Molecular identification of Anisakis species (Nematoda: Anisakidae) in Scorpaena scrofa (Scorpaenidae) from the Aegean Sea

doi:10.21203/rs.3.rs-1647077/v1

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Molecular identification of Anisakis species (Nematoda: Anisakidae) in Scorpaena scrofa (Scorpaenidae) from the Aegean Sea

https://doi.org/10.21203/rs.3.rs-1647077/v1

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The red scorpionfish Scorpaena scrofa (Scorpaenidae) is a high commercial value marine fish species along the Mediterranean coasts. Anisakiasis is a fish‒borne parasitic zoonoses caused by Anisakis larvae in consumers. To date, there are only a few epidemiological studies on the presence and molecular identification of Anisakis larvae infecting the S. scrofa in the Mediterranean Sea. A total of 272 S. scrofa captured from the Aegean Sea (FAO 37.3.1) were examined for Anisakis larvae between March 2019 and March 2020. The prevalence, mean intensity, and mean abundance of Anisakis larvae was 9.6% (95% CI 6.5–13.7%), 2.8 (95% CI 1.88–5.19), and 0.27 (95% CI 0.15–0.56), respectively. Anisakis pegreffii, A. typica, A. ziphidarum, and A. simplex (s.s.)/A.pegreffii hybrid were identified by RFLP analysis of ITS region. Anisakis pegreffii, A. typica and A. ziphidarum larvae were also confirmed by cox2 sequence analysis. A weak positive and statistically significant correlation between total length (ρS 0.204; p = 0.001) and total weight (ρS 0.200; p = 0.001) of S. scrofa and the number of Anisakis larvae was observed. This survey presents the first molecular detection of A. typica and A. ziphidarum in S. scrofa. Thus, this fish species is a new host for A. typica and A. ziphidarum. This is also the first report of A. ziphidarum in the Aegean Sea.

Anisakiasis

cox2

ITS

RFLP

sequencing

red scorpionfish

The red scorpionfish Scorpaena scrofa (Linnaeus, 1758) (Scorpaenidae) is a demersal fish species that is solitary and sedentary over rocky, sandy, or muddy bottoms from 20 to 200 m. This fish species is widely distributed in the Mediterranean Sea, Sea of Marmara, eastern Atlantic which the British Isles south to Cape Verde Islands including Azores and Madeira, Red Sea, the western Indian Ocean which Somalia to South Africa, to Saya de Malha Bank, and Madagascar (Fricke et al. 2021; Hureau and Litvinenko 1986). The red scorpionfish feed mainly on small fish, crustaceans, and mollusks (Hureau and Litvinenko 1986). The conservation status of S. scrofa is “Least Concern (LC)” according to the IUCN Red List (Nunoo et al. 2015). This species is a prevalent, one of the important and high commercial value fish species, in the coastal areas of the Mediterranean Sea (Matić-Skoko et al. 2015; Sureda et al. 2020). The red scorpionfish is much-appreciated in the Mediterranean and Sicilian markets. This species has a good potential of becoming a future candidate for marine aquaculture due to the high quality of its meat, the excellent palatability, and the high market value (Mancuso 2015).

Anisakiasis is a fish‒borne zoonotic disease caused by the larval stages of Anisakis nematodes. The life cycle of these nematodes involves various hosts such as semiplanktonic and planktonic crustaceans (first intermediate), squids and marine fish (intermediate or paratenic), and marine mammals (definitive/final). Consumers can be infected within the life cycle by consuming raw or undercooked intermediate hosts, and they act as accidental hosts (Mattiucci et al. 2018). Nine Anisakis species have been recognized using different genetic markers (Mattiucci et al. 2014, 2018; Valentini et al. 2006). Among recognized species, A. pegreffii and A. simplex (s.s.) have been confirmed as a major etiological agents of human anisakiasis worldwide (Mattiucci et al. 2018). To date, different types of anisakiasis have been confirmed in humans, such as oral, oesophageal, gastric, intestinal, gastro-allergic, and ectopic (Choi et al. 2017; Daschner et al. 2000; Guardone et al. 2018; Iacomino et al. 2020; Ishikura et al. 1993; Kołodziejczyk et al. 2020; Mattiucci et al. 2011, 2013, 2017, 2018; Mladineo et al. 2016; Year et al. 2016). So far, A. pegrefii, A. simplex (s.s.), A. typica, A. physeteris, A. ziphidarum, and A. simplex (s.s.)/A. pegreffii hybrid have been genetically identified in several marine fish from the Mediterranean Sea (FAO Major Fishing Area 37) (Bušelić et al. 2018; Cipriani et al. 2018; Gaglio et al. 2018; Mattiucci and Nascetti 2008; Mattiucci et al. 2018; Molina-Fernández et al. 2018; Pekmezci et al. 2014; Roca-Geronès et al. 2020; Serracca et al. 2014).

