Terrestrial snails are recognised as bioindicators of environmental contamination and may represent a source of dietary exposure to persistent organic pollutants (POPs). However, data on polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs), and polychlorinated biphenyls (PCBs) in edible land snails remain limited. This study investigated PCDD/Fs, dioxin-like PCBs (dl-PCBs), and non-dioxin-like PCBs (ndl-PCBs) in the foot muscle and hepatopancreas of free-living Helix pomatia (H. pomatia) collected from two regions of Poland. A total of 48 snails (24 per region) were analysed as three pooled replicates per tissue type in each region. Thirty-five congeners were determined using high-resolution gas chromatography coupled with high-resolution mass spectrometry (HRGC-HRMS). Overall, only trace concentrations of selected PCDFs and dl-PCBs were detected, exclusively in hepatopancreas samples. Among PCDFs, only 2,3,7,8-TCDF and 1,2,3,7,8-PeCDF were quantified, whereas PCB 77 was the only quantified dl-PCB congener. All analysed PCDD congeners and ndl-PCBs were below the limit of quantification. TEQ values calculated using 2005 WHO-TEFs remained very low in all analysed samples, including under the upper-bound approach. The preferential detection of contaminants in the hepatopancreas was consistent with the higher lipid content of this tissue. The results indicate very low contamination of H. pomatia with dioxins and PCBs and low toxicological relevance under the investigated conditions.
Photoinitiators (PIs) are ubiquitous contaminants with endocrine-disrupting effects, yet their bioaccumulation and biotransformation in aquatic organisms remain unclear. This study reports the first multimedia analysis of 24 PIs in a textile-impacted area in eastern China. Apple snails showed the highest PI levels and diversity (∑19PIs 44.5-353 ng/g dw), exceeding those in fish (∑17PIs 18.3-213 ng/g dw), sediment (∑14PIs 4.1-122 ng/g dw), and river water (∑11PIs 11.7-405 ng/L). In snails, several PIs had median bioaccumulation factors (BAFs) > 1000 L/kg and median biota-sediment accumulation factors >1, suggesting accumulation from both aqueous and sediment-associated sources. Viscera were the primary PI sink, with higher burdens than muscle and gonadal tissues. Muscle accumulation was associated with hydrophobicity, showing a parabolic log BAF-log Kow relationship (R2 = 0.97, p < 0.01). Several metabolites were detected in snail tissues, but low metabolite-to-parent ratios (≤9.8%) for confirmed hydroxylated benzophenones indicated limited biotransformation. Three phosphine oxides, benzophenone, and 2-isopropylthioxanthone were identified as priority risk drivers, with risks concentrated in industrial zones. These findings provide field evidence of PI bioaccumulation and biotransformation in freshwater mollusks, informing aquatic ecological risk assessments of PIs.
Understanding the thermal adaptation of protein function is key to predicting species distributions under climate warming. This study investigated the secondary structure of cytosolic malate dehydrogenase (cMDH) from three intertidal snails with distinct heat tolerance. Despite minimal sequence differences (2-3 amino acids) in cMDHs, their heat tolerance differed significantly. Circular dichroism spectra of wild-type and experimentally-generated mutant cMDHs confirmed that all are α-helix-rich proteins. The cMDH from the extremely heat-tolerant Echinolittorina malaccana had a significantly higher α-helix content than that from heat-tolerant E. radiata. A substitution from glycine to serine at residue 48 significantly reduced the α-helix content of cMDH (p < 0.05), however, substitutions at residues 4 and 114 did not significantly alter α-helix content (p > 0.05). Furthermore, the extremely heat-tolerant cMDH exhibited fewer salt bridges and hydrogen bonds, suggesting greater structural flexibility that may facilitate functional dynamics under heat stress. The findings demonstrate that the secondary structure content, particularly α-helix percentage, may be associated with the temperature adaptation of cMDHs.
After several revisions, Littorinimorpha has been classified as an order-level taxonomic unit under the subclass Heterobranchia of the class Gastropoda, but its suborder classification system remains controversial. In this study, high-throughput sequencing technology was used to obtain the complete mitochondrial genomes of three species from Stromboidea and Tonnoidea: Doxander vittatus, Tonna chinensis, and Phalium glaucum. Genome analysis revealed that the genomes of these three species are highly consistent in structure. They all contain 13 protein-coding genes (PCGs), 22 tRNA genes, and 2 rRNA genes and lack a typical long non-coding control region. The phylogenetic tree constructed based on PCGs shows that Tonnoidea is a monophyletic group within Littorinimorpha, while Stromboidea is a paraphyletic group. Vermetoidea was identified as a separate evolutionary branch under this order. The present study clarifies the mitochondrial genomic characters of three species, D. vittatus, T. chinensis and P. glaucum and their positions in Littorinimorpha, which provides an important basis for evolutionary and phylogenetic studies of this order.
