An application of in situ hybridization for the identification of commercially important fish species

Fisheries Research 170 (2015) 1–8

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An application of in situ hybridization for the identification of commercially important fish species Thorben Hofmann ∗ , Michael J. Raupach, Pedro Martinez Arbízu, Thomas Knebelsberger Senckenberg am Meer, German Centre for Marine Biodiversity Research, Südstrand 44, 26382 Wilhelmshaven, Germany

a r t i c l e

i n f o

Article history: Received 15 May 2014 Received in revised form 20 April 2015 Accepted 4 May 2015 Handled by B. Morales-Nin Keywords: Molecular species identification North Sea Oligonucleotide probes Ribosomes 18S rRNA In situ hybridization

a b s t r a c t In most cases, adult fish can be easily identified based on the analysis of morphological characteristics. In contrast to this, a successful identification of processed specimens or early life cycle stages might be quite challenging. Here we present an in situ hybridization (ISH) approach for reliable species identification. Oligonucleotide probes targeting the small ribosomal subunit (18S rRNA) were developed and tested for the identification of the three commercially important fish species Merluccius merluccius, Scomber scombrus, and Trachurus trachurus. These species are routinely monitored by triennial ichthyoplankton surveys in the Northeast Atlantic. Our results demonstrated a species specific hybridization and staining for all three target species using a combination of helper, competitor and probes. The Trachurus trachurus probe can also be used to identify Trachurus mediterraneus which can be found further South. Additionally, we showed successful hybridization by a subsequent use of probes. The application of in situ hybridization may represent a robust, fast and cheap alternative for the identification of fish species in comparison to other molecular approaches. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Adults of most of the commercially exploited fish species can easily be identified to species level using external morphological features. On the other hand, a reliable species identification of early life cycle stages like eggs or larvae is often difficult as a consequence of similarities in shape and form (Ahlstrom and Moser, 1980; Teletchea, 2009). Such uncertainties in species identification are especially problematic for ichthyoplankton surveys that aim to determine spatial and temporal spawning patterns as well as the biomass of commercially important species. It is obvious that ichthyoplankton surveys require reliable identification of eggs and larvae, as the results of such surveys massively influence strategies in fishery management in terms of the estimation of fishery quotas (Fox et al., 2005; Perez et al., 2005). In the last two decades various molecular methods have been established for the identification of fish species (Ali et al., 2012). These include approaches based on the separation and characterization of specific proteins, e.g. capillary electrophoresis (Kvasnicka, 2005), high performance liquid chromatography (HPLC) (Hubalkova et al., 2007), or immunoassay systems such as enzyme-linked ImmunoSorbet Assay (ELISA) (Asensio et al., 2008).

∗ Corresponding author. Tel.: +49 0 4421 9475 149; fax: +49 0 4421 9475 111. E-mail address: [email protected] (T. Hofmann). http://dx.doi.org/10.1016/j.fishres.2015.05.002 0165-7836/© 2015 Elsevier B.V. All rights reserved.

Furthermore, various DNA-based identification methods have been developed, ranging from the analysis of fragments that are cut by endonucleases at specific restriction sites (Restriction Fragment Length Polymorphism, RFLP) (Wolf et al., 2000) to a simultaneous DNA amplification and quantification (Real-time PCR) (Lockley and Bardsley, 2000), DNA microarrays (Kochzius et al., 2010; Teletchea et al., 2008), high resolution melt (HRM) analysis (Fitzcharles, 2012), or DNA barcoding (Costa et al., 2012; Holmes et al., 2009; Knebelsberger et al., 2014; Ward et al., 2005). In addition to these methods, in situ hybridization (ISH) approaches have become quite popular for the identification and quantification of various marine taxa, including Bacteria and Archea (Amann et al., 1990a; DeLong et al., 1989), diatoms (Scholin et al., 1997), nanoflagellates (Lim et al., 1996), picophytoplankton (Simon et al., 2005), marine invertebrate larvae (Goffredi et al., 2006; Pradillon et al., 2007), mollusks (Thomas et al., 2011), echinoderms (Mountfort et al., 2007), and zooplankton communities (Harvey et al., 2012). The concept of in situ hybridization (ISH) relies on the interaction of small labeled oligonucleotides, the so-called probes, with species-specific corresponding sequences of the ribosomes in single cells or tissue samples, e.g. 16S or 18S rRNAs, during the hybridization step. In general, species-specific oligonucleotide probes used for ISH have a length of 15 to 25 nucleotides. Using software packages such as ARB (Ludwig et al., 2004), species-specific 16S or 18S rRNA probes can be easily designed in silico. It is not surprising that the accessibility of the targeting site on the ribosomes

