Ivermectin (IVM), an antiparasitic drug, has a positive effect against Anisakis simplex s.s. infection and has been used for the treatment and prevention of anisakiasis in humans. However, the molecular mechanism of action of IVM on A. simplex s.s. remains unknown. Herein, tandem mass tag (TMT) labeling and extensive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis were used to identify the effect of IVM on the proteome of A. simplex s.s. in vitro. During the study, 3433 proteins, of which 1247 had at least two protein unique peptides, were identified. Comparative proteomics analysis revealed that 59 proteins were differentially regulated (DRPs) in IVM-treated larvae, of which 14 proteins were upregulated and 38 were downregulated after 12 h of culture, but after 24 h, 12 proteins were upregulated and 22 were downregulated. The transcription level of five randomly selected DRPs was determined by real-time PCR as a supplement to the proteomic data. The functional enrichment analysis showed that most of the DRPs were involved in oxidoreductase activity, immunogenicity, protein degradation, and other biological processes. This study has, for the first time, provided comprehensive proteomics data on A. simplex s.s. response to IVM and might deliver new insight into the molecular mechanism by which IVM acts on invasive larvae of A. simplex s.s.

Ostertagia leptospicularis Assadov, 1953 was formally described in roe deer Capreolus capreolus and has been reported in a wide range of ruminants, including other Cervidae, as well as Bovidae. Nematode specimens derived from various host species exhibit morphological similarity; however, some differences can be observed. It is unclear if this is due to the differential reaction of one nematode species in different host species (i.e., host-induced changes) or because of distinct nematode species in these hosts (i.e., species complex). This paper focuses on specimens resembling O. leptospicularis f. leptospicularis and its closely related species (Ostertagia ostertagi f. ostertagi) collected from various hosts. Morphometric and molecular techniques were applied to assess host-induced changes in nematode morphology and to clarify its systematic classification. There was an overall effect of host species on measurements of nematodes resembling O. leptospicularis (both males and females), but the distinctiveness of the specimens from cattle Bos taurus were highlighted. The results obtained may suggest that the specimens of O. leptospicularis from cattle in Germany and cervids in central Europe belong to different strains. Furthermore, nematodes from the cervid strain appear to circulate within particular host species, which can be seen in the stated morphological variations.

Anisakis simplex is a cosmopolitan parasitic nematode of marine organisms with a complex life cycle. Consuming fish infected with its larvae poses a health risk, as the parasites can penetrate gastrointestinal mucosa, damage stomach and intestinal walls, and trigger allergic reactions. The resulting disease is known as anisakiasis. The European Food Safety Authority (EFSA) classifies A. simplex as a biohazard. Most of its developmental stages (L3, L4, and adults) occur under anaerobic conditions, and larvae derive energy mainly from saccharides. However, the effect of glucose on L3 and L4 larvae—stages pathogenic to humans—has not been described. This study aimed to identify genes and pathways involved in glucose (10 mg/mL) response through transcriptomic and proteomic analyses of L3 and L4 larvae. Differentially expressed genes (DEGs), long non-coding RNAs (DELs), and differentially regulated proteins (DRPs) were identified. DEGs were involved in cuticle structure, lyase activity, and metabolic processes. Comparing L3 CTR and L4 CTR (control) to glucose-treated samples revealed 1,969 DEGs; 259 overlapped between L4 GLU vs. L3 GLU, (glucose-treated) with 11 showing reversed expression. Additionally, 84 DELs were identified in L3 GLU vs. control, 40 in L4 GLU vs. control, and 163 between glucose-treated L4 and L3. Larval and glucose-specific alternative splicing events were also analyzed. Proteomic analysis revealed 35 DRPs—5 more abundant in L4, 30 in L3. The data reveal that developmental stage exerts a more substantial influence than glucose exposure on gene and protein expression profiles. However, glucose still modulates several pathways related to translation, cytoskeletal remodeling, extracellular matrix (ECM) reorganization, and energy metabolism.

Introduction: Glucose transporter (GLUT) research in parasitic nematodes focuses on identifying and characterizing developmentally regulated isoforms, elucidating their regulatory and structural properties, and evaluating their potential as drug targets. While glucose transport mechanisms have been well characterized in the free-living nematode Caenorhabditis elegans, data on parasitic species remain limited. Anisakis simplex s. s., a parasitic nematode, relies on host-derived glucose to maintain energy metabolism. It is hypothesized that A. simplex s. s. utilizes specific glucose transporters to facilitate sugar uptake under varying nutritional conditions.Materials and methods: In silico analysis identified five putative facilitated glucose transporter genes (fgt-1, fgt-2, fgt-3, fgt-5, fgt-9) and one Sugars Will Eventually be Exported Transporter (sweet-1) gene. The FGTs were classified as members of the solute carrier family 2 (SLC2), while sweet-1 belonged to the SWEET transporter family. Full-length cDNA sequences were obtained, and encoded proteins structurally characterized using bioinformatic modeling. Expression of transporter genes was assessed in A. simplex s. s. larvae at stages L3 and L4 cultured in vitro under different glucose concentrations and time points.Results: Structural and phylogenetic analyses revealed that fgt-1 and fgt-3 share high similarity with class I GLUTs found in nematodes and vertebrates. Gene expression profiling demonstrated differential regulation between larval stages. Most notably, FGT genes were stably expressed in L4 larvae, whereas in L3 larvae, gene activation was more variable and dependent on glucose concentration, showing a dynamic transcriptional response to nutrient levels. Sweet-1 was expressed in both stages, but its regulation differed over time and with glucose availability. Glucose supplementation altered trehalose and glycogen levels, and trehalase activity varied across stages and treatments, indicating stage-specific metabolic adaptation.Discussion: The observed transcriptional and biochemical differences between L3 and L4 larvae suggest a shift in glucose uptake mechanisms, from transcuticular absorption in L3 to intestinal glucose uptake in L4 following intestine activation. FGT1 and FGT3 are proposed as key facilitators of glucose uptake, with roles varying across developmental stages. These findings indicate that glucose transporters are regulated in response to changing environmental conditions and may represent targets for rational anthelmintic drug design.