3C involves formaldehyde crosslinking to stabilize transient interactions, followed by restriction digestion, ligation and locus-specific PCR. Accordingly, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Here, we describe a modified 3C protocol designed for detection of transient chromatin interactions in the yeast Saccharomyces cerevisiae. Using this protocol, we are able to detect juxtaposition of promoter and terminator regions of genes with ORFs as short as 1 kb in length. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions and will facilitate the characterization of gene loops and their functional consequences.     

Determining the efficacy of camera traps,live capture traps,and detection dogs for locating cryptic small mammal species

Metal box (e.g., Elliott, Sherman) traps and remote cameras are two of the most commonly employed methods presently used to survey terrestrial mammals. However, their relative efficacy at accurately detecting cryptic small mammals has not been adequately assessed. The present study therefore compared the effectiveness of metal box (Elliott) traps and vertically oriented, close range, white flash camera traps in detecting small mammals occurring in the Scenic Rim of eastern Australia. We also conducted a preliminary survey to determine effectiveness of a conservation detection dog (CDD) for identifying presence of a threatened carnivorous marsupial, Antechinus arktos, in present‐day and historical locations, using camera traps to corroborate detections. 200 Elliott traps and 20 white flash camera traps were set for four deployments per method, across a site where the target small mammals, including A. arktos, are known to occur. Camera traps produced higher detection probabilities than Elliott traps for all four species. Thus, vertically mounted white flash cameras were preferable for detecting the presence of cryptic small mammals in our survey. The CDD, which had been trained to detect A. arktos scat, indicated in total 31 times when deployed in the field survey area, with subsequent camera trap deployments specifically corroborating A. arktos presence at 100% (3) indication locations. Importantly, the dog indicated twice within Border Ranges National Park, where historical (1980s–1990s) specimen‐based records indicate the species was present, but extensive Elliott and camera trapping over the last 5–10 years have resulted in zero A. arktos captures. Camera traps subsequently corroborated A. arktos presence at these sites. This demonstrates that detection dogs can be a highly effective means of locating threatened, cryptic species, especially when traditional methods are unable to detect low‐density mammal populations.     

An approach to rapid processing of camera trap images with minimal human input

1. Camera traps have become an extensively utilized tool in ecological research, but the manual processing of images created by a network of camera traps rapidly becomes an overwhelming task, even for small camera trap studies.
2. We used transfer learning to create convolutional neural network (CNN) models for identification and classification. By utilizing a small dataset with an average of 275 labeled images per species class, the model was able to distinguish between species and remove false triggers.
3. We trained the model to detect 17 object classes with individual species identification, reaching an accuracy up to 92% and an average F1 score of 85%. Previous studies have suggested the need for thousands of images of each object class to reach results comparable to those achieved by human observers; however, we show that such accuracy can be achieved with fewer images.
4. With transfer learning and an ongoing camera trap study, a deep learning model can be successfully created by a small camera trap study. A generalizable model produced from an unbalanced class set can be utilized to extract trap events that can later be confirmed by human processors.

     

Optimising camera trap surveys for the Carpentarian Pseudantechinus (Pseudantechinus mimulus) in northwest Queensland

The Carpentarian Pseudantechinus (Pseudantechinus mimulus) is a poorly studied dasyurid marsupial that inhabits rocky outcrops in the Mount Isa Inlier bioregion in Queensland and the Gulf Coastal and Gulf Fall and Uplands bioregions in the Northern Territory. It is readily detected by passive infrared triggered camera traps (‘camera traps’). Camera trap data can be used to develop detection probability estimates from which activity patterns can be inferred, but no effort has previously been made to determine changes in the detectability of P. mimulus throughout the year. We undertook a 13-month baited camera trap survey across nine sampling periods at 60 locations of known historic presence or nearby suitable habitat to assess the change in detection rates and detection probabilities of P. mimulus across a year. Detection probabilities were calculated from camera trap data within a single-species, multi-season occupancy framework to determine optimal survey timing. Detection probability data were used to calculate the likelihood of false absences to determine optimal survey duration. We recorded 2493 detections of P. mimulus over 10 966 camera days. Detection probability ranged from 0.009 to 0.179 and was significantly higher from April to October than from November to March. The likelihood of false absences varied by sampling period and desired level of confidence. We find that camera trap surveys for P. mimulus are best conducted from April to October, but optimal survey duration is dependent upon the time of year and desired level of confidence that an observed absence from a given site reflects a true absence at that site. Attaining a minimum of 80% confidence of absence requires as few as 9 days of survey effort in May to 16 days of survey effort in October.     