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is most crucial and must be checked accurately. Otherwise, the higher-order structure of the ribosome as result of RNA/RNA or RNA/protein interactions may hinder a successful binding of the probe (Fuchs et al., 1998). To avoid hybridization to a non-target species, species-specific probes should possess at least one mismatch to all non-target groups, which should be located on a central position within the probe. This causes a strong destabilizing during the washing step and removes unspecific bindings at non-target regions. The final step of the probe design includes the establishment of optimal hybridization conditions by performing a series of hybridizations at rising stringency. Typically, the stringency is adjusted by increasing the temperature of the hybridization or by increasing the concentration of a denaturing agent such as formamide in the hybridization buffer. Changes of fluorescent intensities can be displayed in a “melting curve”. In terms of fluorescence ISH (FISH), oligonucleotide probes are labeled directly or indirectly with fluorochromes, specific dyes that reemit light after excitation, e.g. via a laser, whereas probes of non-fluorescent ISH approaches are labeled with horseradish-peroxidase (HRP) or digoxygenin. In these cases, a successful binding is indicated by a color reaction that can be easily observed by eye without additional instruments, e.g. a fluorescent microscope or a flow cytometer. A first study using non-fluorescent ISH to identify eggs and larvae of polychaetes and bivalves demonstrated the potential and broad applicability of this method (Pradillon et al., 2007). In the Northeast Atlantic, populations of the Atlantic mackerel (Scomber scombrus, Scombridae) and the Atlantic horse mackerel (Trachurus trachurus, Carangidae) are routinely studied by triennial egg surveys carried out by participating countries including among others Germany, Iceland, and Spain as part of the International Council for the Exploration of the Seas (ICES). Together with eggs of S. scombrus and T. trachurus, eggs of the European hake (Merluccius merluccius, Merlucciidae) can be found at the same time. The use of diagnostic morphological traits to discriminate the eggs of these three species efficiently is quite difficult and time-consuming. In this context, the so-called ‘surface adhesion test’ (SAT) (Porebski, 1975) helps to separate eggs of M. merluccius from those of other species, but the results are not consistent (ICES, 2010). In this study we developed a non-fluorescent ISH protocol for the identification of S. scombrus, T. trachurus and M. merluccius. Furthermore, we included the Mediterranean horse mackerel (Trachurus mediterraneus, Carangidae). The protocol presented here represents the initial step towards the routine application of ISH for the identification of fish eggs and larvae.

Table 1 Primer (5 to 3 ) used for PCR amplifications * and sequencing † of the complete 18S rRNA gene in this study. Prime name

Sequence

Reference

Deut-F1*† Deut-R1*† SeqF1† SeqF2† SeqR2† SeqR3*† SeqF4*∼†

ACC TGG TTG ATC CTG CCA TGA TCC ATC TGC AGG TTC AGC AGC CGC GGT AAT TCC AGC T GAA ACT TAA AGG AAT TGA CGG AA AGC TGG AAT TAC CGC GGC TGC T GCA TCG TTT ATG GTC GGA ACT AC CGA GGC CCT GTA ATT GGA ATG

Turbeville et al. (1994) Turbeville et al. (1994) Turbeville et al. (1994) Turbeville et al. (1994) Turbeville et al. (1994) Present study Present study

preparation. For the in situ hybridization assay (ISH), muscle tissue preserved in 96% ethanol abs. and stored at 4 ◦ C were used. 2.2. DNA isolation, PCR amplification and sequencing

