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Polymerase chain reaction as a promising tool for DNA-based diet studies of crustaceans

Joana V. Campos1*, Cristiana Moreira1, Sérgia C. Costa-Dias2*, Eunice Ferreira3, Ana B. Matos3, Vitor Vasconcelos1, 3 and Agostinho Antunes1, 3
  • 1 Interdisciplinary Center for Marine and Environmental Research, Abel Salazar Institute of Biomedical Sciences, University of Porto, Portugal
  • 2 Abel Salazar Institute of Biomedical Sciences, University of Porto, Portugal
  • 3 Faculty of Sciences, University of Porto, Portugal

1. Introduction Feeding ecology studies provide relevant information to understand the ecosystem’s trophic web and functioning. Diet investigations based on direct visual inspection of crustaceans’ gut contents can be complicated due to partial ingestion and/or prey crushing, resulting in few identifiable food remains. In contrary, biochemical methods enable alternative, rapid and unambiguous prey identification. These include enzyme electrophoresis, immunological assays, and Polymerase Chain Reaction (PCR)-based methods in detecting prey DNA (Albaina et al.2010, King et al.2008, Symondson 2002). The PCR technique is faster, more sensitive and specific in predator-prey studies than other methodologies, allowing a simple and rapid detection of the presence/absence of a potential prey by application of specific genetic markers. Here, we aimed to assess the utility of PCR as an alternative tool for studies on curstaceans’ diet using the shore crab Carcinus maenas and the brown shrimp Crangon crangon as study models. 2. Material and methods 2.1. Sampling and stomach dissection Crustaceans and potential preys (common goby Pomatoschistus microps, ragworm Hediste diversicolor, flounder Platichthys flesus, peppery furrow shell Scrobicularia plana) were collected in the Minho Estuary (Portugal). Size (mm; total length, TL, for shrimps; carapace width, CW, for crabs).The whole stomach was removed from about 100 shrimps and 25 crabs per campaign, weighted under sterile conditions and stored in Eppendorfs at -20oC. A stomach fullness index (SFI: 0=empty; 1=up to half full; 2=full) was recorded. Only SFI=2 stomachs were used for DNA extraction. . 2.2. DNA extraction Shrimps full stomach was used for DNA extraction, since stomachs were all very small (mean stomach wet weight=0.0069g, sd=0.0052, n=664), while the large amount of the crabs’ stomach content required subsampling by extracting a 1.5 to 25mg portion. Full stomachs and subsamples were homogenized prior to DNA extraction. DNA was extracted using the PureLinkTM Genomic DNA Mini kit (Invitrogen, Carlsbad, California, USA) applying the protocol for mammalian tissues. Each month, up to ten stomachs (SFI=2) of shrimps and crabs were chosen for DNA extraction. The protocol was followed as recommended by the manufacturer, and the final DNA was eluted in 25 and 50 µL of elution buffer provided by the kit, respectively for shrimps and for crabs; 5µL of the extracted DNA were visualized by 1.5% agarose gel electrophoresis in 1X TAE buffer and stained with ethidium bromide to visualize the integrity and quality of the extracted DNA. 2.3. PCR specificity for the potential preys PCR amplification was performed using a muscle sample of each prey species applying specific primers previously described and tested for specificity, for H. diversicolor (Virgilio et al.2009), P. flesus (Espiñeira et al.2008), S. plana (Santos et al.2012) and P. microps (Gysels et al.2004). The cytochrome subunit b (cytb) gene was used for the identification of P. microps, while the mitochondrial cytochrome oxidase c subunit I (COI) gene was used for the identification of the other prey species. All PCR reactions were performed in 20 µl containing 1x PCR Buffer, 2.5 μM of MgCl2, 250 μM of each deoxynucleotide triphosphate, 10 pmol/μL of each primer, 0.5 units/μL of Taq polymerase (Bioline, Luckenwalde, Germany) and 5-10 ng of DNA. Bovine Serum Albumin (BSA) (10mg/mL) was also added to all the PCR reactions to remove any existing Taq inhibitors. All reactions were carried out in a Biometra thermocycler (Biometra GmbH, Goettingen, Germany). All potential prey were identified in the DNA extracted samples to determine their presence or absence in the stomachs. In shrimps, whole stomachs were tested for all preys, while in crabs the larger stomachs’ content allowed to test the presence of more than one prey species per predator. For prey PCR identification primers sets and PCR conditions were applied according to those available in the literature (Espiñeira et al.2008, Gysels et al.2004, Santos et al.2012, Virgilio et al.2009). In H. diversicolor, PCR conditions consisted of a denaturation at 94°C for 4 min, followed by 9 cycles at 94°C for 45 s, 55°C for 1 min and 72°C for 1 min, followed by a next 26 cycles at 93°C for 30 s, 45°C for 45 s and 72°C for 45 s in the end all samples were subjected to 72°C for 5 min (Virgilio et al.2009). In S. plana, PCR conditions consisted of an initial denaturation at 94°C for 5 min followed by 35 cycles at 94°C for 30 s, 40°C for 45 s and 72°C for 45 s, followed by a final extension at 72°C for 7 min (Santos et al.2012). In P. microps PCR conditions consisted in initial denaturation at 97°C for 3 min followed by 35 cycles at 95°C for 45s, 54°C for 45 s, 72°C for 45 s and final elongation at 72°C for 7 min (Gysels et al.2004). PCR conditions of P. flesus consisted in denaturation at 95°C for 3 min, followed by 35 cycles of 30 s at 95°C, 30 s at 54°C, and 30 s at 72°C, followed by a final extension at 72°C for 3 min (Espiñeira et al.2008); 10 µL of each amplified DNA was visualized by 1.5% agarose gel electrophoresis in 1X TAE buffer. The gel was stained with ethidium bromide and photographed under an UV trans-illuminator. 3. Results A total of 699 shrimps (TL: 13.0 to 43.0 mm; 62% females, 33% males, 6% sex undetermined) and 142 crabs (CW: 23.71 to 64.40 mm; 39% females, 61% males; 57% green, 28% red, 15% undetermined) were collected and dissected. More than two thirds of the shrimps (71%) and crabs (85%) had food in the stomach. For both species, the highest percentage of full stomachs was found in February (23 and 56%, respectively, for shrimps and crabs, Figure 1). The primer sets were successfully tested to evaluate their ability to amplify the prey target from the stomach contents (Figure 2a and 2b). Only S. plana showed negative results in both predators, while the other prey species (P. microps, H. diversicolor, P. flesus) were present in the stomach of both crustaceans (Figure 2a and 2b). The stomach content of 29 crabs gave positive amplifications, four of them with two different prey species, while for shrimps, 11 positive amplifications were obtained corresponding to three prey species. In the positive amplifications, a single band was obtained at the corresponding size of the positive control (Figure 2a and 2b). In light of this data we found no need for the immediate DNA sequencing of these positive bands allowing this to be used in future studies. 4. Discussion Molecular techniques have recently been used to study predation by several taxa, but only a few studies have been dedicated to crustaceans. These included two shrimps Crangon crangon (Albaina et al.2010) and Crangon affinis (Asahida et al.1997), two crab species Carcinus maenas (Asahida et al.1997) and Callinectes sapidus (Collier et al.2014), the rock lobster Jasus edwardsii (Redd et al.2014), the amphipod Themisto abyssorum (Olsen et al.2014) and the calanoid copepod Calanus finmarchicus (Durbin et al.2008, Nejstgaard et al.2003, Nejstgaard et al.2008), as predators. Yet, such studies were focused on a single prey species – flatfish (Albaina et al.2010, Asahida et al.1997, Collier et al.2014), or species group – algae (Durbin et al.2008, Nejstgaard et al.2003, Nejstgaard et al.2008). Therefore, the present investigation enlarged the range of prey identified in crustacean stomachs through molecular methodologies. Moreover, these previous studies were mostly performed under controlled conditions, while in our study we investigated predation in the natural habitat. By adding a PCR inhibitor we obtained a more precise identification, diminishing any false negatives attributed to natural samples. Three additional improvements may be implemented to fully investigate crustaceans’ diet through PCR-based methods: 1) a larger pool of primers; 2) the quantification of the relative importance of each prey species; and 3) DNA sequencing of the positive amplicons in order to proceed to other type of studies (e.g.phylogenetic analysis by comparing these with DNA sequences from other geographical populations). Polymerase Chain Reaction (PCR)-based methods proved to be a very powerful alternative tool to study crustaceans’ diet. The results suggest that these methods are reliable not only for detecting prey DNA in the stomach, but also for its identification to the lowest taxonomic level, i.e. the species level. Therefore, once potential prey and their availability in the system are known, this technique can be useful to overcome some of the limitations of traditional methods in the study of the crustaceans’ diet, even when stomach contents include a great part of ‘detritus’. List of figures Figure 1. Stomach fullness index (SFI, % of animals) results for Carcinus maenas (CM) and for Crangon crangon (CC) during the studied period. Figure 2. Electrophoresis of the positive amplifications obtained in the PCR with stomach contents and the prey species’ controls. From left to right: 2a) DNA ladder, one Pomatoschistus microps (Pm), four Platichthys flesus, and three Hediste diversicolor positive results from crabs; 2b) DNA ladder, one P. microps, eight P. flesus and one H. diversicolor (Hd) positive results from shrimps. ‘+’ is the positive control amplification for the prey species immediately on its left side.