Parasites and infections are one of the major costs associated with group living and high host density. Although infection threat can influence the behavior of both uninfected and infected individuals, investigations have focused on the responses of uninfected individuals. Here we describe the effects on, and response to, terrestrial snails, Cepaea nemoralis, treated with either a bacterial or viral pathogenic stimulus. Brief exposure of untreated (uninfected) snails to the mucus associated chemical cues of snails acutely treated with either the bacterial cell wall component, lipopolysaccharide (LPS), or the viral mimetic, polyinosinic:polycytidylic acid (Poly I:C), elicited active behavioral avoidance and antinociceptive responses (decreased sensitivity to noxious stimuli). Treated (infected) snails also avoided and displayed antinociceptive responses to the chemical cues of snails with either a simulated bacterial or viral infection; however, each according to their own treatment (infection) status. LPS (bacterial) treated snails avoided the cues of Poly I:C (viral), but not LPS, treated conspecifics, whereas Poly I:C treated snails primarily avoided the mucus associated cues of LPS, but not Poly I:C, treated individuals. These findings show that chemical mediated behavioral avoidance of bacterial and viral threat is affected by both the nature of the infection threat and the infection status of the individual.
This study investigated the diversity of culturable bacteria in the gut of Bithynia siamensis goniomphalos, the first intermediate host of trematode parasites. It examined the relationship between bacterial communities and trematode infection. Snails were collected from freshwater habitats in Udon Thani and Khon Kaen provinces, Thailand, and classified as infected or uninfected using the cercarial shedding method. A total of 132 bacterial isolates were obtained and identified based on 16S rRNA gene sequencing. Bacterial abundance differed significantly between the two groups, with higher counts observed in uninfected snails (2.06-8.60 × 103 CFU/ml) than in infected snails (1.06-1.60 × 103 CFU/ml) (p < 0.05). Bacterial composition also varied according to infection status. Bacterial isolates from infected snails were predominantly associated with Acinetobacter, Vogesella, and Stenotrophomonas, whereas Exiguobacterium spp. were mainly found in uninfected snails. Several taxa, including Bacillus spp., Chryseobacterium spp., and Micrococcus spp., were detected in both groups. These findings suggest that parasite infection is associated with shifts in both the abundance and composition of gut bacterial communities in B. siamensis goniomphalos. The results provide new insights into host-microbe-parasite interactions and may contribute to a better understanding of parasite transmission dynamics in freshwater ecosystems.
Biomphalaria snails are intermediate hosts of Schistosoma mansoni, and their distribution predicts where intestinal schistosomiasis may occur. Here, we investigated the genetic diversity and distribution patterns of Biomphalaria spp. in the Lake Albert region and assessed their role in the transmission of schistosomes. Snails (n = 6869) were screened for schistosome infections using cercariae shedding and molecular techniques. In total, 29,670 Biomphalaria snails were collected, 90% of which were in the upland sites. For a subset of snails (n = 184) we sequenced a partial cytochrome c oxidase subunit one (COX1) fragment for phylogeographic analyses. Biomphalaria sudanica, Biomphalaria pfeifferi and Biomphalaria stanleyi were identified, with the former two species having haplotypes shared at the lake and upland sites. We also report the presence of a cryptic lineage, tentatively named B. cf. pfeifferi, awaiting further description. Molecular assays revealed higher overall prevalence of S. mansoni (1.9%) and S. rodhaini (1.12%) among Biomphalaria spp. (n = 1619) than inferred from cercarial shedding (0.66%). However, S. mansoni occurred exclusively at lake and nearby stream sites and was hosted predominantly by B. sudanica (24.7%, 22/89) and B. pfeifferi (5.4%; 5/147), while S. rodhaini, hosted mainly by B. cf. pfeifferi, was widely distribution in upland sites allopatric to S. mansoni. The wet season (October and November) had the highest S. mansoni prevalence. We argue that the lack of S. mansoni in upland sites reflects a more nuanced situation than simply the absence of suitable host snails, and suggest taxonomic revision of the B. pfeifferi complex.