2. Materials and methods

For the amplification of the complete 18S rRNA gene, total genomic DNA was extracted from specimens of T. trachurus, T. mediterraneus, S. scombrus and M. merluccius using the DNeasy 96 Blood & Tissue Kit (Qiagen) (Table S1). The primer pair Deut-F1 and Deut-R1 (Medlin et al., 1988; Turbeville et al., 1994) was used for amplification (Table 1). PCR reactions were carried out in a total volume of 40 ␮l, containing 4 ␮l 10× buffer, 4 ␮l dNTPs (2 mM each), 0.4 ␮l of each primer (10 pm ␮l−1 ), 0.2 ␮l Taq Polymerase (Roche, Grenzach-Wyhlen, Germany), 2 ␮l DNA template, and filled up with sterile water. The thermal cycle profile consisted of an initial denaturation at 94 ◦ C for 5 min, followed by 35 cycles of denaturation at 94 ◦ C for 45 s, annealing at 50 ◦ C for 50 s, elongation at 72 ◦ C for 3:20 min, and a final elongation at 72 ◦ C for 10 min. As consequence of amplification problems, the newly designed internal primers Scomber-SeqF4 (5 -CGA GGC CCT GTA ATT GGA ATG-3 ) and Scomber-SeqR3 (GCA TCG TTT ATG GTC GGA ACT AC-3 ) (Table 1) were additionally used for S. scombrus. For all reactions, negative and positive controls were included. Two microliters of the amplified products were verified for the correct insert size by electrophoresis in a 1% agarose gel with GelRed (Biotium, Hayward, USA) using commercial DNA size standards. PCR products were purified using Exo-SAP (Thermo Scientific, Waltham, MA, USA) by adding 1.25 ␮l Exonuclease I (20 U ␮l−1 ) and 5 ␮l thermosensitive alkaline phosphatase (1 U ␮l−1 ). The mixture was heated at 37 ◦ C for 15 min, followed by 75 ◦ C for 20 s. Purified PCR products were outsourced for sequencing to a contract sequencing facility (Macrogen, Amsterdam, Netherlands) or sequenced at the MaxPlanck-Institute for Marine Microbiology (Bremen, Germany). For DNA sequencing various primers were used (Table 1).

2.1. Sample collection

2.3. Sequence alignment and probe design

Specimens of T. trachurus, S. scombrus and M. merluccius were collected during several cruises of the German Small Scale Bottom Trawl Surveys (Ehrich et al., 2007) in different regions of the North Sea between 2010 and 2011 (see Appendix). Additional specimens of T. mediterraneus and S. scombrus were bought at local consumer in Bremerhaven (Germany) whereas fin samples of M. merluccius were obtained from another consumer market in Bremerhaven (Germany). All specimens were directly frozen at −20 ◦ C until

All obtained 18S rRNA sequences were assembled and edited using the Geneious© sequence editor (Geneious version Pro 5.4.6 by Biomatters, available from (www.geneious.com), BioEdit 7.2.5 (Hall, 1999) and MEGA 6 (Tamura et al., 2011). Pair-wise distances (p-distances) were calculated with sequence alignments produced by Muscle (Edgar, 2004) with default settings. For probe design, 18S rRNA sequences were aligned in ARB software suite 5.5 (Ludwig et al., 2004) using the Silva small subunit database (SSU Ref NR

Table 2 List of oligonucleotide probes used in this study, including probe sequences and optimal formamid concentrations. Target organism

Probe name

Probe sequence (5 to 3 )

% Formamid (optimum)

Source

T. trachurus/mediterraneus S. scombrus M. merluccius Eukaryota Negative control

Trach1449 Scom1449 Mer845 Euk516 Non338

GTC GGT CAC GGC CCT GGC GTC GGT AAC GGT CCT GGC CCG CCG GGG AGC TAC CCG ACC AGA TTG CCC TCC ACT CCT ACG GGA GGC AGC

20 20 30 0–50 0–50

Present study Present study Present study Amann et al. (1990a) Wallner et al. (1993)

