First report of adults of Acanthocephaloides irregularis from cuttlefish Sepia officinalis (Cephalopoda) in Tunisia: SEM observations and molecular analysis.

Organochlorine and organobromine compounds in a benthic fish (Solea solea) from Bizerte Lagoon (northern Tunisia): Implications for human exposure

Concentration of polychlorinated biphenyls and organochlorine pesticides in mullet (Mugil cephalus) and sea bass (Dicentrarchus labrax) from Bizerte Lagoon (Northern Tunisia)

Response of potentially toxic Pseudo-nitzschia (Bacillariophyceae) populations and domoic acid to environmental conditions in a eutrophied, SW Mediterranean coastal lagoon (Tunisia)

The three chthamalids Chthamalus stellatus, C. montagui and Euraphia depressa are common inhabitants of the intertidal zone in the Eastern Atlantic, Mediterranean Sea and Black Sea. In this study, we investigated the occurrence of these barnacles in a wide range of their distribution. Population divergences of these two species have been inferred using three molecular markers — internal transcribed spacer (ITS), elongation factor 1α (EF‐1α) and cytochrome oxidase subunit I (COI). ITS sequences of C. stellatus were identical throughout the species range, whereas ITS sequences of C. montagui indicated that the Black Sea and Mediterranean populations are isolated from the Atlantic population. The COI and EF‐1α sequences were the most variable and informative. They indicated a high genetic divergence between Atlantic, Mediterranean and Black Sea populations for C. montagui. In addition significant genetic structure was found among the populations of C. stellatus based on EF‐1α but not COI. Interestingly, our molecular dating analysis correlated the pattern of diversification in C. montagui to major geological changes that occurred in the Mediterranean during the end of the Messinian and Pleiocene periods. We suggest that palaeohistory shaped the divergences between Chthamalus populations that have probably been maintained by current hydrographic conditions. Finally, COI phylogenetic analysis placed the genus Euraphia within the Chthamalus clade, suggesting the need for a taxonomic revision of Euraphia. This study represents the most detailed phylogeographical analysis of intertidal Mediterranean species to date, and shows that geological events have strongly shaped the current diversity pattern of this fauna.

The Bizerte Lagoon is classified in the category of medium salinity environments. The lagoon geochemistry, with regard to the major ions, is essentially controlled by the continental contributions and interactions with the marine environment. The geochemical analyses of the major ions as well as their behaviour in the lagoon water permitted to describe the eventual ability of a solution chemical composition, to precipitate different minerals. Sampling and geochemical analysis of the principal ions (Ca++, K+, Na+, Mg++, Cl-, SO42- and HCO3-) at the water surface (0 to 2 m) and at the bottom (over 2 m) of the lagoon (9 stations), in the Channel (3 stations) and in the bay of Bizerte (3 stations), was performed monthly during one year. The results for the Bizerte Lagoon waters show that only carbonates and gypsum can precipitate. Key words: Water, Bizerte lagoon, ionic ratio, precipitation.

This study investigates the reproductive cycle, size at first sexual maturity and biochem- ical composition of Hexaplex trunculus from the Bizerte lagoon (northern Tunisia). Overall, females predominated over males, resulting in an unbalanced sex ratio of 1.2:1. Males dominated in the smaller size classes ( 50 mm SL). The gonad developmental stages and the gonadosomatic index (GSI) indicated that H. trunculus apparently has an annual reproductive cycle with extended periods of gonadal activity. In both sexes, examination of the appearance of the gonads showed that gamete release occurred mainly from March to May (with an apparent spawn- ing between March and April), followed by a period of empty gonads between June and August. Sexual maturity occurred at smaller sizes in males (SL50 = 41.02 mm SL) than in females (SL50 = 50.47 mm SL). The contents of protein, lipids and carbohydrates were invariably greater in the gonads than in the foot, confirming the key role of the reproductive tissues in energetic storage. The monthly variation in protein, lipids and carbohydrates in the gonads followed the oscillation in the relative proportion of developed gonads and in the GSI, reflecting energetic mobilisation during mat- uration and spawning. Overall, the data gathered in this study constitutes valuable baseline informa- tion for making preliminary recommendations to the fishing community targeting H. trunculus in the Bizerte lagoon. Further studies and more solid data on the reproductive cycle of the species are required before definitive management measures for this locally important artisanal fishery can be proposed.

