Findings in this study suggest that breech strike resistance is unlikely to be associated with differences in skin protein expression during a short infestation, and larval ES products also showed no significant differences between resistant and non-selected sheep.
Abstract
Background
Blowfly strike (myiasis), caused mainly by Lucilia spp., poses a serious animal welfare concern and results in substantial economic losses to the global sheep industry. However, the early molecular and proteomic responses of sheep skin to larval infestation are not well characterised.
Methods
The early proteomic response in sheep skin following infestation by blowfly Lucilia cuprina larvae was assessed in 14 Merino ewes, 7 from a group selected for breech strike resistance and 7 from a non-selected group. Control sites were also established using mock dental plugs without blowfly eggs. After ~ 22 h post-egg implantation, larvae were removed to collect excretory-secretory (ES) products. Skin washings were also collected to recover sheep proteins secreted in response to larval activity. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), comparisons were made between (1) larval-challenged and mock control sites to identify sheep proteins induced by larval activity and (2) resistant and non-selected sheep. While this paper focuses mainly on the sheep proteins produced in response to larval activity, excretory-secretory (ES) proteins from the larvae were also detected.
Results
In total, 105 proteins were significantly enriched at larval-challenged sites. Following challenge, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed strong enrichment of the complement and coagulation cascades as well as the neutrophil extracellular trap (NET) formation pathway. Protein-protein interaction analysis identified a major cluster with alpha-2-macroglobulin (A2M) as the seed protein, while the top 10 hub proteins included A2M, serine protease inhibitors (Alfa-1 antitrypsin, Antithrombin III), fibrinogens and plasma proteins such as albumin and haptoglobin. No proteins in the skin exudate differed significantly between resistant and non-selected sheep, and larval ES products also showed no significant differences between these groups.
Conclusions
Early infestation by Lucilia cuprina larvae induces a strong, non-specific proteomic response in sheep skin, characterised by activation of the complement and coagulation cascades and NET formation. However, findings in this study suggests that breech strike resistance is unlikely to be associated with differences in skin protein expression during a short infestation.
The blow fly Lucilia sericata is a species of major medical and veterinary importance, functioning both as a causative agent of myiasis and as a beneficial organism in maggot debridement therapy. Increasing resistance to conventional insecticides highlights the need for alternative compounds targeting fundamental physiological processes. In this study, we investigated the insecticidal and physiological effects of 2,6-dimethylphenol (2,6-DMP), a lipophilic phenolic compound, with particular emphasis on its impact on cuticular lipid homeostasis and immune cell integrity. Topical application of 2,6-DMP significantly reduced larval survival and, more prominently, suppressed adult emergence to 5-20% of control levels, indicating disruption of metamorphosis beyond acute toxicity. Dose-response analysis revealed similar median lethal doses (LD50) for larvae and adults (∼1.85-1.95 μg/mg body mass), suggesting comparable susceptibility across developmental stages. Gas chromatography-mass spectrometry demonstrated pronounced, stage-specific remodeling of cuticular free fatty acids (FFAs). In larvae, exposure induced a strong, dose-dependent accumulation of long-chain FFAs, particularly C16:0 and C18:1. In contrast, adults exhibited a biphasic response: sublethal exposure resulted in a >3-fold increase in total FFAs, whereas lethal exposure caused near-complete lipid depletion. Cholesterol was consistently depleted in all treated groups. Principal component analysis confirmed that variation in lipid profiles was driven primarily by changes in total FFA abundance and sterol composition. In parallel, hemocyte analysis revealed clear immunotoxic effects, including reduced granulocyte abundance, impaired aggregation, and progressive morphological disruption in both in vivo and in vitro models. Collectively, these findings demonstrate that disruption of lipid homeostasis contributes to the physiological and immunotoxic effects of 2,6-DMP in L. sericata. Beyond revealing a potential mode of insecticidal action, our findings raise concerns about the potential ecotoxicological impact of 2,6-DMP as an environmental contaminant, particularly with respect to non-target insects, and identify lipid homeostasis as a sensitive mechanistic endpoint for environmental risk assessment.