Nickel ferrite nanoparticles (NiFNPs) have been indicated as a sustainable nanotechnology for biomedical, electronic, and nanoremediation fields. However, their release and presence in aquatic environments raise concerns about their potential effects on organisms and their offspring. Thus, the current study aimed to evaluate the effects of NiFNPs and nickel chloride (NiCl2) on young-to-adult freshwater snail Biomphalaria glabrata (mortality, growth, and fecundity) during 30 days of exposure, as well as the possible carry-over effects on the first generation (mortality, embryo development, hatching rate, heart rate, locomotor behavior, and touch sensitivity behavior) sampled after 7 and 28 days of parental exposure. B. glabrata adults were not significantly affected by the direct exposure to either Ni forms. Similarly, mortality, embryo development, and hatching rate of their offspring were not significantly impacted by parental exposure to either NiFNPs or NiCl2. Nevertheless, an increase in heart rate was observed in embryos sampled after 7-day parental exposure for both Ni forms compared with controls, whereas the opposite pattern was observed in embryos sampled after 28-day parental exposure. Further studies are needed to explain this contrasting outcome. In addition, parental exposure to NiFNPs produced snails with reduced thigmotaxis, which would make these snails more susceptible to predation in a NiFNPs-contaminated environment. The present study showed that parental exposure to sub-lethal concentrations of NiFNPs and NiCl2 can cause effects on unexposed offspring, highlighting the relevance of intergenerational studies in nanoecotoxicology.
Polysorbate 20 (Tween 20) is a widely used non-ionic surfactant applied in food, pharmaceutical, and experimental research fields. However, information on its potential effects on freshwater organisms across different life stages remains limited. In this study, the freshwater gastropod Biomphalaria glabrata was used to assess the acute and chronic toxicity of Polysorbate 20 across three developmental stages: embryos, newly hatched snails, and adults. Results showed that Polysorbate 20 induced life-stage-dependent toxicity in B. glabrata. Embryos and newly hatched snails were more sensitive to acute exposure, with LC50 values of 43.57 mg/L at 216 h and 48.21 mg/L at 96 h, respectively, whereas adults showed a higher 96 h LC50 of 324.5 mg/L. Chronic exposure to sublethal concentrations impaired reproduction and growth, as reflected by reduced egg mass production, inhibited shell growth, and altered body weight change. Histological analyses further revealed alterations in the gonads and hepatopancreas, indicating adverse effects on reproductive and digestive tissues. Overall, these findings show that Polysorbate 20 can adversely affect development, reproduction, growth, and tissue organization in B. glabrata, suggesting that its ecological effects on freshwater organisms warrant further attention.
Parasite recolonization and succession following disturbance are inherently linked to the recovery of free-living species, yet patterns governing parasite recolonization in freshwater ecosystems remain largely unexplored. The Emscher River in western Germany was recently restored after a century of degradation as an open sewer system. As only few aquatic organisms persisted under these conditions, free-living organisms and parasites recolonized the system following restoration, providing a unique opportunity to study early successional dynamics. Trematodes infecting first intermediate snail hosts were surveyed through seasonal samplings during the initial years following restoration. Snails and trematodes were identified using combined morphological and molecular approaches. Successional dynamics of snail and trematode communities were analyzed on temporal and spatial scales using abundance, α-diversity (within-site species richness), and β-diversity (among-site dissimilarity). Trematode recolonization potential was assessed based on life-history traits, including dispersal capacity, life cycle complexity, and phylogenetic and geographic host specificity. In total, 1511 snails of 12 species were examined, harboring 16 trematode species at an overall prevalence of 7.8%. The onset of trematode infections coincided with the establishment of native snail hosts, particularly Ampullaceana balthica, which harbored the highest trematode richness and overall prevalence. Although snail host abundance varied with season and distance to the river mouth, recolonization did not follow distinct spatial or temporal patterns. Both snail and trematode communities showed high spatial heterogeneity with pronounced species turnover between sites. Trematode assemblages were dominated by taxa from the regional species pool distributed via mobile definitive hosts. Species with truncated life cycles were underrepresented in terms of species richness but accounted for a high share of overall prevalence. High phylogenetic host specificity toward first intermediate hosts was observed, however, several species exhibited geographic host turnover among localities. These findings demonstrate rapid trematode recolonization following freshwater restoration, and suggest that early trematode assemblages show high spatial heterogeneity and are primarily shaped by host dispersal capacity and the regional species pool, rather than host specificity or life cycle complexity of trematodes. Continued monitoring will be required to assess whether directional changes in infection patterns emerge as the restored habitats mature.