Accuracy of identifications of mammal species from camera trap images: A northern Australian case study

Camera traps are a powerful and increasingly popular tool for mammal research, but like all survey methods, they have limitations. Identifying animal species from images is a critical component of camera trap studies, yet while researchers recognize constraints with experimental design or camera technology, image misidentification is still not well understood. We evaluated the effects of a species’ attributes (body mass and distinctiveness) and individual observer variables (experience and confidence) on the accuracy of mammal identifications from camera trap images. We conducted an Internet‐based survey containing 20 questions about observer experience and 60 camera trap images to identify. Images were sourced from surveys in northern Australia and included 25 species, ranging in body mass from the delicate mouse (Pseudomys delicatulus, 10 g) to the agile wallaby (Macropus agilis, >10 kg). There was a weak relationship between the accuracy of mammal identifications and observer experience. However, accuracy was highest (100%) for distinctive species (e.g. Short‐beaked echidna [Tachyglossus aculeatus]) and lowest (36%) for superficially non‐distinctive mammals (e.g. rodents like the Pale field‐rat [Rattus tunneyi]). There was a positive relationship between the accuracy of identifications and body mass. Participant confidence was highest for large and distinctive mammals, but was not related to participant experience level. Identifications made with greater confidence were more likely to be accurate. Unreliability in identifications of mammal species is a significant limitation to camera trap studies, particularly where small mammals are the focus, or where similar‐looking species co‐occur. Integration of camera traps with conventional survey techniques (e.g. live‐trapping), use of a reference library or computer‐automated programs are likely to aid positive identifications, while employing a confidence rating system and/or multiple observers may lead to a collection of more robust data. Although our study focussed on Australian species, our findings apply to camera trap studies globally.     

A bioluminescent HL‐60 cell line to assay anti‐leukaemia therapeutics under physiological conditions

Screens for compounds and proteins with anti‐cancer activity employ viability assays using relevant cancer cell lines. For leukaemia studies, the human leukaemia cell line, HL‐60, is often used as a model system. To facilitate the discovery and investigation of anti‐leukaemia therapeutics under physiological conditions, we have engineered HL‐60 cells that stably express firefly luciferase and produce light that can be detected using an in vivo imaging system (IVIS). Bioluminescent HL‐60luc cells could be rapidly detected in whole blood with a sensitivity of approximately 1000 viable cells/200 µl blood. Treatment of HL‐60luc cells with the drug chlorambucil revealed that the bioluminescent viability assay is able to detect cell death earlier than the Trypan blue dye exclusion assay. HL‐60luc cells administered intraperitoneally (i.p.) or intravenously (i.v.) were visualized in living mice. The rapidity and ease of detecting HL‐60luc cells in biological fluid indicates that this cell line could be used in high‐throughput screens for the identification of drugs with anti‐leukaemia activity under physiological conditions. Copyright © 2007 John Wiley & Sons, Ltd.     