Dysaphis (Pomaphis) pyri (Boyer de Fonscolombe, 1841) and Dysaphis (Pomaphis) reaumuri (Mordvilko, 1928) are two holocyclic aphid species alternating between Pyrus (Rosaceae) and Galium (Rubiaceae). Comparative phylogenetic analysis was performed using partial mitochondrial cytochrome oxidase subunit I (COI) and nuclear elongation factor 1 alpha (EF-1α) sequences. Partial COI data indicate the possibility of the early divergence in the D. pyri–D. reaumuri stem, which might have occurred even before the splitting of the common ancestral species of the D. reaumuri–D. plantaginea complex. Such a conclusion seems to be compatible with the available data on host specificity, life cycles and distribution of both species. This introductory phylogenetic analysis based on partial COI and EF-1α sequences indicates the need for reconsideration of the subgeneric structure in the genus Dysaphis.

In this paper, we investigated seasonal variations in concentrations of microplastics (MPs) in surface sediments of a lagoon heavily impacted by human activities, located in northern Tunisia (the Bizerte lagoon, southern Mediterranean Sea). Analyses of 112 sediment samples collected from 28 stations between May 2019 and February 2020 revealed significant seasonal variation in concentrations of total MPs, with the highest levels recorded in August 2019 (109.6 ± 59.8 items kg-1 DS (dry sediment)) and the lowest in February 2020 (33.2 ± 22.0 items kg-1 DS). In terms of polymer types, polyethylene particles were the most abundant throughout the year, followed by polypropylene, polyvinyl chloride, and polyethylene terephthalate. Spatial variations in total MP concentrations were found to depend on several environmental factors, including proximity to the coastline, level of anthropogenic pressure, location inside the lagoon, and presence/absence of vegetation. The upper 5-cm surface sediment layer of the lagoon was found to contain ~ 9.96 × 1010 MP particles, equal to ~ 248.97 t of plastic. Similar patterns of microplastic composition and structure were found throughout the year, revealing the same plastic pollution hotspots during all seasons. This indicates that sources of plastic pollution are land-based and originate from coastal urban, industrial, and agricultural areas, as well as from major freshwater streams. The findings of the present work can help to develop an efficient environmental management plan aiming to reduce and/or stop the spread of plastic pollution and its impacts on the socially and economically important ecosystem of the Bizerte lagoon.

First evaluation of microplastics in two commercial fish species from the lagoons of Bizerte and Ghar El Melh (Northern Tunisia)

The Japanese sea cucumber Apostichopus japonicus (Selenka, 1867), native to the East Asian coasts, was recorded for the first time in the Mediterranean Sea along the east Aegean coast (Izmir, Turkey). Fifty-eight specimens were collected by scuba diving at depths up to 10 m between July and August 2024. Species identity was confirmed by morphological examination and molecular analyses using partial regions of the cytochrome oxidase subunit I (COI) and 16S ribosomal RNA (16S rRNA) genes. Phylogenetic analyses based on COI and 16S rRNA sequences confirmed species identity, with haplotypes closely matching those of A. japonicus from its native range. Specimens ranged from 72.5 to 160.0 mm in total length and 21.79 to 88.30 g in gutted body weight, with a slightly negative allometric growth pattern (b = 1.821). Examination of ossicles, including reduced tables, rods, and elongated button-like structures on the body wall and papillae, confirmed the specimens as A. japonicus. The presence of mature individuals and a high proportion of juveniles suggests successful reproduction and establishment in this region. The abiotic data indicated that the environmental conditions at the sampling site were within the tolerance range. This study documented the first occurrence of A. japonicus in the Mediterranean Sea, highlighting its potential to expand its range and to adapt to new environments.