Mucus is known as a viscous fluid; yet, snails manufacture various mucus-based materials with much higher cohesion and tailored to different and even antagonistic functions, including lubrication, adhesion, protection, and defense. To gain insight into this versatility, we use a multidisciplinary approach to investigate five different mucus-based materials produced by the snail Cepaea nemoralis. Our results demonstrate that snails use collagen VI as a main structural component and add amorphous calcium carbonate (ACC) during mucus secretion. ACC functions as an ion source in wet mucus types, most likely for cross-linking, or as a mineral precursor in dry mucus types. These findings shed light on the versatility of these viscoelastic materials, which are able to switch between very different properties on the basis of calcium and protein content.
The coupled function of physiology and behavior is crucial for generating survival responses. Generally, the cardiovascular system undergoes rapid adjustments during scape behavior to increase oxygen delivery to muscle tissues. In contrast, passive antipredator responses such as retraction in snails may impose mechanical constraints on the circulatory system. Here, we evaluated the cardiovascular response underlying the scape response (through induced retraction) and the physical activity in the invasive land snail Cornu aspersum. We quantified heart rate and heart rate variability in adult snails using laser optocardiography under the two conditions. We found that heart rate increased from retracted to the moving state and was accompanied by changes in heart rate variability and reduction of cardiac irregularities. Also, we found that locomotion intensity and body size did not influence these cardiac parameters. Our results suggest that metabolic demands, neural regulation and mechanical configuration collectively alter heart rate and heart rate variability. Thus, cardiovascular function is strongly dependent on behaviorally induced mechanical shifts in the circulatory system and on a "fight-or-flight"-like response.
Rapid urbanisation provides remarkable opportunities to study how sudden, extreme changes impact wildlife. Compared to natural areas, cities are characterised by factors affecting abiotic (e.g., climate, habitat fragmentation) and biotic (e.g., species composition, phenology) components of the ecosystem, ultimately changing the ecological and evolutionary dynamics of those habitats. Similarly to many other taxonomic groups, urban birds differ from rural birds in morphology, behaviour and reproductive patterns. Yet potential associations between urbanisation and avian egg traits-a key aspect of birds life-cycle-remain under-researched. Given the limited availability of primary natural calcium sources (snails) in cities, eggs from heavily urbanised areas were expected to be smaller and more pigmented, indicating thinner shells and lower overall egg quality. To better understand how urbanisation affects egg traits, data on 718 great tit (Parus major) eggs from 90 clutches, spread across eight study sites in a city mosaic, were collected for two breeding seasons. All clutches were photographed and analysed using digital imaging and visual scoring to assess egg volume and pigmentation patterns. Urbanisation was quantified as the percentage of Impervious Surface Area (ISA) in the vicinity of each clutch via satellite imagery. In line with some studies conducted on semi-natural bird communities, egg volume covaried with lay date and female body condition, while for both egg volume and egg pigmentation (spots percentage) a year effect was detected. However, in contrast with the predictions, there was no association between urbanisation and the examined egg traits. While urban clutches are consistently smaller, this study shows that eggs as such are similar to those found in rural habitats in terms of volume and pigmentation patterns. Thus, urban-driven environmental pressures may not be as strong or directional during the egg laying phase as they are at later stages of reproduction.