Figure 1
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Figure 2
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Acknowledgements

Santander Totta through IJUP2011 program (Project IJUP-44), UID/Multi/04423/2019 (J.Campos, C.Moreira), Project RecBio - Operation MAR-01.04.02-FEAMP-0025 (S.Costa-Dias), Cláudia Moreira, and Eduardo Martins.

References

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Keywords: Shore crab, carcinus maenas, Brown shrimp, Crangon crangon, Stomach content, Prey identification, PCR

Conference: XX Iberian Symposium on Marine Biology Studies (SIEBM XX) , Braga, Portugal, 9 Sep - 12 Sep, 2019.

Presentation Type: Poster Presentation

Topic: Ecology, Biodiversity and Vulnerable Ecosystems

Citation: Campos JV, Moreira C, Costa-Dias SC, Ferreira E, Matos AB, Vasconcelos V and Antunes A (2019). Polymerase chain reaction as a promising tool for DNA-based diet studies of crustaceans. Front. Mar. Sci. Conference Abstract: XX Iberian Symposium on Marine Biology Studies (SIEBM XX) . doi: 10.3389/conf.fmars.2019.08.00104

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Received: 13 May 2019; Published Online: 27 Sep 2019.

* Correspondence:
PhD. Joana V Campos, Interdisciplinary Center for Marine and Environmental Research, Abel Salazar Institute of Biomedical Sciences, University of Porto, Matosinhos, 4450-208, Portugal, jcampos@ciimar.up.pt
PhD. Sérgia C Costa-Dias, Abel Salazar Institute of Biomedical Sciences, University of Porto, Porto, Portugal, scdias@icbas.up.pt