Characterization of microplastics in the surface waters of an urban lagoon (Bizerte lagoon, Southern Mediterranean Sea): Composition, density, distribution, and influence of environmental factors

Dear Editor, Multiple infections with small liver flukes and minute intestinal flukes are the serious public health concern in the lower Mekong basin [1,2]. Although the epidemiological survey for those trematode infections are primarily carried out based on copro-parasitological examination, detection/identification of fecal eggs/worms is a tedious job and often problematic because of the morphological similarities of eggs/worms. Along with the popularization of PCR-sequencing methods, copro-DNA diagnosis and molecular phylogenetic identification/speciation have been introduced in epidemiological studies. Among various genes and non-coding lesions of nuclear and mitochondrial DNAs, mitochondrial cytochrome c oxidase subunit I (COXI) is one of the most widely used inter- and intra-species marker. Using COXI and some other markers, Lee and his colleagues performed molecular phylogenetic analyses on small liver flukes (Lee SU, Huh S. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis. Korean J Parasitol 2004; 42: 145-148) and on minute intestinal flukes (Lee SU, Huh S, Sohn WM, Chai JY. Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai. Korean J Parasitol 2004; 42: 129-135). The COX1 gene sequences appeared in those articles are; Clonorchis sinensis ({type:entrez-nucleotide,attrs:{text:AF184619,term_id:296940320,term_text:AF184619}}AF184619, {type:entrez-nucleotide,attrs:{text:AF181889,term_id:285026682,term_text:AF181889}}AF181889, {type:entrez-nucleotide,attrs:{text:AF188122,term_id:285809842,term_text:AF188122}}AF188122), Metagonimus yokogawai ({type:entrez-nucleotide,attrs:{text:AF096230,term_id:297039733,term_text:AF096230}}AF096230), Metagonimus takahashii ({type:entrez-nucleotide,attrs:{text:AF096231,term_id:297039734,term_text:AF096231}}AF096231), Metagonimus miyatai ({type:entrez-nucleotide,attrs:{text:AF096232,term_id:297039735,term_text:AF096232}}AF096232), Pygidiopsis summa ({type:entrez-nucleotide,attrs:{text:AF181884,term_id:288563287,term_text:AF181884}}AF181884), and Stellantchasmus falcatus ({type:entrez-nucleotide,attrs:{text:AF181887,term_id:285804435,term_text:AF181887}}AF181887). In addition, Park [3] compared his COXI sequence of Opisthorchis viverrini Laotian isolate (AY055 382) to those of Gymnophalloides seoi ({type:entrez-nucleotide,attrs:{text:AF096234,term_id:297039736,term_text:AF096234}}AF096234) and Neodiplostomum seoulense ({type:entrez-nucleotide,attrs:{text:AF096233,term_id:285026845,term_text:AF096233}}AF096233) registered in the DNA database (Lee et al. unpublished). For the phylogenetic analyses of COXI gene of minute intestinal flukes of our own data, we have downloaded all those above mentioned COXI of Lee et al. and aligned them including our own COXI sequence of Haplorchis taichui ({type:entrez-nucleotide,attrs:{text:EF055885,term_id:119855482,term_text:EF055885}}EF055885) [4] and Paragonimus bangkokensis ({type:entrez-nucleotide,attrs:{text:AB354227,term_id:155369203,term_text:AB354227}}AB354227) [5]. Surprisingly, those sequence data were divided into 2 distinct groups without any similarities (Fig. 1). Eventually, we realized that this astonishing result is due to the reverse complementary sequences of COXI data deposited by Lee et al. (in the bottom half of the figure). We also noticed similar mixed-up deposition of the forward and reverse sequences of COXI gene of Fasciola spp., which were also included in Fig. 1 ({type:entrez-nucleotide,attrs:{text:AJ628024,term_id:88319716,term_text:AJ628024}}AJ628024, {type:entrez-nucleotide,attrs:{text:AJ628039,term_id:88319746,term_text:AJ628039}}AJ628039, {type:entrez-nucleotide,attrs:{text:FJ469984,term_id:238631966,term_text:FJ469984}}FJ469984; Zhu XQ et al. unpublished). Fig. 1 The DNA sequence alignment of the partial COXI gene of some trematodes obtained from the GenBank. Seven sequences from the top are the forward strands with JB3 primer sequence, and 2 in the middle are the forward strands without primer. Eight sequences ... For the determination of partial COX1 sequences of Platyhelminthes, the primer set of JB3 (5'-TTT TTT GGG CAT CCT GAG GTT TAT-3') and JB4.5 (5'-TAA AGA AAG AAC ATA ATG AAA ATG-3') [6] was widely used for investigating the inter- and intra-species variations of trematodes and cestodes. We noticed the mixed-up of the forward and reverse COXI sequences by Lee et al. as well as Zhu et al. because of the presence of the characteristic feature of this primer set (boxed in Fig. 1) in the sequences. The primer sequence should be deleted from the sequence data because it is not always identical with the real DNA sequence of the gene and the inclusion of the primer sequences sometimes causes the misreading in phylogenetic analyses [7]. In 3 reverse sequences, {type:entrez-nucleotide,attrs:{text:AF181884,term_id:288563287,term_text:AF181884}}AF181884, {type:entrez-nucleotide,attrs:{text:AY055380,term_id:22203992,term_text:AY055380}}AY055380, and {type:entrez-nucleotide,attrs:{text:AF096233,term_id:285026845,term_text:AF096233}}AF096233 seems to contain also the partial sequence of the cloning vector, which should be trimmed off before deposition. In general, raw data of forward and reverse sequences obtained from the sequencer should be aligned manually by cross-checking of the wave patterns because some 10-20 bases downstream from the forward primer and upstream from the reverse primer often contain erroneous base pairs [8]. Deposition of the reverse sequence means that those sequences were not aligned against forward sequence and not quite reliable. Since each sequence data in GenBank are opened for the public use, an accuracy of the sequence data is critically important for the mutual reliability of the scientists. The scientists should aware how to deposit accurate sequence data to the DNA data base. The reappraisal and correction of those sequences mentioned above is urgently necessary.

