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Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples
Published on: July 28, 2016
Increased bacterial load in shrimp hemolymph in the absence of prophenoloxidase
Fernand F Fagutao1, Takashi Koyama, Akihiro Kaizu
1Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Japan.
Abstract:
Invertebrates rely on their innate immune responses to protect themselves from pathogens, one of which is melanization of bacteria mediated by the activation of phenoloxidase (PO). Furthermore, invertebrate hemolymph, even that of healthy individuals, has been shown to contain bacterial species. The mechanisms that prevent these bacteria from proliferating and becoming deleterious to the host are, however, poorly understood. Here, we show that knocking down the activity of the inactive precursor of PO [prophenoloxidase (proPO)] by RNA interference resulted in a significant increase in the bacterial load of kuruma shrimp, Marsupenaeus japonicus, even in the absence of a bacterial or viral challenge. Silencing of proPO also led to a sharp increase in shrimp mortality. In addition, the hemolymph of proPO-depleted shrimp had significantly lower hemocyte counts and PO activity than control samples. Microarray analysis after proPO silencing also showed a decrease in the expression of a few antimicrobial peptides, but no effect on the expression of the genes involved in the clotting system. Treatment with antibiotics prior to and after proPO dsRNA injection, to counteract the loss of proPO, resulted in a significant increase in shrimp survival. Our results therefore show that the absence of proPO renders the shrimp incapable of controlling bacteria present in the hemolymph, and that proPO is therefore essential for its survival.
Insights
Prophenoloxidase (proPO) is crucial for shrimp immunity. Depleting proPO increases bacterial load and mortality, highlighting its essential role in controlling hemolymph bacteria and ensuring host survival.
Area of Science:
- Invertebrate Immunology
- Crustacean Physiology
- Microbial Pathogenesis
Background:
- Invertebrates possess innate immunity, including melanization via phenoloxidase (PO), to combat pathogens.
- Hemolymph of invertebrates often harbors bacteria, but mechanisms preventing proliferation are unclear.
- The role of prophenoloxidase (proPO) in controlling endogenous bacterial loads is largely unknown.
Purpose of the Study:
- To investigate the role of prophenoloxidase (proPO) in controlling bacterial load and survival in kuruma shrimp (Marsupenaeus japonicus).
- To determine the impact of proPO depletion on hemocyte counts, PO activity, and gene expression.
Main Methods:
- RNA interference (RNAi) was used to knock down prophenoloxidase (proPO) activity in kuruma shrimp.
- Bacterial load, hemocyte counts, PO activity, and gene expression (antimicrobial peptides, clotting system) were analyzed.
- Antibiotic treatments were employed to assess the direct impact of bacterial control on survival.
Main Results:
- Silencing proPO significantly increased hemolymph bacterial load and shrimp mortality, even without external challenge.
- ProPO-depleted shrimp exhibited reduced hemocyte counts and lower PO activity.
- Microarray analysis revealed decreased expression of some antimicrobial peptides but no change in clotting factor genes.
- Antibiotic treatment partially rescued survival in proPO-depleted shrimp.
Conclusions:
- Prophenoloxidase (proPO) is essential for controlling endogenous bacterial populations in kuruma shrimp hemolymph.
- The absence of proPO impairs the shrimp's ability to manage hemolymph bacteria, leading to increased mortality.
- ProPO plays a vital role in innate immunity and host survival beyond its known role in melanization.
