A common theme in extracellular fluids of beetles: extracellular superoxide dismutases crucial for balancing ROS in response to microbial challenge

Abstract

Extracellular Cu/Zn superoxide dismutases (SODs) are critical for balancing the level of reactive oxygen species in the extracellular matrix of eukaryotes. In the present study we have detected constitutive SOD activity in the haemolymph and defensive secretions of different leaf beetle species. Exemplarily, we have chosen the mustard leaf beetle, Phaedon cochleariae, as representative model organism to investigate the role of extracellular SODs in antimicrobial defence. Qualitative and quantitative proteome analyses resulted in the identification of two extracellular Cu/Zn SODs in the haemolymph and one in the defensive secretions of juvenile P. cochleariae. Furthermore, quantitative expression studies indicated fat body tissue and defensive glands as the main synthesis sites of these SODs. Silencing of the two SODs revealed one of them, PcSOD3.1, as the only relevant enzyme facilitating SOD activity in haemolymph and defensive secretions in vivo. Upon challenge with the entomopathogenic fungus, Metarhizium anisopliae, PcSOD3.1-deficient larvae exhibited a significantly higher mortality compared to other SOD-silenced groups. Hence, our results serve as a basis for further research on SOD regulated host-pathogen interactions. In defensive secretions PcSOD3.1-silencing affected neither deterrent production nor activity against fungal growth. Instead, we propose another antifungal mechanism based on MRJP/yellow proteins in the defensive exudates.Using oxygen in aerobic metabolism provides more energy per glucose than glycolysis does and hence constitutes an evolutionary advantage. Anyhow, molecular oxygen can be reduced to highly reactive by-products, termed reactive oxygen species (ROS), that can inflict damage on almost all molecules 1 . Oxygen is therefore a stringent selection criterion acting on organisms to sustain its reactive variants 2-4 . O 2 • − is considered the "primary" ROS, which can further interact with other molecules to generate "secondary" ROS including hydroxyl radicals (• OH), hydroperoxyl radicals (HOO• ) or hydrogen peroxide (H 2 O 2 ) 5,6 . While organisms evolved antioxidant activity to counteract the deleterious effects (termed oxidative stress) of these oxygen variants, they have also developed mechanisms to use ROS as vital components in cellular signalling pathways or immune response 5,7,8 .Superoxide dismutases (SOD, E.C. 1.15.1.1), common to all kingdoms of life, serve as first-line antioxidant enzymes 6,9 . They dismutate O 2 • − into O 2 and H 2 O 2 with diffusion-limited rate constants. SODs are classified according to their metal co-factors and localization 10 . In eukaryotes two intracellular forms are known. The dimeric Cu/Zn-binding SODs (SOD1; 32 kDa) are localized in the cytoplasm, outer mitochondrial space and nucleus 11 , whereas the tetrameric MnSODs (SOD2; 89 kDa) is exclusively present in the inner mitochondrial space 12 . The most recently discovered members of the SOD family are the tetrameric Cu/Zn SODs (SOD3; EC-SOD; 135 kDa) [13][14][15][16] functioning in the ext...

Exploring the Caste-Specific Multi-Layer Defense Mechanism of Formosan Subterranean Termites, Coptotermes formosanus Shiraki

“…Among them, Cu/Zn-SOD2 showed the highest fold expression in winged imagoes against the most pathogenic isolate M. anisopliae 02049. The high upregulation is in agreement with other studies on the first-line insect antioxidant defense genes against fungal infection [ 34 ].…”

Host-pathogen interaction for screening potential of Metarhizium anisopliae isolates against the date-palm dust mite, Oligonychus afrasiaticus (McGregor) (Acari: Tetranychidae)

“…Results showed significant differences in the SOD activities of date-palm dust mites calculated at different times (F = 48.55; df = 12, 24; P < 0.0001), with different fungal isolates (F = 85.27; df = 2, 24; P < 0.0001), and their interaction (F = 356.29; df = 4, 24; P < 0.0001) as shown in Table 2. The highest SOD activity, as depicted in the present results, among datepalm dust mites at the start of infection agrees with the results of Gretscher et al (2016) and Hussain et al (2017). However, a decline in SOD activities with time from the most virulent isolate 2049, corroborates with AlJabr et al (2018), who suggested that decline in SOD activities at a lateral infection stage from virulent isolates led to ROS elimination failure.…”

Cu,Zn Superoxide Dismutase Genes in Tribolium castaneum: Evolution, Molecular Characterisation, and Gene Expression during Immune Priming

“…A second hypothesis arises from the intriguing finding in our phylogenetic analysis, revealing that the two variants of tc-sodb are most closely related to the SOD3 isoforms of the mustard leaf beetle P. cochlearis . Interestingly, in this coleopteran species, substantial EC-SOD activity is found in the haemolymph as well as in the defensive secretions, and SOD-deficient larvae showed increased mortality when challenged with the entomopathogenic fungus Metarhizium anisopliae ( 59 ). Defensive secretions also play an important role in T. castaneum for the regulation of the flour microbiota ( 60 , 61 ), but a potential role of SOD in these fluids needs to be further explored.…”