Large-scale treatment with praziquantel (PZQ) monotherapy is used to control schistosomiasis, leading to concerns about the emergence of PZQ-resistance. In Western Kenya, schistosome-infected patients frequently remain egg-positive following PZQ treatment, and several "hotspot" villages have been observed where transmission remains high, despite annual mass PZQ treatments. This project asks (i) whether PZQ-resistant parasites are found in Western Kenya and (ii) whether "hotspot" villages can be explained by a higher prevalence of PZQ-resistant parasites. We established a simple platform for directly assaying worm motility following in vitro PZQ-exposure in adult schistosomes isolated from a field setting. To do this, we established snail and hamster breeding colonies, and generated large populations of field-derived adult worms, by (i) harvesting S. mansoni eggs from multiple infected patients; (ii) infecting Biomphalaria spp snails with miracidia; (iii) infecting hamsters with released cercariae; (iv) perfusing adult worms from hamsters, and (iv) examining drug response following exposure to PZQ (1 µg/ml for 1 day) in individual S. mansoni worms using an automated movement assay. We measured PZQ-response in 1,800 adult male parasites, representing an estimated 185 parasite genotypes. We identified a single worm that remained motile after PZQ-exposure among the 185 parasite genotypes surveyed (frequency = 0.54%; 95% CI 0.01 - 2.97%, exact binomial) consistent with PZQ-resistant worms being extremely rare or absent. Our direct phenotypic screening results suggests that (i) PZQ-resistance is not currently an obstacle for S. mansoni control in Western Kenya, and (ii) that other factors explain the existence of persistent hotspots.
Background/Objectives: Schistosomiasis, a parasitic disease caused by Schistosoma worms with freshwater snails as intermediate hosts, affects over 250 million people. The current control relies solely on praziquantel, which raises concerns on drug resistance and highlights the need for new therapeutic alternatives. Our bioprospection studies have focused on marine macroalgae as an unexplored source of antischistosomal metabolites with promising results. Guided by WHO recommendations to target both the parasite and its transmission vectors, this study aimed to investigate Ochtodes secundiramea to: (i) isolate active metabolites; (ii) evaluate the isolated compounds against adult worms and oviposition to identify leads for drug development; and (iii) perform an independent screening of their effects against the environmental transmission stages on cercariae and B. glabrata embryos. Methods: A dichloromethane extract of O. secundiramea was submitted to an NMR-biomonitored guided fractionation against Schistosoma mansoni adult worms. Active fractions were further purified through HPLC and characterized by 1H and 13C NMR spectroscopy to identify the isolated compounds. Results: Three halogenated monoterpenes were isolated: ochtodene 1 (4-bromo-1,6,8-trichloro-2,3-ochtodene), ochtodene 2 (2-chloro-1,6,8-tribromo-3,8-ochtodene), and the novel natural product ochtodene 3 [2,6-dibromo-4-(2-chloroethylidene)-1,1dimethylcyclohexane]. Ochtodene 1 was the primary active metabolite against Schistosoma mansoni adult worms, with IC50/96 h values of 47.2 and 46.1 µM for male and female worms respectively, and totally suppressed egg laying with 60 µM, while showing no toxicity toward human fibroblasts. Notably, all metabolites, including the novel ochtodene 3, caused 100% mortality in cercariae and embryos at low concentrations. Conclusions: The discovery of the novel ochtodene 3 and the identification of distinct leads for host treatment and transmission elimination position O. secundiramea as a promising source for integrated schistosomiasis control.
Replicated hybrid zones between ecotypes established over environmental gradients provide an opportunity to study the genomic architecture of barriers to gene flow. The marine snail Littorina fabalis segregates locally into dwarf and large ecotypes over wave-exposure gradients on northwestern European shores. Previous work focusing on a hybrid zone in Sweden revealed strong genetic differentiation between the ecotypes, concentrated in 12 putative chromosomal inversions. Here we compare the Swedish hybrid zone with samples from France distributed across a similar wave-exposure gradient. Our aims were to test if similar exposure gradients promote parallel ecotype distributions and hybrid zones, and if similar genomic architectures contribute to divergence and adaptation across the gradients. Unpredictably, we found that the shell size cline was reversed in France compared to Sweden, with small individuals occupying the more sheltered end of the environmental gradient in Sweden but the more exposed end in France. We also observed a cline in shell colour in France, whereas nearly all Swedish snails were yellow. Using whole-genome sequencing, we found similar levels of genetic differentiation between ecotypes in both places. Most of the differences were accounted for by the same 15 inversions, and the arrangement clines showed similar associations to the wave-exposure gradient in both hybrid zones. These inversions were enriched in SNPs differentiating the ecotypes that were either specific to one hybrid zone or showed reversed cline patterns between zones. Genome-wide association studies (GWAS) detected significant associations between genomic regions within inversions and shell size in Sweden, while one inversion was associated with colour in France. Our results show that the same inversions play a dual role: they support ecotype differences across similar environmental gradients in distant locations while also habouring site-specific variation that contributes to local adaptation.