A specimen of dusky spinefoot Siganus luridus (Ruppel, 1829) was recorded in the peri-Mediterranean Lagoon of Bizerte located in northern Tunisia. This record constitutes the northernmost extension range of the species in the Tunisian waters, and the second record of a teleost Lessepsian species, known to date in a restricted brackish area. The specimen is described including morphometric measurements and meristic counts. Additionally, the distribution of S. Luridus , herbivorous species, in the Lagoon of Bizerte, the Tunisian waters in the area and in the Mediterranean Sea is commented and discussed.

Lake Baikal is inhabited by a relatively large number of cyclopid species, many of which are endemics. Two genera, Diacyclops Kiefer, 1927 and Acanthocyclops Kiefer, 1927, are the most specious in the lake. Taxonomic discrimination of the majority of representatives of these genera is difficult owing to their high morphological similarities and poor standard description. In this study, a molecular phylogenetic analysis of Lake Baikal members of the Diacyclops/Acanthocyclops group is performed on the basis of mitochondrial cytochrome c oxidase subunit I (COI) gene. It is shown that a fragment of COI 1000 bp long is sufficient for intragenus discrimination of the cyclopids of Lake Baikal. The issues of Diacyclops/Acanthocyclops taxonomy are reflected in the obtained molecular data. Two distinct phylogenetic groups of Diacyclops genus with uncertain taxonomic status are revealed.