DISTRIBUTION AND GENETIC DIVERSITY OF THE LUCIFERASE GENE WITHIN MARINE DINOFLAGELLATES1

Dinoflagellates are the most abundant protists that produce bioluminescence. Currently, there is an incomplete knowledge of the identity of bioluminescent species arising from inter‐ and intraspecific variability in bioluminescence properties. In this study, PCR primers were designed to amplify the dinoflagellate luciferase gene (lcf) from genetically distant bioluminescent species. One of the primer pairs was “universal,” whereas others amplified longer gene sequences from subsets of taxa. The primers were used to study the distribution of lcf and assess bioluminescence potential in dinoflagellate strains representing a wide variety of taxa as well as multiple strains of selected species. Strains of normally bioluminescent species always contained lcf even when they were found not to produce light, thus demonstrating the utility of this methodology as a powerful tool for identifying bioluminescent species. Bioluminescence and lcf were confined to the Gonyaulacales, Noctilucales, and Peridiniales. Considerable variation was observed among genera, or even species within some genera, that contained this gene. Partial sequences of lcf were obtained for the genera Ceratocorys, Ceratium, Fragilidium, and Protoperidinium as well as from previously untested species or gene regions of Alexandrium and Gonyaulax. The sequences revealed high variation among gene copies that obscured the boundaries between species or even genera, some of which could be explained by the presence of two genetic variants within the same species of Alexandrium. Highly divergent sequences within Alexandrium and Ceratium show a more diverse composition of lcf than previously known.     

Rapid methods of detecting the target molecule in immunohistology using a bioluminescence probe

We demonstrate a novel rapid direct detection method for immunohistochemistry, using a bioluminescent probe. An anti‐CEA antibody‐fused far‐red bioluminescent protein can monitor the accumulation of this type of probe in tumour tissues. The bimodal spectrum (λ_max = 460 and 675 nm) of this bioluminescent probe is extremely stable under different conditions of pH and ion concentration. The sensitivity of our bioluminescent labelling was at the same level of enzymatic labelling, e.g. peroxidase, as an indirect system. Our novel technique is simple and can shorten the pretreatment time of paraffin sections to around 30 min. The utility of our bioluminescent labelling covers all imaging in vitro, in vivo and ex vivo, suggesting that our antibody‐fused bioluminescent probe has the potential to detect tumour antigens with a high sensitivity in routine immune histological examinations. Copyright © 2012 John Wiley & Sons, Ltd.     

Stability of d‐luciferin for bioluminescence to detect gene expression in freely moving mice for long durations

Circadian disturbance of clock gene expression is a risk factor for diseases such as obesity, cancer, and sleep disorders. To study these diseases, it is necessary to monitor and analyze the expression rhythm of clock genes in the whole body for a long duration. The bioluminescent reporter enzyme firefly luciferase and its substrate d ‐luciferin have been used to generate optical signals from tissues in vivo with high sensitivity. However, little information is known about the stability of d ‐luciferin to detect gene expression in living animals for a long duration. In the present study, we examined the stability of a luciferin solution over 21 days. l ‐Luciferin, which is synthesized using racemization of d ‐luciferin, was at high concentrations after 21 days. In addition, we showed that bioluminescence of Period1 (Per1) expression in the liver was significantly decreased compared with the day 1 solution, although locomotor activity rhythm was not affected. These results showed that d ‐luciferin should be applied to the mouse within, at most, 7 days to detect bioluminescence of Per1 gene expression rhythm in vivo.     

Bias associated with baited camera sites for assessing population characteristics of deer

Camera surveys often involve placing bait in front of the camera to capture animals more frequently, which could introduce biases in parameter estimates. From September 2008 to March 2009, we monitored cameras placed at random, along game trails, and at feed stations to determine if camera placement influenced measures of population demographics in a herd of white-tailed deer (Odocoileus virginianus). There was no time period in which cameras placed at feed stations provided sex ratio and recruitment estimates similar to those acquired from randomly placed cameras. Trail-based camera surveys provided population estimates similar to those from random sites and may provide a feasible alternative to using baited camera stations. © 2011 The Wildlife Society.     