Although the red scorpionfish is a high commercial value fish and has a wide distribution on the coasts of the Mediterranean Sea, eastern Atlantic, and the western Indian Ocean, to date, there are only a few epidemiological studies on the presence and molecular identification of Anisakis larvae infecting the red scorpionfish in the Mediterranean Sea and Spanish NE Atlantic waters (Abollo et al. 2001; Costa et al. 2018; Ferrantelli et al. 2015; Petter and Maillard 1988) (Table 1). Therefore, the aim of the present study was to investigate the prevalence, mean intensity, and mean abundance of Anisakis larvae in the red scorpionfish from the Aegean Sea coasts of Turkey (FAO area 37.3.1) and molecularly identify the Anisakis larvae at the species level for the first time.

Table 1

*Anisakis* infection levels in red scorpionfish *Scorpaena scrofa*.
No. infected/ sampled	Prevalence of Anisakis larvae (%)	Mean intensity/ (range)	Mean abundance	Origin‒FAO fishing zone	Anisakis species	Reference
-	-	-/-	-	Western Mediterranean Sea ^a	Anisakis type I larvae Anisakis type II larvae	Petter and Maillard (1988)
25/25	100	6.4/-	6.4	North-east Atlantic Ocean‒27.8.c	A. pegreffii A. simplex (s.s.)	Abollo et al. (2001)
3/11	27	-/-	-	Western Mediterranean Sea‒37.1.3	Anisakis spp. ^b	Ferrantelli et al. (2015)
4/8	50	-/-	-	Ionian Sea‒37.2.2, Eastern Mediterranean Sea‒37.3	A. pegreffii	Costa et al. (2018)
26/272	9.6	2.8/ (1‒21)	0.2	Aegean Sea‒37.3.1	A. pegreffii, A. typica, A. ziphidarum	Present Study
					Hybrid
^a: FAO fishing zone was not determined
^b: Anisakis species was not genetically identified in S. scrofa
Hybrid: hybrid of A. simplex (s.s.) and A. pegreffii

Fish sampling and parasitological analysis

The parasitological survey was performed to detect Anisakis larvae in red scorpionfish Scorpaena scrofa (Scorpaenidae) between March 2019 and March 2020. A total of 272 fresh specimen of S. scrofa were obtained from the Gulf of Izmir in the Turkish Aegean coasts (FAO 37.3.1) (38°29'32" N 26°48'57" E) (Fig. 1). After captured, S. scrofa immediately transferred in ice box to the laboratory and examined within the next one or two days. All specimens were measured (total body length‒TL, 1 mm accuracy), weighed (total body weight‒TW, 1 g accuracy), and then examined for the presence of Anisakis larvae. Fish were carefully dissected and examined for the presence of Anisakis larvae in the viscera based on the incubation method (Shamsi and Suthar, 2016). For checking Anisakis larvae, the eviscerated fish was carefully skinned and filleted, yielding two dorsal and two ventral fillets. Each fillet was carefully sliced into thin sections (< 3 mm), and initially candled over a white light transilluminator and then a standard UV‒press method used (EFSA 2010; Karl and Leinemann 1993; Levsen et al. 2018). All detected larvae were counted, washed in saline solution, and stored at ethanol for further morphological and molecular identification.