T. Hofmann et al. / Fisheries Research 170 (2015) 1–8

111, www.arb-silva.de (Pruesse et al., 2007)). The alignment was inspected by eye and refined by hand. Oligonucleotide probes were designed either with the probe design function in ARB or by visually identifying a suitable target site in the ARB alignment. Probes were named by the first letters of the analyzed genus and the position targeted within the 18S rRNA (Table 2). All designed probes were identical to the targeted region of the 18S sequence of the target species and had at least one mismatch to sequences from the other species (Fig. 1). The target positions were chosen to guarantee the best possible accessibility according to previous studies (Behrens et al., 2003). In order to increase the in situ accessibility, unlabeled helper oligonucleotides (Fuchs et al., 2000) were designed. Furthermore, unlabeled competitor-probes were used to suppress unspecific bindings (Manz et al., 1992).

2.4. Standard ISH protocol A standard protocol for in situ hybridizations of fish tissue was developed, based on a modified version of the described protocol for whole larvae (Pradillon et al., 2007). In this context, the permeabilization, the inactivation of endogenous peroxidases, the hybridization, the washing and the visualization with TMB were modified. Tests were conducted with the general eukaryotic probe EUK338 (Amann et al., 1990a) and the NON338 probe as negative control (Amann et al., 1990b). All species specific probes were labeled with horseradish peroxidase (HRP) (Biomers, Ulm, Germany). Reactions were carried out in 1.5 ml tubes. Tissue was rehydrated in 70% and 50% ethanol in PBS (phosphate-buffers saline: 145 mM NaCl, 1.4 mM NaH2 PO4 , 8 mM Na2 HPO4 , pH 7.5) and in 1× PBS before ISH, respectively. For permeabilization, 20 min incubation in 0.5% SDS and for inactivation of endogenous

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Table 3 Summary of steps for standard ISH and multiple ISH on tissue of fish samples with HRP oligonucleotide probes. All steps were conducted at room temperature except when specific temperature is mentioned (see Material and Methods for details). Standard ISH on tissue Fixation Rehydration

Permeabilization Inactivation of endogenous peroxidases Hybridization

Washing

Visualization Multiple ISH Removing of bound probes

Second hybridization

96% ethanol 10 min 70% ethanol 10 min 50% ethanol 10 min 1× PBS 20 min 0.5% SDS (facultative) 10 min 1× PBS 20 min 0.3% H2O2 in methanol 10 min 1× PBS 100 ␮l hybridization buffer containing probe at 125–250 pg ␮l−1 (sufficient for 1 cm2 tissue sample) Incubation in 0.5 ml tubes or equivalent for 3 h at 46 ◦ C Remove of excess hybridization buffer with filter paper 3 × 30 min in pre warmed washing buffer at 48 ◦ C 10 min 1× PBS 4–20 min incubation in TMB 16 h in high-stringency washing buffer (without NaCl) 10 min 1× PBS Check with TMB 10 min 1× PBS Proceed with hybridization

peroxidases 20 min incubation in 0.2% H2 O2 in Methanol showed best results. For hybridization, HRP-labeled probes were dissolved in hybridization buffer (0.9 M NaCl, 20 mM Tris–HCl [pH 8], 0.02%

Fig. 1. Screenshot of the alignment of the 18S rDNA target sequence region for the probes Trach1449 (above), Scom1449 (central) and Mer845 (below). Black marked sequences indicate the specific target regions, numbers are alignment positions.

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Fig. 2. (a) Display of the arbitrary units for the increasing TMB signal intensity from 1 (no signal) to 10 (dark blue). (b) Melting curves for the specific probes Trach1449, (c) Scom1449, and (d) Mer845, with increasing formamid concentration on tissue samples of T. trachurus (black line), S. scombrus (light gray) and M. merluccius (dark gray). Error bars: ± standard deviation of separate hybridizations. The signal intensity was made by eye observation.