The selfie trap: A novel camera trap design for accurate small mammal identification

Camera traps are a popular tool for monitoring wildlife though they can fail to capture enough morphological detail for accurate small mammal species identification. Camera trapping small mammals is often limited by the inability of camera models to: (i) record at close distances; and (ii) provide standardised photos. This study aims to provide a camera trapping method that captures standardised images of the faces of small mammals for accurate species identification, with further potential for individual identification. A novel camera trap design coined the ‘selfie trap’ was developed. The selfie trap is a camera contained within an enclosed PVC pipe with a modified lens that produces standardised close images of small mammal species encountered in this study, including: Brown Antechinus (Antechinus stuartii), Bush Rat (Rattus fuscipes) and Sugar Glider (Petaurus breviceps). Individual identification was tested on the common arboreal Sugar Glider. Five individual Sugar Gliders were identified based on unique head stripe pelage. The selfie trap is an accurate camera trapping method for capturing detailed and standardised images of small mammal species. The design described may be useful for wildlife management as a reliable method for surveying small mammal species. However, intraspecies individual identification using the selfie trap requires further testing.     

Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiation

A faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m⁻²) and solar irradiation (≈620 W m⁻²). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py⁺-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions.     

Boreal predator co‐occurrences reveal shared use of seismic lines in a working landscape

Interspecific interactions are an integral aspect of ecosystem functioning that may be disrupted in an increasingly anthropocentric world. Industrial landscape change creates a novel playing field on which these interactions take place, and a key question for wildlife managers is whether and how species are able to coexist in such working landscapes. Using camera traps deployed in northern Alberta, we surveyed boreal predators to determine whether interspecific interactions affected occurrences of black bears (Ursus americanus), coyotes (Canis latrans), and lynx (Lynx canadensis) within a landscape disturbed by networks of seismic lines (corridors cut for seismic exploration of oil and gas reserves). We tested hypotheses of species interactions across one spatial‐only and two spatiotemporal (daily and weekly) scales. Specifically, we hypothesized that (1) predators avoid competition with the apex predator, gray wolf (Canis lupus), (2) they avoid competition with each other as intraguild competitors, and (3) they overlap with their prey. All three predators overlapped with wolves on at least one scale, although models at the daily and weekly scale had substantial unexplained variance. None of the predators showed avoidance of intraguild competitors or overlap with prey. These results show patterns in predator space use that are consistent with both facilitative interactions or shared responses to unmeasured ecological cues. Our study provides insight into how predator species use the working boreal landscape in relation to each other, and highlights that predator management may indirectly influence multiple species through their interactions.     

A camera trap assessment of terrestrial vertebrates in Bwindi Impenetrable National Park,Uganda

We placed camera traps for a month at sixty locations in Bwindi Impenetrable National Park to determine the species composition and distribution of medium‐to‐large terrestrial vertebrates. A total of 15912 images were recorded from 1800 camera trap days. These provided a total of 625 and 338 camera events when filtered by hour and day, respectively. Twenty mammal species were recorded from 594 and 314 camera events by hour and day, respectively. Four bird species were recorded from 31 and 24 camera events by hour and day, respectively. The African golden cat Profelis aurata Temminck was recorded from 27 and nineteen camera events by hour and day, respectively. The black‐fronted duiker Cephalophus nigrifrons Gray was most frequently photographed with 179 and 65 camera events by hour and day, respectively. Analyses reveal two species possessed a significantly interior‐biased distribution. One species showed an edge‐biased pattern. Five species were detected to have significantly biased altitudinal distributions with higher elevations. Distance to park edge and elevation can significantly influence species distribution. The selective use of the park limits the area that each species utilizes, with implications for maximum population sizes and viability. Our observations provide a baseline for long‐term terrestrial vertebrate monitoring in Bwindi.     

