Investigation of the autofluorescence of various abalone (Haliotis midae) tissues and the implications for future use of fluorescent molecules.
|Publication:||Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000|
|Issue:||Date: August, 2012 Source Volume: 31 Source Issue: 3|
|Topic:||Event Code: 310 Science & research|
|Geographic:||Geographic Scope: South Africa Geographic Code: 6SOUT South Africa|
ABSTRACT Fluorescent molecules have revolutionized the field of
molecular biology and biotechnology, and could be of benefit to research
conducted on economically important haliotid species (abalone) for
applications such as protein analysis, cell metabolism studies,
fluorescence resonance energy transfer, cell imaging, and reporter genes
for gene transfer. Many marine organisms exhibit autofluorescence;
therefore, this raises the question whether autofluorescence also occurs
in abalone and whether it could hinder fluorescence studies. We examined
ova, sperm, larvae, juveniles, and hemocyte cell cultures of the
southern African abalone (Haliotis midae Linnaeus, 1758) and established
that ova, larvae, and juveniles exhibited autofluorescence at most
wavelengths tested, especially in the mid-wavelength range of the
visible spectrum. Autofluorescence was particularly prominent at the
same wavelengths as that of green fluorescent proteins. Therefore, we
caution studies using fluorescent molecules with emissions in the
mid-wavelength range and suggest the use of molecules with emissions at
the extremes of the light spectra.
KEY WORDS: abalone, autofluorescence, fluorescent reporters, Haliotis midae
As a result of diminishing natural abalone resources and market demand, farming of these marine molluscs has increased markedly with a concomitant increase in the overall production of abalone worldwide (Cook & Gordon 2010). Scientific research advancing the understanding of the genus Haliotis is integral in keeping abalone industries competitive and sustainable in a global market. As a result of the relatively recent domestication of abalone (Troell et al. 2006), research concerning the molecular biology, genetics, and cellular functioning of abalone species is still ongoing.
Fluorescent molecules have revolutionized the field of molecular biology and biotechnology, have proved to be truly versatile, and are used in several different in vivo and in vitro applications (Cubitt et al. 1995, March et al. 2003, Metha & Zhang 2011). Abalone research could benefit from utilizing fluorescent molecules for protein analysis, cell metabolism studies, fluorescence resonance energy transfer techniques, cell imaging, as well as reporter genes for potential gene transfer applications (Shaner et al. 2005, Muller-Taubenberger & Anderson 2007, Fernandez-Suarez & Ting 2008).
It has been noted that many aquatic organisms have the ability to fluoresce (Weissleder & Ntziachristos 2003, Widder 2010). Prescott and Salih (2009) reported that several marine organisms such as coral, anemones, and zoanthids have fluorescent emissions in the cyan (470-499 nm), green (500-520 nm), and red (520 nm and longer) wavelengths. Mollusc species such as the Eastern oyster Crassostrea virginica (Buchanan et al. 2001), freshwater pond snail Lymnaea stagnalis (Abe et al. 2009), and the boring clam Pholas dactylus (Shimomura 2009) have been reported to exhibit autofluorescence. Although autofluorescence of haliotid shells has been reported (Proudfoot et al. 2008), there have been no reports detailing autofluorescence encountered in haliotid tissues, with only one article mentioning autofluorescence of Haliotis asinina ova during investigation of the egg coat of this species (Suphamungmee et al. 2010). The only reported study using a fluorescent reporter protein (enhanced green fluorescent protein (EGFP)) in a haliotid species was during the transfection of ova, embryos, and sperm in Haliotis discus hannai by Wang et al. (2004), with no mention made of autofluorescence. Therefore, the current study investigated whether autofluorescence, which is commonplace in aquatic organisms, was present in various types of Haliotis midae tissue and whether this autoftuorescence could influence future studies relying on fluorescent molecules.
MATERIALS AND METHODS
Abalone (H. midae) gametes, embryos, larvae, juvenile, and hemocyte tissue cultures were examined using fluorescent microscopy. Gametes were obtained from a commercial abalone farm on the south coast of South Africa during routine spawning events. At least 3 male and 3 female individuals contributed to the pool of gametes that was examined. Gametes were examined fluorescently within 60 min of spawning. Fertilization of ova and settling of juveniles was carried out by farm personnel as part of the commercial production process. Samples were obtained within an hour postfertilization and were examined fluorescently. Developing embryos and larvae were maintained in filtered seawater at 18[degrees]C. A sample of 2-wk-old settled juveniles was also obtained and investigated fluorescently. A number of gamete and larval samples were preserved in 100% ethanol to determine whether ethanol storage would alter the autofluorescence of tissues.
Hemolymph was bled from animals and seeded on well plates according to Van der Merwe et al. (2010). All samples (gametes, embryos, larvae, settled juveniles, and hemolymph) were examined using an Olympus IX51 fluorescent microscope (Olympus, Germany) with green fluorescent protein (GFP; [[lambda].sub.ex], 450-460 nm; [[lambda].sub.em], 500-550 nm) and fluorescein isothiocyanate (FITC; [[lambda].sub.ex], 460-500 nm; [[lambda].sub.em], 510-560 nm) filters. Fertilized ova were also examined with several filters using an Olympus IX81 motorized inverted microscope. Two Fura filters ([[lambda].sub.ex], 340 nm/380 nm; [[lambda].sub.em], 510 nm); 4' 6-diamindino-2-phenylindole dihydrochloride hydrate (DAPI; [[lambda].sub.ex], 310-380 nm; [[lambda].sub.em], 430-480 nm); cyan, yellow, and green fluorescent proteins (CFP: [[lambda].sub.ex], 425-445 nm, [[lambda].sub.em]. 460-500 nm; GFP: [[lambda].sub.ex], 450-460 nm, [[lambda].sub.em], 500-550 nm; YFP: [[lambda].sub.ex], 480-510 nm, [[lambda].sub.em], 520-550 nm); FITC ([[lambda].sub.ex], 460-500 nm; [[lambda].sub.em], 510-560 nm); and Texas-Red ([[lambda].sub.ex], 555 595 nm, [[lambda].sub.em], 600-630 nm) filters were used for fluorescent imaging. Images were acquired using a monochromatic F-view-II cooled CCD camera (Soft Imaging Systems) with the Cell^R system (Olympus) with background subtraction.