[w/v] SDS, 10% [w/v] dextran sulphate, 1% [w/v] blocking reagent (Roche), 0 to 70% [v/v] formamide (depending on probe) in concentrations between 125 and 250 pg ␮l−1 ). Probe specific formamide concentrations were determined using melting curves (see section below), summarized in Table 2. Following the hybridization, tissue samples were washed 3 × 30 min in pre-warmed washing buffer (0.5 ml 0.5 M EDTA pH 8, 1 ml 1 M Tris–HCl pH 8, variable volumes of NaCl (depending on probe), dH2 O to final volume of 50 ml, and 50 ␮l 10% SDS) at 48 ◦ C with different concentrations of NaCl according to the formamide concentration in the hybridization buffer (Thiele et al., 2011). Following a final wash step for 10 min in 1× PBS, 1.25 mM TMB (3,3 ,5,5 -tetramethylbenzidine) (Fitzgerald, Acton, MA, USA) was added to the tissue. The substrate was oxidized by the HRP, developing a deep blue color within 20 min at room temperature in case of a positive hybridization. Images were recorded with

Fig. 3. Comparison of the TMB signal intensity (in arbitrary units) at increasing incubation time for specific probes: (a) Trach1449, (b) Scom1449, and (c) Mer845 on 100% complementary targets and mismatch targets. Tests were conducted on tissue of T. trachurus (black line), S. scombrus (light gray line) and M. merluccius (dark gray line). Error bars: ± standard deviation. The signal intensity was made by eye observation.

a Canon 60D digital camera and edited with Lightroom 4 (Adobe). Table 3 shows the final ISH procedure for fish tissue. 2.5. Probe testing Probes were tested with and without helper probes as well as with and without competitor probes to confirm the positive

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effects of these additional oligonucleotides. The concentration of the helper probe, the competitor probe and the specific probe was identical in all performed hybridizations. In order to find the optimal hybridization conditions that guaranteed high specificity and signal intensity for the newly designed species-specific probes including both helper and competitor probes, a series of hybridizations at increasing formamide concentrations ranging from 10% to 70% were performed. For all experiments, the hybridization time was 3 h and the TMB incubation time 8 min. Signal intensity was evaluated by eye, varying in ten defined levels from no visible signal (1) to very dark blue (10). 2.6. Multiple ISH Additional tests were carried out to evaluate the possibility of consecutively staining of the tissue samples with different probes. Therefore, tissue of T. trachurus and S. scombrus were first hybridized with the T. trachurus specific probe Trach1449. After this first hybridization, tissue samples were washed for 10 min in 1× PBS, followed by an overnight incubation in washing buffer without NaCl (high stringency) at 48 ◦ C. TMB incubations were intended to ensure that no HRP probes remained before a second hybridization was conducted with the Scomber specific probe Scom1449. 3. Results & discussion 3.1. Sequence divergence Five M. merluccius (accession number KF986698–KF986702), five T. trachurus (KF986703–KF986707), three T. mediterraneus

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(KF986708–KF986710) and four S. scombrus (KF986711– KF986714) 18S rRNA gene sequences were obtained (Table S1). Sequences ranged from 1067 to 1802 bp (mean 1676 bp) and the length of the alignment was 1802 bp. The sequence analysis of the 18S rRNA gene showed no differences between T. trachurus and T. mediterraneus. Sequence divergence based on p-distances was 0.51% between T. trachurus/mediterraneus and S. scombrus, 5.13% between T. trachurus/mediterraneus and M. merluccius, and 5.23% between S. scombrus and M. merluccius. We also found seven substitutions and two additional nucleotides between T. trachurus/mediterraneus and S. scombrus. Such low variability of 18S rRNA gene sequences already have been found in other studies focusing on various fish species (Zhang and Hanner, 2011), limiting its use as a marker for sequence based identification approaches (e.g. DNA barcoding) in fish.

3.2. Probe design Species-specific probes were designed for S. scombrus (Scom1449) and M. merluccius (Mer845). As consequence of the identical 18S rRNA of T. trachurus and T. mediterraneus, the probe Trach1449 was designed for both species (Table 2). All probes had a length of 18 bases. Probe target regions were located within the V5 (Mer845) or the V9 (Scom1449, Trach1449) expansion segment of the 18S rRNA (Neefs et al., 1993). All probes were tested individually on tissue samples of their target species. We found strong signals for Mer845 whereas the signal intensities of Trach1449 and Scom1449 were significantly weaker. This can be interpreted as a consequence of the different accessibilities of the probes to the individual target regions (Behrens et al., 2003).