Morphological and molecular identification of Anisakis larvae

The collected all larvae were individually cut into three parts. Middle parts were used for molecular identification. Anterior and posterior parts were cleared in Amman's lactophenol and morphologically identified under the light microscope according to identification keys (Berland 1961; Petter and Maillard 1988). Genomic DNA was extracted from the individual middle part of larvae using the DNA extraction kit (GeneJET Genomic DNA Purification Kit, Thermo Scientific, Waltham, MA, USA). The rDNA ITS region (ITS‒1, 5.8S subunit, ITS‒2) and the mitochondrial cytochrome C oxidase subunit II (cox2) gene were amplified using the primer sets NC5/NC2 (Zhu et al. 1998) and 211F/210R (Nadler and Hudspeth 2000), respectively. PCR conditions followed the protocol given by Pekmezci et al. (2014). The amplified ITS region PCR products were analyzed by restriction fragment length polymorphism (RFLP) technique using the Fast Digest Hinf I and Hha I restriction enzymes (Thermo Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Amplicons were analysed in 1.5% agarose gel. RFLP results were evaluated according to Abollo et al. (2003), D'Amelio et al. (2000), and Pontes et al. (2005). Some representative specimens were randomly chosen from identified Anisakis species based on RFLP results and sequenced to confirm the species identification using the cox2 gene. The cox2 PCR products were bidirectionally sequenced using ABI PRISM 310 genetic analyzer (Applied Biosystems). The quality of the sequences was checked using Geneious R11 (Kearse et al. 2012). Sequences were verified by forward and reverse comparisons, assembled, and edited with Geneious R11 (Kearse et al. 2012). The consensus sequences were compared with those already obtained for the same gene in previous works (Valentini et al. 2006; Mattiucci et al. 2009, 2014) and deposited in GenBank: A. simplex (s.s.) (DQ116426), A. typica (DQ116427), A. pegreffii (DQ116428), A. berlandi (KC809999), A. ziphidarum (DQ116430), A. physeteris (DQ116432), A. brevispiculata (DQ116433), A. paggiae (DQ116434), and A. nascettii (FJ685642) using the BLAST via GenBank (Altschul et al. 1990).

Epidemiological Data And Statistical Analyses

The prevalence (P, %), mean intensity (mI), and mean abundance (mA) were calculated by Quantitative Parasitology 3.0 (Rózsa et al. 2002; Reiczigel and Rózsa 2005). These parameters were estimated with 95% confidence intervals (CI). Sterne's exact 95% confidence limits (or adjusted Wald's for N > 1000) were calculated for the P and bootstrap 95% confidence limits (2000 bootstrap replications) for the mI and mA. Spearman rank test was run to assess the relationship between the number of Anisakis larvae and biometric measures (the TL and TW of fish hosts) using SPSS v22 software.

Morphological identification, epidemiological and statistical data of Anisakis type I larvae

The mean TL ± SD (min‒max) and mean TW ± SD (min‒max) of the red scorpionfish were 23.4 ± 4.8 (12.4‒34.9) cm and 246 ± 128 (26‒722) g for the 272 specimens from FAO 37.3.1. A total of 74 Anisakis larvae were collected from the viscera and body cavity of S. scrofa, although no Anisakis larvae were detected in the fish fillets. All Anisakis larvae were morphologically identified as third-stage of Anisakis type I larvae (sensu Berland 1961) using light microscopy. The P, mI, and mA of Anisakis type I larvae was 9.6% (95% CI 6.5–13.7%), 2.8 (95% CI 1.88–5.19), and 0.27 (95% CI 0.15–0.56), respectively. Spearman's correlation analysis revealed a weak positive and statistically significant correlation between the number of Anisakis type I larvae and TL (ρS 0.204; p = 0.001) and TW (ρS 0.200; p = 0.001).

The RFLP results of ITS regions and cox 2 sequencing of Anisakis species

The amplified ITS region of all Anisakis type I larvae was produced ~ 1000 bp length in the PCR analyses and cut successfully with Hha I and Hinf I restriction enzymes by RFLP analyses. By considering RFLP profiles, larvae were identified as A. pegreffii (Hha I: ~550 and ~ 430 bp; Hinf I: ~370 bp, ~ 300 bp, and ~ 250 bp), A. typica (Hha I: ~320 bp, ~ 240 bp, ~ 180 bp, and ~ 160 bp; Hinf I: ~620bp and ~ 350 bp), A. ziphidarum (Hha I: ~550 and ~ 430 bp; Hinf I: ~370 bp, ~ 320 bp, and ~ 290 bp), and A. simplex (s.s.)/A. pegreffii hybrid genotype (Hha I: ~550 and ~ 430 bp; Hinf I: ~620 bp, ~ 370 bp, ~ 300 bp, ~ 250 bp) (Fig. 2A‒B). Moreover, 37 out of 74 (51.3%) of Anisakis type I larvae were also detected as A. pegreffii, 22 (30.5%) as A. typica, 11 (15.2%) as A. ziphidarum, and 4 (5.5%) as hybrid genotype.

