Hormonal Control of the Making of Crab Shell, a Biological ...
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Hormonal Control of the Making of Crab Shell, a Biological concrete Enmin Zou Department of Biological Sciences, Nicholls State University Abstract This endeavor sought to determine the hormonal mechanism driving post-ecdysial shell calcification in crustaceans. Carbonic anhydrase (CA) catalyzes the carbon dioxide hydration reaction, which generates bicarbonate ions essential for calcium carbonate formation. Using metal analysis, enzymatic assay, tissue culture, and real-time PCR, my lab has found that epidermal CA activity is correlated to exoskeletal calcium content and that both epidermal CA activity and CA gene expression are inducible by the molting hormone in the blue crab Callinectes sapidus. These results have led to the new paradigm of post- ecdysial shell mineralization being controlled by the molting hormone. Intriguingly, while molting hormone injection elevated epidermal CA activity, the exogenous molting hormone was found to suppress shell calcification, implicating that sclerotization and mineralization of the new shell must be coordinated and enhanced deposition of calcium carbonate as a result of increased epidermal CA activity following hormonal treatment would be inhibited to avoid the formation of a structurally defective exoskeleton. Background • Crab shell, a biological concrete, is made up of an organic matrix of chitin and proteins (“steel rods”) mineralized by inorganics, primarily carbonate salts (“cement”). • After molting, crustaceans are covered by a soft shell, which must harden rapidly. Shell hardening consists of sclerotization (“tightening of steel rods”) and mineralization (“addition of cement”). References [1] Chung, J.S., Katayama, H., Dircksen, H., 2012. New functions of arthropod bursicon: inducing deposition and thickening of new cuticle and hemocyte granulation in the blue crab, Callinectes sapidus. PLoS One 7(9), e46299. [2] Calhoun, S., Zou, E. (2016) Epidermal carbonic anhydrase activity and exoskeletal metal content during the molting cycle of the blue crab, Callinectes sapidus. J. Exp. Zool. 325A:200–208. [3] Booth, A., Zou, E. (2016) Impact of molt-disrupting BDE-47 on epidermal ecdysteroid signaling in the blue crab, Callinectes sapidus, in vitro. Aquatic Toxicology 177:373-379. Hypothesis Given the similarity between the patterns of changes in molting hormone concentration and epidermal carbonic anhydrase (CA) activity during the molting cycle [2], it was hypothesized that the molting hormone regulates mineralization through controlling epidermal CA due to the following reactions. CO 2 + H 2 O H + + HCO 3 - Carbonic Anhydrase Results and Discussion 1. Significant correlation between epidermal CA activity and calcium content (Fig. 1) suggests that CA mediates calcium incorporation into the exoskeleton. 3. Interestingly, injection of the exogenous molting hormone inhibited shell calcium content (Fig. 4). Figure 1. Correlation between epidermal CA activity and exoskeletal calcium content in post-ecdysial blue crabs. Future Investigation Further RNAi experiments will be conducted to ascertain the role of epidermal CA in post-ecdysial deposition of calcium to the shell. Acknowledgements This study was sponsored by Louisiana Board of Regents RCS Pfund and Nicholls State University Research Council. I am grateful to my graduate students, Stacy Calhoun, Ashley Booth and Andria Ostrowski, and undergraduate students, Madelyn Beyer and Cam Fontenot, for their contributions to this project. Scientific Question to Be Addressed While it well known that sclerotization is controlled by the neurohormone bursicon [1], the hormonal mechanism driving mineralization remains unresolved. This project attempted to address the question of which hormone controls mineralization in crustaceans , using the blue crab, Callinectes sapidus, as the model crustacean. Crustacean exoskeleton (A biological concrete) Chitin (glucosamine polymer) Proteins (such as collagens) Inorganics (such as CaCO 3 ) Shell hardening Sclerotization or Tanning of organic matrix (“Tightening of steel rods”) Mineralization (“Cement addition”) HCO 3 - + Ca 2+ H + + CaCO 3 Methodologies 1. The “buster” blue crabs purchased from local softshell crab makers were used to produce postmolt crabs in the laboratory. 