Lepeophtheirus salmonis is a crustacean ectoparasite that poses a significant threat to the salmon aquaculture industry, causing substantial economic losses. The copepodite stage is critical for successful host infestation, as failure to attach to the host results in the parasite's death. Once attached, copepodites must evade the host immune system to establish infestation and grow. Secretory/excretory proteins (SEPs) released by copepodites are hypothesized to play a role in immune evasion and host manipulation. In this study, SEPs were collected from 1000 copepodites (n = 3) incubated in artificial seawater (ASW) and natural seawater (NSW), both with and without dopamine stimulation. Using LC-MS/MS-based proteomics, we identified 1815 non-redundant proteins, representing the largest dataset of L. salmonis SEPs reported to date. A higher number of proteins were identified in dopamine stimulated SEPs from ASW compared to NSW. Key protein classes included proteases (metalloproteases, serine proteases, cysteine proteases, aspartic proteases), protease inhibitors, peroxidases, and oxidoreductases. Gene ontology (GO) enrichment analysis revealed key molecular functions, including oxidoreductase, peptidase and catalytic activity, biological processes related to energy metabolism and cellular components such as proteasomes and ribosomes. These findings highlight the metabolic shifts and immune modulation strategies employed by copepodites during host attachment. Our findings demonstrate the utility of ASW for in vitro SEP profiling, with lower background noise compared to NSW and underscore the importance of dopamine stimulation in enhancing SEP release. This study provides valuable insights into the molecular mechanisms underlying L. salmonis parasitism. However, further characterization of these proteins is necessary to identify their specific roles in host immune evasion, infestation success and might offer strategies to mitigate its impact on aquaculture.
Vismai Naik Thuppe, J. Bruun, T. A. Grønset et al.· Journal of Fish Diseases· 0 citations
Complement inhibition is a key strategy used by many blood-feeding arthropods to ensure successful feeding. Mosquitoes, ticks, sand flies, and other hematophagous species secrete salivary proteins that block different steps of the complement cascade. These inhibitors prevent complement-mediated lysis and inflammation at the feeding site, allowing the arthropod to feed efficiently while also creating a more permissive environment for pathogen transmission
Here, we characterize the anti-complement activity of Sicpin, a salivary protein from the blackfly Simulium nigrimanum. Sicpin was found to inhibit all three pathways of complement activation. Surface plasmon resonance analysis revealed a strong and specific interaction with C3, with no detectable binding to other complement components common to the pathways.
The binding induces a conformational change in C3 that blocks the interaction with the C3 convertase, consequently inhibiting the deposition of downstream complement proteins. Additionally, in both acute lung injury and sepsis models, Sicpin enhances survival and exhibits anti-inflammatory activity.
These findings support that Sicpin modulates the complement system, with potential implications for pathogen transmission, blood feeding, and therapeutic development
NIH
Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Paola Carolina Valenzuela Leon, Molly Ring, Brian Bonilla et al.· Journal of Immunology· 0 citations
Spodoptera frugiperda (fall armyworm) is a globally distributed and highly destructive migratory pest, while Serratia marcescens is a widespread entomopathogenic bacterium capable of infecting diverse insects. In this study, oral infection with S. marcescens strain SM001 exhibited potent virulence, with an LC50 of 7.40 × 106 CFU/mL at 168 h post-infection (hpi). Logistic regression modeling demonstrated that the bacterium enters an exponential proliferation phase within the hemolymph approximately 48-54 h after infection. Infection significantly upregulated Duox and Nos, induced severe oxidative stress, and activated an apoptotic cascade involving Cyt c, p53, and caspase, culminating in extensive midgut epithelial cell apoptosis. Mitigating oxidative stress via targeted Vitamin C supplementation or Duox knockdown alleviated midgut tissue damage and delayed bacterial dissemination. However, exogenous Vitamin C treatment decreased larval mortality, whereas Duox knockdown failed to improve overall survival, revealing a critical physiological trade-off between ROS-mediated pathogen clearance and host immunopathology. Overall, SM001 disrupts midgut redox homeostasis, drives excessive H2O2 accumulation, and breaches the intestinal barrier to facilitate hemocoel translocation, ultimately leading to lethal systemic septicemia. These findings highlight the pivotal role of host-mediated immunopathology in bacterial pathogenesis and provide a robust theoretical foundation for developing advanced microbial control strategies against S. frugiperda.