Biohybrid composites have emerged as highly effective and biocompatible materials of growing interest for applications in environmental remediation. In this work, the potential of porous collagen-/keratin-based capsules, which are naturally released by marine murex snails after their eggs hatch, was explored as an organic matrix for pollutant adsorption. In contrast to previous approaches, an all-in-water linker-mediated bis-condensation was performed directly on the egg capsules in order to synthesize a polyfunctional dendrimer version of polyphenol moieties, starting from gallic acid. The efficacy of this novel material to adsorb various organic pharmaceutical pollutants was evaluated by means of micro-Raman spectroscopy, micro-Fourier transform infrared (μFTIR) mapping, and ultraviolet (UV)-visible/fluorescence microscopy.
Cercariae of three Paramphistomoidea species were identified in the trematode fauna parasitizing the freshwater snails Gabbia fuchsiana from Vietnam and Helicorbis sujfunensis and Gyraulus centrifugops from the southern part of the Russian Far East. To elucidate their taxonomic status, the study included a morphological description accompanied by a molecular analysis using both nuclear and mitochondrial markers. An analysis of the evidence obtained showed that taxonomic identification of paramphistomes can only be possible on the basis of both morphological and molecular data, which are not always available for most representatives of the superfamily Paramphistomoidea. Therefore, genera could be identified for only two trematode species considered in this study. The issue of incorrect taxonomic identification of some trematodes from this superfamily was also highlighted. Due to the challenges encountered in the practical application of the previously obtained sequences to the development of diagnostic PCRs, the superfamily Paramphistomoidea requires careful taxonomic revision.
Cone snails (Conus spp.) produce complex venoms rich in conotoxins, a diverse group of cysteine-rich peptides with high specificity toward ion channels, receptors, and transporters, making them valuable candidates for drug discovery. Despite the pharmacological potential of cone snail venoms, the venom composition of Conus inscriptus remains largely unexplored. In this study, we present the first comprehensive venom gland transcriptome of C. inscriptus collected from the southwest coast of India. High-throughput Illumina sequencing generated 179.6 million paired-end reads, which were assembled into 259,828 transcripts and 75,366 predicted coding sequences (CDS). Functional annotation revealed enrichment of genes involved in cellular processes, metabolism, protein processing, and signal transduction, reflecting the active biosynthetic nature of the venom gland. A total of 6,066 putative conotoxin genes were identified, of which 4,921 were classified into 23 recognised conotoxin superfamilies. The A, O1, and M superfamilies were the most abundant. Additionally, 1,145 transcripts were assigned to conflict groups due to overlapping superfamily characteristics. Analysis of conflict-associated transcripts revealed remarkable cystine framework diversity, including several previously unreported cysteine-rich architectures containing up to 20 cysteine residues. Structural characterisation using AlphaFold and FoldSeek identified both conserved proteins and a large proportion of highly novel proteins lacking recognisable structural homologs. Many of these proteins exhibited high intrinsic disorder, suggesting the presence of previously undescribed peptide scaffolds and lineage-specific venom components. Overall, the transcriptome of C. inscriptus reveals an extensive and previously undocumented repertoire of conotoxins and structurally unique proteins. These findings provide new insights into cone snail venom evolution and establish C. inscriptus as a promising source of novel bioactive peptides with potential applications in marine biotechnology, neuropharmacology, and peptide-based drug development.
Conotoxins are peptide toxins that are found in marine cone snails and inhibit ion channels with high affinity and selectivity. Pl14A is a conotoxin belonging to the J-superfamily, and is one of a few conotoxins that can inhibit two types of receptors, the Kv1.6 subtype of voltage-gated potassium channels as well as nicotinic acetylcholine receptors (nAChRs). In this work, we use classical molecular dynamics simulations to examine the mechanism of inhibition and structural interactions between Pl14A and the Kv1.6 channel. Our study reveals that Pl14A binds stably in the extracellular vestibule of Kv1.6, engaging key residues within the selectivity filter and turret region through a combination of electrostatic and hydrophobic contacts. The peptide's basic residues, mainly the arginine residue and putative dyad motif Lys18 and Tyr19, are implicated in its subtype specificity and pore-blocking activity. Structural analysis and free energy calculations provide a quantitative view of the conformational changes in both the peptide and the channel, consistent with a pore blocking mode of inhibition. These findings help us to understand the modulation of the Kv1.6 channel by conotoxin Pl14A and offer insight for designing selective peptide inhibitors with therapeutic potential against Kv1.6 related pathologies.