2. Calcium content in the shell was analyzed using ICP-OES. 3. Epidermal CA activity was assayed according to Calhoun and Zou [2]. 4. The in vitro molting hormone 20-hydroxyecdsone (20-HE) treatment of epidermal tissues followed Booth and Zou [3], while for in vivo treatment 20-HE was injected to post- ecdysial crabs. 5. The expression of epidermal CA genes (CA mRNA) was quantified using quantitative real-time PCR. 6. Epidermal CA gene expression of knocked down using RNAi (through injection of dsRNA to softshell crabs). “Busters” in cages A molting crab ICP-OES for metal analysis 2. Significant in vitro induction of epidermal CA mRNA (Fig. 2) and significant in vivo induction of epidermal CA activity (Fig. 3) suggest that epidermal CA is controlled by the molting hormone. Epidermal tissue culture 0 7 14 21 28 35 42 Control 10nM 100nM 1uM Relative Abundance of Epidermal CasCAg mRNA 20-HE Treatment Concentration * Figure 2. In vitro induction of epidermal CA gene (CasCAg mRNA) by 20-HE. N = 5, * P < 0.05 relative to control. 0 4 8 12 16 20 24 10% Ethanol 15ng/g 20-HE Mean Epidermal Carbonic Anhydrase Activity (units/mg protein) Treatment * N N Figure 3. In vivo induction of epidermal CA activity by 20-HE. * P < 0.05. 0 20 40 60 80 100 120 Control 15 ng/g 20-HE Mean Calcium Content mg/g Dried Exoskeleton Treatment * N = 8 N = 9 Figure 4. Effect of 20-HE injection on shell calcium content. * P < 0.05. This result implicates that sclerotization and mineralization of the new shell must be coordinated and enhanced deposition of calcium carbonate as a result of increased epidermal CA activity following hormonal treatment would be inhibited to avoid the formation of a structurally defective exoskeleton. Conclusion Epidermal CA is controlled by the molting hormone, suggesting that the molting hormone is involved in the regulation of post- ecdysial shell mineralization in Crustacea. Controlled by bursicon Controlled by ???? 0 50 100 150 200 250 300 350 400 Control dsRNA Ca Content (mg/g dry weight) N = 6 N = 5 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Control dsRNA Epidermal CA Activity (units/mg protein) N = 6 N = 5 4. Preliminary RNAi results Figure 5. Knock-down of epidermal CA activity by 10 μg CasCAg dsRNA/crab. Figure 6. Effect of CasCAg dsRNA injection on exoskeletal Ca content.
Transcript of Hormonal Control of the Making of Crab Shell, a Biological ...
Hormonal Control of the Making of Crab Shell, a Biological concrete
Enmin ZouDepartment of Biological Sciences, Nicholls State University
AbstractThis endeavor sought to determine the hormonal mechanism driving post-ecdysial shell calcification in crustaceans. Carbonic anhydrase (CA) catalyzes the carbon dioxide hydration reaction, which generates bicarbonate ions essential for calcium carbonate formation. Using metal analysis, enzymatic assay, tissue
culture, and real-time PCR, my lab has found that epidermal CA activity is correlated to exoskeletal calcium content and that both epidermal CA activity and CA gene expression are inducible by the molting hormone in the blue crab Callinectes sapidus. These results have led to the new paradigm of post-
ecdysial shell mineralization being controlled by the molting hormone. Intriguingly, while molting hormone injection elevated epidermal CA activity, the exogenous molting hormone was found to suppress shell calcification, implicating that sclerotization and mineralization of the new shell must be coordinated and enhanced deposition of calcium carbonate as a result of increased epidermal CA activity following hormonal treatment would be inhibited to avoid the
formation of a structurally defective exoskeleton. Background
• Crab shell, a biological concrete, is made up of an organic matrix of chitin and proteins (“steel rods”) mineralized by inorganics, primarily carbonate salts (“cement”).
• After molting, crustaceans are covered by a soft shell, which must harden rapidly. Shell hardening consists of sclerotization(“tightening of steel rods”) and mineralization (“addition of cement”).