Theileria equi is an etiological agent of equine piroplasmosis, a tick-borne disease that significantly affects equine health and the global equine industry. In Brazil, Rhipicephalus microplus is the only tick species with confirmed vector competence for T. equi. However, the immunological mechanisms underlying pathogen-vector interactions remain poorly understood. This study aimed to evaluate the differential expression of key genes involved in major immune signaling pathways in the larval and nymphal stages, as well as in the gut and salivary glands of engorged female ticks, following T. equi infection. Our results showed that in infected ticks, the Toll receptor and its associated transcription factor (Dorsal) were upregulated in the gut and salivary glands. The gene encoding the IMD pathway receptor (PGRP) was upregulated in the infected gut, whereas the transcription factor Relish was upregulated in the salivary glands, suggesting tissue-specific activation and alternative regulation of this pathway. Additionally, genes encoding the antimicrobial peptides Microplusin and Defensin were upregulated in both tissues in response to infection. In contrast, the JAK/STAT pathway was downregulated in the infected gut, which may explain the lack of significant differential expression of the Ixodidin gene in this tissue. In the larval and nymphal stages, the transcriptional response was more restricted, with significant downregulation of PGRP in larvae and JAK in nymphs. These findings expand current knowledge of the R. microplus-T. equi immunological interface and highlight potential molecular targets for the development of novel biotechnological strategies for the control and prevention of equine piroplasmosis.
Carla Alves Rabello, K. Galdino, P. Paulino et al.· Acta Tropica· 0 citations
Parasite-induced immunosuppression increases the host’s susceptibility to secondary infection, thereby decreasing its fitness. Parasites primarily mediate host immunomodulation via the production of excretory secretory products (ESPs). The cestode, Schistocephalus solidus, modulates the immune response, behavior, and physiology of its host, the threespine stickleback fish, via ESPs. However, as previous work has almost exclusively focused on the host responses during late-stage S. solidus infection, the anatomical origins and timing of ESP production are unknown.
To address this gap, we used differential staining techniques to detect S. solidus secretory organs at multiple developmental stages: day 21 (immature, unsegmented) and 42 (mature, segmented) plerocercoids. These techniques involved Hematoxylin and Eosin (H&E) and immunohistochemical analysis through lectin staining.
H&E staining revealed that d21 samples had a nucleated tegument, and did not contain secretory vesicles. Conversely, d42 samples showed an acellular tegument, and secretory vesicles. Lectin staining indicated the presence of abundant glycoprotein-containing secretory vesicles in the parenchyma of d42 samples, which were absent at d21. These results suggest that ESPs may be produced and stored in the secretory vesicles of the parenchyma, and that significant ESP production, and therefore immunomodulation, may not occur until later stages of plerocercoid development.
Supporting this finding, we observe significant, and wide-reaching immune suppression in d42-infected fish, with reductions in splenic melanomacrophages, IgM+ B cells, and CD4+ T cells. These preliminary findings and tools lay the foundation for future work, including confirmation of secretory vesicle contents and the characterization of ESP composition across cestode development.
University of Massachusetts Lowell
Microbial, Parasitic, and Fungal Immunology (MPF)
Stacia Szafran-Lally, Alexandra Collias, Natalie C. Steinel· Journal of Immunology· 0 citations
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