The identified three Anisakis species using RFLP analysis was also confirmed by DNA sequencing of the cox2 gene. The cox2 gene of A. ziphidarum (n = 3), A. typica (n = 3), and A. pegreffii (n = 2) was produced ~ 600 bp length, and consensus sequences of Anisakis species were also obtained as 582 bp. The cox 2 sequences of Anisakis species were deposited in GenBank under the accession numbers from MZ297324 to MZ297327 for A. ziphidarum, from MZ297328 to MZ297330 for A. typica, and from MZ297334 to MZ297335 for A. pegreffii.

The cox2 sequences of four specimens of A. ziphidarum (MZ297324 to MZ297327) herein obtained from Aegean Sea showed 98.97‒100% of identity with the sequence of adults of A. ziphidarum in the Southeast Atlantic Ocean (DQ116430) for cox2 sequence (Valentini et al. 2006). Sequences of the cox2 gene of three A. typica from the Aegean Sea (MZ297328 to MZ297330) matched 98.80‒99.31% with the previously reported the cox2 sequence from adult of A. typica (DQ116427) in the Southeast Atlantic Ocean (Valentini et al. 2006). Finally, two specimens of A. pegreffii in the Aegean coast of Turkey were 97.77‒99.48% match with the sequence deposited in GenBank for the species adult A. pegreffii at the cox2 gene from Delphinus delphis in the Northeast Atlantic (Valentini et al. 2006).

In the present study, data on the presence, parasitological indices of Anisakis larvae, and molecular identification of A. pegreffii, A. typica, A. ziphidarum, and A. simplex (s.s.)/A.pegreffii hybrid in S. scrofa from the Aegean Sea (Eastern Mediterranean Sea, FAO 37.3.1) are presented. The results present the first molecular detection of A. typica and A. ziphidarum in S. scrofa, and thus this fish species is a new host for two Anisakis species. This is also the first comprehensive survey on the Anisakis parasites of S. scrofa in the Mediterranean Sea (Table 1).

Anisakis type I (sensu Berland 1961) and type II (sensu Berland 1961) larvae have been previously reported from S. scrofa in the western Mediterranean Sea (Petter and Maillard 1988). In the Northeast Atlantic Ocean (Galician waters, Northwest Spain), Abollo et al. (2001) reported 100% prevalence of Anisakis larvae in S. scrofa, and A. pegreffii and A. simplex (s.s) species were genetically identified. The presence of Anisakis spp. in S. scrofa was detected as 27% (3/11) in the Western Mediterranean Sea (FAO 37.1.3) (Ferrantelli et al. 2015). Recently, Costa et al. (2018) recorded a high prevalence (P = 50%, 4/8) of A. pegreffii in S. scrofa from the Ionian Sea (FAO 37.2.2) and the Eastern Mediterranean Sea (FAO 37.3). The overall prevalence of Anisakis larvae in S. scrofa in the present study (P = 9.6%, 26/272) was lower than those studies in the same host species sampling from the Mediterranean Sea and the NE Atlantic Ocean (Abollo et al. 2001; Costa et al. 2018; Ferrantelli et al. 2015). Since the overall sample size of these previous studies on Anisakis spp. infecting S. scrofa were considerably lower compared to our study, it is possible that their prevalence of Anisakis larvae may have been detected at relatively higher rates. In fact, relatively high rates of Anisakis prevalence could be affected by the small sample size (Farjallah et al. 2008; Pekmezci et al. 2014; Pulleiro-Potel et al. 2015; Serracca et al. 2013). Moreover, a sample size of more than 15 specimens is enough to obtain a good estimate of prevalence (Jovani and Tella 2006).