Fig. 4. Signal intensities evaluation of three specific probes with their supporting helper and competitor probes. (a) Overview of the analyzed samples after a staining of 8 min in TMB. Signal intensities (in arbitrary units) of the specific probes Mer845 (light gray rectangle), Scom1449 (dark gray triangle), and Trach1449 (black circle) on tissue samples of (b) M. merluccius, (c) S. scombrus, and (d) T. trachurus.

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Table 4 List of oligonucleotide helper and competitor probes with sequences used in this study. Probetype

Name

Sequence (5 to 3 )

Source

Helper

H1437 H1453 H835 H862 cTrach1449 cScom1449

CCT CGG ATC GGC CCC GCC GGG CCT CGG ATC GGC CCC GCC GGG GTC ATG GGA ATA ACG CCG CCG AAC CCA AAG ACT TTG GTT TCC GTC GGT AAC GGT CCT GGC GTC GGT CAC GGC CCT GGC

Present study Present study Present study Present study Present study Present study

Competitor

Whereas the V5 expansion segment represents an easily accessible region in general (brightness class I & III of the evaluation system defined by Behrens), the V9 expansion segment can be classified as so-called highly inaccessible region to probe binding (class V) (Behrens et al., 2003). Due to the fact that the V9 segment was the only suitable region for the design of specific probes discriminating Trachurus and Scomber, we improved the accessibility by designing additional unlabeled helper probes which flank the target site and enhance a successful binding of the speciesspecific probe (Table 4) (Fuchs et al., 2000). Whereas Trach1449 and Scom1449 had the same target sites, two helper probes (H1437, H1453) were designed for both species-specific probe s. For Mer845, two helper probes (H835, H862) were designed to test if the presence of additional probes would affect the signal intensity also. All helper probes were evaluated using tissue samples (approx. 20 mg) of the target organisms T. trachurus, S. scombrus and M. merluccius. Efficiency was determined in multiple hybridizations with and without helper probes. For Scom1449 and Trach1449 the use of both helper probes improved the binding and signal intensity. For the Mer845 probe, our tests showed strong signals with as well as without the presence of the helper probes, resulting in an optional use of this helper probes. Owing to the almost identical target region of Trach1449 and Scom1449 that differ only in two base pairs, we found strong unspecific probe signals of Trach1449 at S. scombrus tissue as well of Scom1449 at T. trachurus tissue. In order to decrease such unspecific binding reactions we also employed supplementary competitor probes which were fully complementary to the mismatch-containing non-target sequence and therefore increase the discriminatory effect by blocking the target area of nontarget species (Manz et al., 1992) (Table 4). The additional use of such competitor probes increased the specificity of the speciesspecific probes significantly. Only very weak unspecific signals were observed.

oxidation reaction. In such a case, the substrate will change its color to yellow (450 nm) and will be stable for at least 1 h. However, this approach has not been tested yet. 3.4. Application of probes All complete probe/helper/competitor combinations were tested separately on tissue samples of ten specimens of T. mediterraneus, S. scombrus and M. merluccius (Fig. 4). The Trachurus-specific combination consisted of the probe Trach1449, the helper probes H1437 and H1453, and the competitor cTrach1449. The specific signal on T. mediterraneus tissue had a mean intensity of 7.7 ± 0.8, whereas the unspecific signal on S. scombrus tissue samples was 1.1 ± 0.3 and on M. merluccius tissue samples was 1.8 ± 0.8 (Fig. 4b). For S. scombrus, the used probe combination contained the probe Scom1449, the helper H1437 and H1457, and the competitor cScom1449. The mean specific signal intensity on the analyzed S. scombrus tissue samples was 7.9 ± 1.0; the unspecific signal