References[1] Chung, J.S., Katayama, H., Dircksen, H., 2012. New functions of arthropod bursicon: inducing deposition and thickening of new cuticle and hemocyte granulation in the blue crab, Callinectes sapidus. PLoS One 7(9), e46299.[2] Calhoun, S., Zou, E. (2016) Epidermal carbonic anhydrase activity and exoskeletal metal content during the molting cycle of the blue crab, Callinectes sapidus. J. Exp. Zool. 325A:200–208. [3] Booth, A., Zou, E. (2016) Impact of molt-disrupting BDE-47 on epidermal ecdysteroid signaling in the blue crab, Callinectes sapidus, in vitro. Aquatic Toxicology 177:373-379.
HypothesisGiven the similarity between the patterns of changes in molting hormone concentration and epidermal carbonic anhydrase (CA) activity during the molting cycle [2], it was hypothesized that the molting hormone regulates mineralization through controlling epidermal CA due to the following reactions.
CO2 + H2O H+ + HCO3-
Carbonic Anhydrase
Results and Discussion
1. Significant correlation between epidermal CA activity and calcium content (Fig. 1) suggests that CA mediates calcium incorporation into the exoskeleton.
3. Interestingly, injection of the exogenous molting hormone inhibited shell calcium content (Fig. 4).
Figure 1. Correlation between epidermal CA activity and exoskeletal calcium content in post-ecdysial blue crabs.
Future InvestigationFurther RNAi experiments will be conducted to ascertain the role of epidermal CA in post-ecdysial deposition of calcium to the shell.
AcknowledgementsThis study was sponsored by Louisiana Board of Regents RCS Pfund and Nicholls State University Research Council. I am grateful to my graduate students, Stacy Calhoun, Ashley Booth and Andria Ostrowski, and undergraduate students, Madelyn Beyer and Cam Fontenot, for their contributions to this project.
Scientific Question to Be AddressedWhile it well known that sclerotization is controlled by the neurohormone bursicon [1], the hormonal mechanism driving mineralization remains unresolved. This project attempted to address the question of which hormone controls mineralization in crustaceans , using the blue crab, Callinectessapidus, as the model crustacean.
Crustacean exoskeleton(A biological concrete)
Chitin (glucosamine polymer)
Proteins (such as collagens)
Inorganics (such as CaCO3)
Shell hardening
Sclerotization or Tanning oforganic matrix(“Tightening of steel rods”)
Mineralization(“Cement addition”)
HCO3- + Ca2+ H+ + CaCO3
Methodologies
1. The “buster” blue crabs purchased from local softshell crab makers were used to produce postmolt crabs in the laboratory.
2. Calcium content in the shell was analyzed using ICP-OES.3. Epidermal CA activity was assayed according to Calhoun and Zou [2].4. The in vitro molting hormone 20-hydroxyecdsone (20-HE) treatment of epidermal tissues followed Booth and Zou [3], while for in vivo treatment 20-HE was injected to post-ecdysial crabs.5. The expression of epidermal CA genes (CA mRNA) was quantified using quantitative real-time PCR. 6. Epidermal CA gene expression of knocked down using RNAi (through injection of dsRNA to softshell crabs).
“Busters” in cages A molting crab ICP-OES for metal analysis
2. Significant in vitro induction of epidermal CA mRNA (Fig. 2) and significant in vivo induction of epidermal CA activity (Fig. 3) suggest that epidermal CA is controlled by the molting hormone.
Epidermal tissueculture
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Figure 2. In vitro induction of epidermal CA gene (CasCAgmRNA) by 20-HE. N = 5, * P < 0.05 relative to control.
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Figure 3. In vivo induction of epidermal CA activity by 20-HE. * P < 0.05.
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Figure 4. Effect of 20-HE injection on shell calcium content. * P < 0.05.
This result implicates that sclerotization and mineralization of the new shell must be coordinated and enhanced deposition of calcium carbonate as a result of increased epidermal CA activity following hormonal treatment would be inhibited to avoid the formation of a structurally defective exoskeleton.
ConclusionEpidermal CA is controlled by the molting hormone, suggesting that the molting hormone is involved in the regulation of post-ecdysial shell mineralization in Crustacea.
Controlled by bursicon
Controlled by ????
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4. Preliminary RNAi results
Figure 5. Knock-down of epidermal CA activity by 10 μg CasCAg dsRNA/crab.
Figure 6. Effect of CasCAg dsRNA injection on exoskeletal Ca content.