Anisakis pegreffii is the dominant species in the Mediterranean Sea (Mattiucci et al. 2013; 2017; 2018). In the present study, 51.3% of all larvae collected from S. scrofa were genetically identified as A. pegreffii. This molecular finding confirmed A. pegreffii as the dominant species in the Mediterranean Sea. To date, there are still insufficient data on the presence of A. ziphidarum larvae in fish species caught in the Mediterranean Sea (Mattiucci et al. 2018). Anisakis ziphidarum larvae have been previously detected in Diaphus metopoclampus, Lepidopus caudatus, Merluccius merluccius, and Xiphias gladius from the Alborean, the Balearic, the Crete, the Ionian, the Ligurian, and the Tyrrhenian Sea (Gaglio et al. 2018; Mattiucci et al. 2007). The S. scrofa in the present study is a new intermediate/paratenic host record for A. ziphidarum. In addition, A. ziphidarum is reported for the first time in the Aegean Sea, providing a new geographical record for this species. Anisakis typica occurs in warm temperate and tropical waters between 35‒40° N and 36° S (Mattiucci et al. 2018), and it has been often reported from M. merluccius, Micromesistius poutassou, Phycis phycis, Scomber japonicus, S. scombrus, and Trachurus trachurus (Farjallah et al. 2008; Mattiucci et al. 2002, 2004, 2008; Pekmezci et al. 2014) in the Mediterranean Sea. The spread of A. typica to the Mediterranean Sea has been related to the lessepsian migration of its paratenic/intermediate hosts from the Indian Ocean (Mattiucci et al. 2004). The present study also presents the first report of A. typica larvae in S. scrofa as a new host in the Mediterranean Sea. It is known that while A. pegreffii is dominant species in the Mediterranean Sea, A. simplex (s.s.) is dominant in the Atlantic Ocean (Mattiucci et al. 2018). In the present study, A. simplex (s.s.)/A.pegreffii hybrid in S. scrofa was identified by RFLP analysis of the ITS region previously reported Abollo et al. (2003) and D'Amelio et al. (2000). Anisakis simplex (s.s.)/A.pegreffii hybrid was also previously identified by ITS-RFLP analysis in the Aegean Sea (Chaligiannis et al. 2012; Pekmezci et al. 2014). However, ITS‒RFLP analysis could provide an overestimation of hybrid (Mattiucci et al. 2016). Recently, A. simplex (s.s.)/A.pegreffii hybrid was confirmed by the elongation factor 1 alpha 1 (EF1 α-1) nDNA gene sequencing (Mattiucci et al. 2016; Roca-Geronès et al. 2021). Although this species was identified by RFLP analysis of the ITS region, it could be confirmed by sequencing of EF1 α-1 nDNA gene in the future studies. The occurrence of A. simplex (s.s.)/A. pegreffii hybrid has been previously reported from Engraulis encrasicolus, L. caudatus, Lophius piscatorius, M. merluccius, M. poutassou, Pagellus erithrinus, Sardina pilchardus, T. picturatus, T. trachurus, T. mediterraneus, S. colias, S. japonicus, S. scombrus, and Zeus faber in the Aegean and Mediterranean Sea (Abollo et al. 2003; Cavallero et al. 2012; Chaligiannis et al. 2012; Costa et al. 2016; Farjallah et al. 2008; Gazzonis et al. 2017; Meloni et al. 2011; Molina-Fernández et al. 2018; Pekmezci et al. 2014; Roca-Geronès et al. 2021). Our findings also report the presence of A. simplex (s.s.)/A. pegreffii hybrid in S. scrofa.

The present study provides the first comprehensive data regarding the presence of Anisakis larvae infecting S. scrofa in the Aegean Sea. Anisakis typica and A. ziphidarum have been genetically identified in S. scrofa for the first time. Therefore S. scrofa is a new host record for mentioned Anisakis species.

Funding

This study was supported by the Scientific Research Council of Ondokuz Mayis University, Samsun, Turkey, under project no PYO.VET.1904.19.005.

Conflicts of interest/Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Not applicable.

Ethics approval

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Authors’ contributions

This work is summarized from the doctoral thesis of Coskun Aydin, Ondokuz Mayis University, Samsun, Turkey, in 2021. The study was supervised and coordinated by DVM, Ph.D., Associate Professor Gokmen Zafer Pekmezci. Gokmen Zafer Pekmezci came up with the study idea and drafted the initial proposal. Both authors performed the study design and contributed to all previous versions of the manuscript. The authors have also read and approved the final manuscript.

Consent to participate

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Molecular identification of Anisakis species (Nematoda: Anisakidae) in Scorpaena scrofa (Scorpaenidae) from the Aegean Sea

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Fish sampling and parasitological analysis

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