3.3. Probe specificity Melting curves based on increasing formamid concentrations were performed to specify optimal hybridization conditions and to maximize the signal/noise ratio (Fig. 2). Our results revealed that the specificity of Trach1449 and Scom1449 was highest at 20% formamid (Fig. 2b and c), and of Mer845 at 30% (Fig. 2d). Optimal formamide concentrations are shown in Table 1. During the staining process of all hybridization series for all probes, the first specific signals were observed after 2 min (data not shown). All three probes produced visible but sometimes weak specific signals as well as weak unspecific signals after 4 min incubation. Highest signal/noise ratios were found after 8 min whereas lowest ratios were observed after 20 min (Fig. 3). We assume that the observed unspecific signals were mainly caused by unbound but active probes that were not fully removed after the washing steps. It was not possible to remove unspecific staining effects at all, even after extending the washing time or increasing the number of washing steps. According to manufacturer’s instruction, the addition of 0.2 M sulfuric acid will stop the

Fig. 5. Visualization of the TMB signal intensity from multiple ISH on tissue of T. mediterraneus and S. scombrus. (a) After hybridization with Trach1449; (b) Signal after a 1 h high stringent washing step; (c) Signal after 16 h incubation in high stringent washing buffer; (d) Signal after hybridization with Scom1449.

T. Hofmann et al. / Fisheries Research 170 (2015) 1–8

intensity was 1.1 ± 0.3 both for the T. mediterraneus and the M. merluccius tissue samples (Fig. 4c). Finally, the M. merluccius specific combination comprised of the probe Mer845 and the Helper H835 and H862. In this case no competitor probe was needed. The specific signal had a mean intensity of 8.3 ± 0.9 whereas unspecific signals had values of 1.0 ± 0 (T. mediterraneus) and 1.7 ± 0.7 (S. scombrus) (Fig. 4d). All three probes produced strong specific signals accompanied with only very weak unspecific signals allowing a clear differentiation in all analyses. In order to evaluate the possibility of successional hybridizations employing different probes on the same tissue sample, a modification of the standard ISH protocol (so-called multiple ISH) was also tested (Fig. 5). All experiments were conducted on two tissue samples of T. mediterraneus and S. scombrus, using the probe Trach1449 (incl. helper and competitor) for the first hybridization, followed by a second hybridization with the probe Scom1449 (incl. helper and competitor). As expected, the Trach1449 probe produced high specific signals on the T. mediterraneus tissue and almost no unspecific signals on the S. scombrus tissue (Fig. 5a). After washing for one hour in washing buffer and incubation in TMB, specific signals were still present (Fig. 5b). As consequence, the washing time was increased to 16 h, leading to a complete fading of all signals (Fig. 5c). Following this, a second hybridization using the Scom1449 probe was performed. A specific staining was observed, with a somewhat slightly weaker intensity in comparison to the first hybridization (Fig. 5d). As a consequence of this doubled hybridization steps, the analyzed tissue samples became highly degraded (Fig. 5b,d), representing a problem when analyzing small tissue samples. Nevertheless, the observed degradation did not suppress the efficacy of the applied ISH method, indicating that even the presence of a small number of cohesive fibers allowed clear species-specific signals.

4. Conclusion In situ hybridization techniques are used in microbial ecology since the late 80s (DeLong et al., 1989) and are still very common (Petersen et al., 2011; Thiele et al., 2015; Tischer et al., 2012; Wagner and Haider, 2012; Zimmermann et al., 2014). Summing up our results, we demonstrated the efficient use of in situ hybridizations as an alternative and promising method for species identification of fish tissue for the first time. The developed probes produced specific signals in combination with helper and competitor probes. The unlabeled helper and competitor oligonucleotides are used for more than two decades (Fuchs et al., 2000; Manz et al., 1992) and were here applied on Metazoan taxa for the first time. The specific probes developed for the identification of T. trachurus/mediterraneus, S. scombrus and M. merluccius in this study can now be tested for identification of fish eggs from the Mackerel and Atlantic horse mackerel survey in the Northeast Atlantic. In this region only T. trachurus may be expected, as T. mediterraneus occurs more in the south. Due to the conserved nature of the 18S rRNA gene at species level (Eickbush and Eickbush, 2007; Liao, 1999) the probes can be used for identification purposes throughout the whole geographical range of the target species. However, we are fully aware of the fact that the 18S rRNA is in many cases highly similar or even identical between closely related fish species, like in the case of T. trachurus and T. mediterraneus, limiting the use of probes in such cases. Nevertheless, our study demonstrated that even one or two different base pairs between the 18S rRNA sequence of two species allow specific hybridization when combined with helper and competitor probes. Based on the presented protocol, species-specific probes for the identification of fish concerning e.g. ecologically or economically relevant species like tuna could be developed. In this context, it is

