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Background: The European hake ( Merluccius merluccius ) is a commercially valuable demersal species widely distributed in the Mediterranean Sea. Assessing the condition of fish populations in their natural habitats is challenging due to the lack of reliable reference points., Objective: This study aimed to utilize hematological analysis as an economical method to evaluate the physiological and health status of European hake, addressing the gap in hematological data for this species., Methods: Blood samples were collected from the caudal vein of 40 adult European hakes caught from the Toroneos Gulf (northern Greece) using a commercial bottom otter trawl. An automated hematological analyzer was used to assess hematological parameters alongside biometric and biological indices., Results: Female hakes showed significantly higher white blood cell (WBC) counts, thrombocyte (TC) counts, and red cell distribution width (RDW) than their male counterparts. Strong correlations were observed among various hematological parameters, notably between WBC and red blood cells (RBCs), hematocrit (Ht), and hemoglobin (Hb); between RBC and both Ht and Hb; between TC and both mean platelet volume and platelet distribution width (PDW); and between mean corpuscular Hb concentration and RDW. Significant differences were noted in RBCs, Hb, and Ht compared to data from wild-caught European hake populations in Argentina and Denmark. Both trawling depth and duration were found to significantly affect RBC, WBC, Hb, and Ht values, while having no notable impact on TC. Fish captured at an average depth of 80 m and with a trawling duration of 30 min exhibited significantly elevated hematological indices., Conclusion: This study demonstrates that hematological analysis is a valuable, cost-effective tool for assessing the physiological and health status of European hake populations in the Mediterranean. Notable differences in hematological parameters based on sex, as well as significant correlations among key blood metrics, underscore the importance of understanding species-specific hematological profiles. The influence of trawling depth and duration on certain blood parameters highlights the need for standardized sampling protocols in population health assessments. These findings contribute essential baseline hematological data for European hake, facilitating more informed fisheries management and conservation strategies., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (© 2024 The Journal of Biological Methods, All rights reserved.)

The red porgy ( Pagrus pagrus ) and the common dentex ( Dentex dentex ) are Sparidae species of high commercial value, traded in the Greek market. In some cases, fish species identification from Greek fisheries is difficult for the consumer due to the strong morphological similarities with their imported counterparts or closely related species such as Pagrus major , Pagrus caeroleustictus , Dentex gibbosus and Pagellus erythrinus , especially when specimens are frozen, filleted or cooked. Techniques based on DNA sequencing, such as COI barcoding, accurately identify species substitution incidents; however, they are time consuming and expensive. In this study, regions of mtDNA were analyzed with RFLPs, multiplex PCR and HRM in order to develop a rapid method for species identification within the Sparidae family. HRM analysis of a 113 bp region of cytb and/or a 156 bp region of 16s could discriminate raw or cooked samples of P. pagrus and D. dentex from the aforementioned closely related species and P. pagrus specimens sampled in the Mediterranean Sea when compared to those fished in the eastern Atlantic. HRM analysis exhibited high accuracy and repeatability, revealing incidents of mislabeling. Multiple samples can be analyzed within three hours, rendering this method a useful tool in fish fraud monitoring.

The authentication of food products and the verification of their identity are of major importance for consumers. Food fraud through mislabeling is an illegal practice consisting of the substitution of an expensive food product by a relatively cheaper one, misleading false labelling of their origin and adulteration in processed or frozen products. This issue is particularly of high importance concerning fish and seafood, which are easily adulterated primarily due to difficult morphological identification. Fish species of the Mullidae family are considered among the most high-valued seafood products traded in Greece and Eastern Mediterranean in general, in terms of the price and demand. Specifically, the red mullet ( Mullus barbatus ) and the striped red mullet ( Mullus surmuletus ) are both indigenous in the Aegean (FAO Division 37.3.1) and the Ionian (FAO Division 37.2.2) Seas, with high levels of consumers' preferences. However, they could be easily adulterated or misidentified by the invasive Aegean Sea Lessepsian migrator goldband goatfish ( Upeneus moluccensis ) as well as by the imported West African goatfish ( Pseudupeneus prayensis ). Keeping this in mind, we designed two novel, time-saving and easy-to-apply multiplex PCR assays and one multiple Melt-Curve analysis real-time PCR for the identification of these four species. These methodologies are based on species-specific primers targeting single nucleotide polymorphisms (SNPs) detected via sequencing analysis of the mitochondrial cytochrome C oxidase subunit I (CO1) and of the cytochrome b (CYTB) genes in newly collected individuals, with additional comparison with congeneric and conspecific haplotypes obtained from the GenBank database. Both methodologies, targeting CO1 or CYTB, utilize one common and four diagnostic primers, producing amplicons of different length that are easily and reliably separated on agarose gel electrophoresis, yielding a single clear band of diagnostic size for each species or a certain Melt-Curve profile. The applicability of this cost-effective and fast methodology was tested in 328 collected specimens, including 10 cooked samples obtained from restaurants. In the vast majority (327 out of the 328) of the specimens tested, one single band was produced, in agreement with the expected products with a single exception a M. barbatus sample that was identified as M. surmuletus , the identity of which was confirmed using sequencing, indicating erroneous morphological identification. The developed methodologies are expected to contribute to the detection of commercial fraud in fish authentication.