Photon Hunting in the Twilight Zone: Visual Features of Mesopelagic Bioluminescent Sharks

The mesopelagic zone is a visual scene continuum in which organisms have developed various strategies to optimize photon capture. Here, we used light microscopy, stereology-assisted retinal topographic mapping, spectrophotometry and microspectrophotometry to investigate the visual ecology of deep-sea bioluminescent sharks [four etmopterid species (Etmopterus lucifer, E. splendidus, E. spinax and Trigonognathus kabeyai) and one dalatiid species (Squaliolus aliae)]. We highlighted a novel structure, a translucent area present in the upper eye orbit of Etmopteridae, which might be part of a reference system for counterillumination adjustment or acts as a spectral filter for camouflage breaking, as well as several ocular specialisations such as aphakic gaps and semicircular tapeta previously unknown in elasmobranchs. All species showed pure rod hexagonal mosaics with a high topographic diversity. Retinal specialisations, formed by shallow cell density gradients, may aid in prey detection and reflect lifestyle differences; pelagic species display areae centrales while benthopelagic and benthic species display wide and narrow horizontal streaks, respectively. One species (E. lucifer) displays two areae within its horizontal streak that likely allows detection of conspecifics'' elongated bioluminescent flank markings. Ganglion cell topography reveals less variation with all species showing a temporal area for acute frontal binocular vision. This area is dorsally extended in T. kabeyai, allowing this species to adjust the strike of its peculiar jaws in the ventro-frontal visual field. Etmopterus lucifer showed an additional nasal area matching a high rod density area. Peak spectral sensitivities of the rod visual pigments (λ_max) fall within the range 484–491 nm, allowing these sharks to detect a high proportion of photons present in their habitat. Comparisons with previously published data reveal ocular differences between bioluminescent and non-bioluminescent deep-sea sharks. In particular, bioluminescent sharks possess higher rod densities, which might provide them with improved temporal resolution particularly useful for bioluminescent communication during social interactions.     

Manual fish length measurement accuracy for adult river fish using an acoustic camera (DIDSON)

Total lengths (L_T) of 50 free-swimming fish in a tank, silver carp Hypophthalmichthys molitrix and rainbow trout Oncorhynchus mykiss, were measured using a DIDSON (Dual-frequency IDentification SONar) camera. Using Sound Metrics software, multiple measurements of each fish (L_T, side aspect angle and distance from the camera) at different times were analysed by two experienced operators while a subset of data was analysed by two inexperienced operators. The main result showed high variability in intra-fish L_T measurements. The number of measurements required to minimise errors and to obtain robust fish measurements (true L_T ± 3 cm) was estimated by a bootstrap method. Three to five measurements per fish were recommended for fish surveys in rivers. In this experimental study, aiming to reproduce river conditions, no evidence of fish position (side aspect angle and distance from the camera) effect was detected, but an operator effect (partially explained by training) was observed. General linear mixed models also showed that lengths of the smallest fish (L_T < 57 cm) were overestimated and lengths of the largest fish (L_T > 57 cm) were underestimated in comparison with their true lengths. In conclusion, we highlight that this technology, like any monitoring methods, returns imperfect observations. We advise DIDSON users to ensure that measurements are carried out correctly in order to draw accurate conclusion from this new technology.     

A cell array biosensor for environmental toxicity analysis

In this study, a cell-based array technology that uses recombinant bioluminescent bacteria to detect and classify environmental toxicity has been implemented to develop two biosensor arrays, i.e., a chip and a plate array. Twenty recombinant bioluminescent bacteria, having different promoters fused with the bacterial lux genes, were immobilized within LB-agar. About 2 microl of the cell-agar mixture was deposited into the wells of either a cell chip or a 384-well plate. The bioluminescence (BL) from the cell arrays was measured with the use of highly sensitive cooled CCD camera that measured the bioluminescent signal from the immobilized cells and then quantified the pixel density using image analysis software. The responses from the cell arrays were characterized using three chemicals that cause either superoxide damage (paraquat), DNA damage (mitomycin C) or protein/membrane damage (salicylic acid). The responses were found to be dependent upon the promoter fused upstream of the lux operon within each strain. Therefore, a sample's toxicity can be analyzed and classified through the changes in the BL expression from each well. Moreover, a time of only 2 h was needed for analysis, making either of these arrays a fast, portable and economical high-throughput biosensor system for detecting environmental toxicities.