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important to know that ISH is a relatively cheap method for routine applications. For example, the total costs of 100 ␮l hybridization buffer, which is sufficient for one to three tissue samples, including the specific probe were about 0.20 D . It should be also noted that a large number of samples can be easily processed using a standard ISH protocol in about seven hours (from rehydrating of the sample to the final visualization) with a minimum amount of equipment, making this approach much more attractive for its use onboard than DNA barcoding or other sequencing-based methods. Acknowledgments The authors would like to thank N. Dubilier and the Department of Symbiosis from the Max-Planck-Institute for Marine Microbiology, Bremen, for their constant help, their expertise and helpful comments on the manuscript. We are also grateful to M. Kloppmann and J. Ulleweit from the Thünen-Institute of Sea Fisheries, Hamburg. The project was funded by the Federal Ministry of Education and Research (Grant no. 03F0499A) and the Land Niedersachsen. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fishres.2015.05. 002 References Ahlstrom, E.H., Moser, H.G., 1980. Characters useful in identification of pelagic marine fish eggs. Calif. Coop. Ocean. Fish. Invest. Rep. 21, 121–131. Ali, M.E., Kashif, M., Uddin, K., Hashim, U., Mustafa, S., Man, Y.B., 2012. Species authentication methods in foods and feeds: the present, past, and future of halal forensics. Food Anal. Method. 5, 935–955. Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R., Stahl, D.A., 1990a. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919–1925. Amann, R.I., Krumholz, L., Stahl, D.A., 1990b. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172, 762–770. Asensio, L., Gonzalez, I., Garcia, T., Martin, R., 2008. Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control. 19, 1–8. Behrens, S., Ruhland, C., Inacio, J., Huber, H., Fonseca, A., Spencer-Martins, I., Fuchs, B.M., Amann, R., 2003. In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes. Appl. Environ. Microbiol. 69, 1748–1758. Costa, F.O., Landi, M., Martins, R., Costa, M.H., Costa, M.E., Carneiro, M., Alves, M.J., Steinke, D., Carvalho, G.R., 2012. A ranking system for reference libraries of DNA barcodes: application to marine fish species from Portugal. PLoS ONE 7, e35858. DeLong, E.F., Wickham, G.S., Pace, N.R., 1989. Phylogenetic stains: ribosomal RNAbased probes for the identification of single cells. Science 243, 1360–1363. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Ehrich, S., Adlerstein, S., Brockmann, U., Floeter, J., Garthe, S., Hinz, H., Kröncke, I., Neumann, H., Reiss, H., Sell, A.F., 2007. 20 years of the German small-scale bottom trawl survey (GSBTS): a review. Senckenbergiana Marit. 37, 13–82. Eickbush, T.H., Eickbush, D.G., 2007. Finely orchestrated movements: evolution of the ribosomal RNA genes. Genetics 175, 477–485. Fitzcharles, E.M., 2012. Rapid discrimination between four Antarctic fish species, genus Macrourus, using HRM analysis. Fish. Res. 127, 166–170. Fox, C.J., Taylor, M.I., Pereyra, R., Villasana, M.I., Rico, C., TaqMan, D.N.A., 2005. technology confirms likely overestimation of cod (Gadus morhua L.) egg abundance in the Irish Sea: implications for the assessment of the cod stock and mapping of spawning areas using egg-based methods. Mol. Ecol. 14, 879–884. Fuchs, B.M., Glockner, F.O., Wulf, J., Amann, R., 2000. Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 66, 3603–3607. Fuchs, B.M., Wallner, G., Beisker, W., Schwippl, I., Ludwig, W., Amann, R., 1998. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 64, 4973–4982. Goffredi, S.K., Jones, W.J., Scholin, C.A., Marin 3rd, R., Vrijenhoek, R.C., 2006. Molecular detection of marine invertebrate larvae. Mar. Biotechnol. 8, 149–160. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98.

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