The striped false limpet Siphonaria pectinata (Linnaeus, 1758) has traditionally been considered a pulmonate gastropod (but see current phylogenetic work for the placement of Siphonariidae; e.g. Grande, Templado & Zardoya, 2008; Jorger et al., 2010) with a limpet-like shell adapted to life on wave-swept rocky intertidal shores. With its 12 synonyms, S. pectinata is known from the Atlantic coast of Portugal, European and African Mediterranean and West African coast to Angola, Macaronesian archipelagos in the eastern Atlantic and from the Caribbean and nearby Atlantic, including eastern Florida, Texas and Mexico (Abbott, 1974; Rolan, 2005). Although its presence in the western Atlantic has been attributed to a Nineteenth Century invasion from the Mediterranean Sea (Morrison, 1963, 1972), this view was questioned by Carlton (1992) based on habitat and broad Western Atlantic distribution, and it is thus considered by some a cryptogenic species (Carlton, 1992; Baker, Baker & Fajans, 2004) – a species that may be either native or introduced. The potential invasive capacity of Mediterranean Siphonaria pectinata has recently been demonstrated by a considerable range extension to the Gulf of Tunis (northeastern Tunisia), where it was established after 2005 (Antit, Gofas & Azzouna, 2007) and Croatia (unpublished sequence data available in GenBank, accessioned October 2010). The dietary specialization of the species on soft microalgae that may serve to reduce competition with other grazers that consume harder encrusting material (Ocana & Fa, 2003) has been interpreted as one of the advantages of this species for colonizing new localities. In order to test the potential invasion of Siphonaria pectinata into (or from) the western Atlantic, we collected samples from near the extreme of the species distribution in localities in the province of Cadiz, southeastern Spain (La Caleta and el Puerto de Santa Maria), southern Cameroon (Kribi) and the Atlantic coast of Florida (Fort Pierce) (Table 1), totalling 23 individuals, collected between 1993 and 2010 (see Fig. 1 for some of the sampled specimens). The oldest specimens were preserved in 70% ethanol and transferred to 96% ethanol a few years after collection; newer specimens were preserved in 96% ethanol and stored at –808C until processed for DNA extraction. Specimens were retained as vouchers and, together with their DNA extractions, are deposited in the Museum of Comparative Zoology, Department of Invertebrate Zoology DNA collection under numbers MCZ DNA100660, DNA104633, DNA104886 and DNA105664. Total DNA was extracted from a small tissue sample from the foot using the DNeasy Tissue Kit from QIAGEN# and the protocol provided by the manufacturer. The purified DNA was used as a template for PCR amplification of fragments of the mitochondrial genes cytochrome c oxidase subunit I (COI), and 16S rRNA. A 658-bp fragment of the COI gene was amplified and sequenced using the primer pair LCO1490–HCO2198 (Folmer et al., 1994), and part of the 16S rRNA gene, between 436 and 437 bp, was amplified and sequenced using primer pair 16Sa–16Sb (Xiong & Kocher, 1991; Edgecombe, Giribet & Wheeler, 2002). PCRs (25 ml) included 1 ml of the template DNA, 1 ml of each primer, 2.5 ml EconoTaq 10X PCR buffer containing 15 mM MgCl2 (Lucigen), 0.25 ml of dNTPs 100 mM, 1.25 U of EconoTaq DNA polymerase (Lucigen). The PCRs were carried out using an Eppendorf Mastercycler epgradient thermal cycler, and involved, for COI, an initial denaturation at 958C for 2 min, followed by 36 cycles of denaturation step at 958C (45 s), annealing at 41–448C (COI) or 48–538C (16S rRNA) (1 min) and elongation at 728C (90 s). The final elongation step at 728C (4 min) and a rapid thermal ramp for 48C were applied to finalize the process. The double-stranded PCR products were visualized by agarose-gel electrophoresis (1.5% agarose), cleaned up with 2 ml of diluted (1:3) ExoSAP-IT (USB Corp., Cleveland, OH, USA) in a volume of 22 ml PCR product and performed at 378C for 30 min followed by enzyme inactivation at 808C for 15 min. Sequencing reactions were performed in a 10-ml reaction volume using 3.2 ml primer (1 mM), a 1 ml of ABI BigDye Terminator v. 3.0 (Applied Biosystems), 0.5 ml BigDye 5 Sequencing Buffer (Applied Biosystems) and 3.3 ml of cleaned PCR product. The sequencing reaction, performed by using the thermal cycler described above, involved an initial denaturation step for 3 min at 958C, 25 cycles (958C for 10 s, 508C for 5 s and 608C for 4 min) and a rapid thermal ramp to 48C. The BigDye-labelled PCR products were cleaned using Performa DTR Plates (Edge Biosystems, Gaithersburg, MD, USA). The chromatograms were visualized, edited and assembled using Sequencher (Gene Codes Corporation#1991–2006). External primers were cropped and discarded from the edited sequences. Subsequently, the sequences were edited using the sequence alignment editor Se-Al v. 2.0a11. The same program was used to translate COI sequences to ensure that stop codons were not present. The sequences generated in this study have been deposited in GenBank under accession numbers HQ386632–HQ386679. Using Siphonaria concinna G.B. Sowerby I, 1824 (GenBank accession numbers EF489378.1 and EF489300.1 for COI and 16S rRNA, respectively), S. serrata (Fischer, 1807) (GenBank accession numbers EF489380.1 and EF489302.1) and the western Atlantic species S. alternata Say, 1826 (new data for voucher MCZ DNA105833; GenBank accession numbers HQ386678 and HQ386679) as outgroups we performed a maximum-likelihood (ML) phylogenetic tree search. Phylogenetic analyses were conducted in RAxML 7.0.4 (Stamatakis, 2006). Nodal support was estimated via bootstrapping (1,000 replicates) (Stamatakis, Hoover & Rougemont, 2008). Because COI and 16S rRNA can be considered part of the same locus, all analyses were conducted for both genes independently and as a concatenated dataset. The