[FIGURE 1 OMITTED]
To compare the autofluorescence of abalone tissues with transfected tissues expressing fluorescent proteins, an EGFP-containing construct (pTracer-CMV2; Invitrogen, UK) and a DsRed-containing construct (CMV-DsRed-Express; Clontech, USA) were used to transfect Hep2G (ATCC, USA) cells. These transfected cells were then monitored microscopically using GFP and FITC filters, and then compared with abalone tissues.
[FIGURE 2 OMITTED]
Autofluorescence was evident when examining both fresh and ethanol-preserved ova, embryos, and larvae. Ova and larvae (within 24 h postfertilization) examined at additional excitation and emission wavelengths also demonstrated fluorescence at a wide spectrum of excitation and emission wavelengths, with the most intense fluorescent signal observed in the same emission range as that of DAPI, CFP, GFP, FITC, and YFP (Figs. 1 and 2). Samples exhibited no or a very low fluorescent signal when excited by light with very short or long wavelengths. A faint fluorescent signal was detected with the 340-nm and 380-nm excitation filters, and when using the Texas-Red filter (Fig. 2).
When larvae were allowed to develop for more than 24 h postfertilization, a change in the color and localization of the fluorescent signal occurred. As larvae developed, the green fluorescence observed in the fertilized ova was replaced by yellow-green fluorescence and later by yellow-green and red fluorescence (under FITC filter). The color and localization of the fluorescent signal, detected using a GFP filter, remained unchanged throughout larval development (Fig. 1).
The juveniles viewed under an FITC filter exhibited localization of areas of yellow and red fluorescence, similar to that observed in the larvae. The areas in which the yellow fluorescence was exhibited (under FITC filter) in larvae encompassed most of the tissue enveloped by the newly formed shell in the juveniles. The red fluorescence in the juveniles was confined to the area surrounding the dorsal ganglia (Fig. 1).
Hep2G cells transfected with EGFP and DsRed exhibited a bright-green and yellow fluorescence when viewed under a GFP and FITC filter, respectively. This fluorescence observed in the transfected Hep2G cells was similar to the autofluorescence exhibited by ova, larvae, and juveniles (Fig. 1). Sperm cells and cultured hemocytes did not yield any fluorescent signal at any wavelength tested (data not shown).
Flavin proteins are well known for their fluorescent qualities, usually fluorescing in the yellow-green range (500-600 nm) and are the main autofluorescent species in an assortment of cell types (Benson et al. 1979, Van den Berg et al. 2001, Shapiro 2003). The abalone ova provide the lecithotrophic larvae with the nutrient-rich and high-in-protein yolk, which sustains its energy requirements until feeding (Takami et al. 2002). Seguineau et al. (2001) demonstrated that the ova and larvae of the scallop Pecten maximus contain high concentrations of riboflavin, which is used in larval development. The yolk of H. midae ova may also contain a high concentration of flavin proteins, thereby producing the observed autofluorescence. Altering the excitation wavelength when investigating the fluorescence of a single fluorophore should not alter the emission wavelength (Rost 1992). Because shifting of the excitation wavelength altered the emission wavelength of ova and larvae (<24 h postfertilization), the presence of more than 1 fluorophore is assumed. Thus, flavin proteins are not likely to be the sole fluorescent molecule responsible for the autofluorescence, but at least one other fluorescent molecule might be at play.
The biological function of fluorescent proteins has not been elucidated, although several suggestions have been made for their function in other species. These suggestions range from being involved in predator--prey behavior to the reduction of photobleaching of light-sensitive organisms (Prescott & Salih 2009). These suggestions are based mainly on data collected from coral; the function of fluorescent proteins in abalone, however, remains obscure.
Because of the autofluorescence exhibited by H. midae ova, embryos, larvae, and juveniles, the use of fluorescent protein genes such as EGFP and DsRed, and most probably other fluorescent proteins or probes with excitation and emission maxima in the mid-wavelength range of the visible spectrum, would be ineffective for visual reporting. The difficulty in discerning a fluorescent reporter from autofluorescence could be circumvented by using a reporter that has emission maxima at the extremes of the light spectra. Fluorescent proteins such as mCherry and mStrawberry (Clontech, USA) have emission spectra at longer wavelengths than that of DsRed and could serve as reporter genes in studies of H. midae. These proteins also run the risk of being obscured by the autofluorescence and could potentially be indiscernible when viewed in conjunction with background autofluorescence. Excitation of fluorescent molecules and autofluorescent molecules that differ in their emission maxima, by using dual filters, could be a solution (Litaker et al. 2002). This method could, however, still be influenced by the extent of autofluorescence, therefore making the choice of fluorescent molecule very complex.
Similar studies conducted on other abalone species are necessary to establish whether this pattern of autofluorescence occurs across all haliotids or whether autofluorescence is species-specific.
This research was funded by a grant from the NRF Innovation Fund (South Africa). Research facilities were provided by Stellenbosch University.
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LISE SANDENBERGH AND ROUVAY ROODT-WILDING *
Molecular Aquatic Research Group, Department of Genetics, Stellenbosch University, Private Bag XI, Matieland, 7602, South Africa
* Corresponding author. E-mail: firstname.lastname@example.org
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