Proceedings of the National Academy of Sciences, 2018
Significance Some aquatic animals use reflectors in their eyes either to form images or to increa... more Significance Some aquatic animals use reflectors in their eyes either to form images or to increase photon capture. Guanine is the most widespread molecular component of these reflectors. Here, we show that crystals of isoxanthopterin, a pteridine analog of guanine, form both the image-forming “distal” mirror and the intensity-enhancing tapetum reflector in the compound eyes of some decapod crustaceans. The crystal structure of isoxanthopterin was determined, providing an explanation for why these crystals are so well suited for efficient reflection. Pteridines were previously known only as pigments, and our discovery raises the question of which other organic molecules may be used to form crystals with superior reflective properties either in organisms or in artificial optical devices.
The “eyestalk-androgenic gland (AG)-testis” endocrine axis is involved in male sexual differentia... more The “eyestalk-androgenic gland (AG)-testis” endocrine axis is involved in male sexual differentiation of crustaceans. The insulin-like androgenic gland hormone (IAG), secreted from the AG, plays a central role in this axis, however key factors upstream the IAG are still poorly understood. Here, two crustacean hyperglycemic hormone (CHH) genes (LvCHH1 and LvCHH2) and their putative receptor guanylate cyclase (LvGC) were identified in Litopenaeus vannamei. LvCHH1 and LvCHH2 belonged to CHH subfamily I members and LvGC was a membrane-bound guanylate cyclase. They were all differentially expressed in eyestalks and gonads of males and females. RNA interference (RNAi) of either LvCHH1 or LvCHH2 increased LvIAG expression, while injection of their recombinant protein decreased LvIAG expression, indicating that LvCHH1 and LvCHH2 are inhibitory factors of LvIAG expression. Yeast two-hybrid assay showed that both LvCHH1 and LvCHH2 interacted with LvGC and their RNAi and recombinant protein in...
Since its role in the prevention of osteoporosis in humans was proven some 30 years ago, calcium ... more Since its role in the prevention of osteoporosis in humans was proven some 30 years ago, calcium bioavailability has been the subject of numerous scientific studies. Recent technology allowing the production of a stable amorphous calcium carbonate (ACC) now enables a bioavailability analysis of this unique form of calcium. This study thus compares the solubility and fractional absorption of ACC, ACC with chitosan (ACC-C), and crystalline calcium carbonate (CCC). Solubility was evaluated by dissolving these preparations in dilute phosphoric acid. The results demonstrated that both ACC and ACC-C are more soluble than CCC. Fractional absorption was evaluated by intrinsically labeling calcium carbonate preparations with 45Ca, orally administrated to rats using gelatin capsules. Fractional absorption was determined by evaluating the percentage of the administrated radioactive dose per milliliter that was measured in the serum, calcium absorption in the femur, and whole-body retention ove...
The insulin-like androgenic gland hormone (IAG) plays a key role in male sexual differentiation a... more The insulin-like androgenic gland hormone (IAG) plays a key role in male sexual differentiation and spermatogenesis in crustaceans. The expression of IAG is usually negatively regulated by neuropeptide hormones through the “eyestalk-AG” endocrine axis. However, the underlying mechanism is still largely unknown. In the present study, we performed a comparative transcriptome analysis on the androgenic gland (AG), the main secretory organ of IAG, of L. vannamei before and after unilateral eyestalk ablation. A total of 67 differentially expressed genes (DEGs) were identified, including some putative genes involved in sexual development. Interestingly, several genes related to molting and endocrine processes were found differentially expressed between the two treatments. Further investigation on the expression profiles of these genes and Lv-IAG showed that their expression patterns were closely correlated throughout the molting cycle. In addition, injection of 20-hydroxyecdysone (20E) co...
Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network tha... more Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network that constitutes the scaffold upon which cuticles form. The chitin fibers that form this network are closely associated with specific structural proteins, while the cuticular matrix contains many additional structural, enzymatic and other proteins. We study the crayfish gastrolith as a simple model for the assembly of calcified cuticular structures, with particular focus on the proteins involved in this process. The present study integrates a gastrolith-forming epithelium transcriptomic library with data from mass spectrometry analysis of proteins extracted from the gastrolith matrix to obtain a near-complete picture of gastrolith protein content. Using native protein separation we identified 24 matrix proteins, of which 14 are novel. Further analysis led to discovery of three putative protein complexes, all containing GAP 65 the most abundant gastrolith structural protein. Using immunological methods we further studied the role of GAP 65 in the gastrolith matrix and forming epithelium, as well as in the newly identified protein complexes. We propose that gastrolith matrix construction is a sequential process in which protein complexes are dynamically assembled and disassembled around GAP 65, thus changing their functional properties to perform each step in the construction process. The scientific interest on which this study is based arises from three main features of gastroliths: (1) Gastroliths possess partial analogy to cuticles both in structural and molecular properties, and may be regarded, with the appropriate reservations (see Introduction), as simple models for cuticle assembly. At the same time, gastroliths are terminally assembled during a well-defined period, which can be controlled in the laboratory, making them significantly easier to study than cuticles. (2) Gastroliths, like the crayfish exoskeleton, contain stable amorphous calcium carbonate (ACC) rather than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification).
In the Australian red-claw crayfish Cherax quadricarinatus (von Martens) (Decapoda, Parastacidae)... more In the Australian red-claw crayfish Cherax quadricarinatus (von Martens) (Decapoda, Parastacidae), a gonochoristic species, seven different combinations of intersex individuals (with both male and female genital openings) have been described. However, to date, the genetic basis for this phenomenon has not been investigated. This study was designed to test a simple chromosome-based sex-determination model for C. quadricarinatus that assumes the male to be the homogametic (ZZ) sex. According to our model, intersex individuals that are functionally males are genetically females (WZ). Individual crosses were performed between intersex and female crayfish, with control crosses being performed between normal males and females. The control crosses yielded, in most cases, the expected 1[ratio ]1 sex ratio in the F1 progeny. Crosses between intersex individuals and females yielded a 1[ratio ]3 (male[ratio ]female) sex ratio in most crosses. According to our hypothesis, one-third of the femal...
Purification and characterization of the female-specific protein (vitellogenin) from the hemolymp... more Purification and characterization of the female-specific protein (vitellogenin) from the hemolymph of mature female prawn, Macrobrachium rosenbergii, were the objectives of this study. The comparison of biochemical characteristics between vitellogenin and ovarian vitellin was also conducted. Hemolymph vitellogenin was purified with DEAE, hydroxylapatite, and another DEAE chromatographic column. The specific protein (vitellogenin) was shown in the fractions of chromatographic columns on the basis of ELISA, Western blotting, and immunoprecipitation. A purified vitellogenin was obtained with an apparent molecular weight of 700 kDa as determined by PAGE. The purified vitellogenin was considered as a lipoglycoprotein on the basis of staining data. Three subunits (170, 100, and 89 kDa) in purified vitellogenin and two subunits (100 and 89 kDa) in vitellin were detected with SDS-PAGE. Nondisulfide bonds were found in the binding of polypeptide subunits. Only the 89-kDa subunit was a glycopolypeptide in both vitellogenin and vitellin. The amino acid composition of vitellogenin differed from that of vitellin in a few amino acids. Eight amino acid sequences from the N-terminal end of 89- and 100-kDa subunits were determined and they were identical between vitellogenin and vitellin. Seven amino acid sequence from the N-terminal end of the 170-kDa subunit were also identical to the 100-kDa subunit. Purified vitellogenin was more susceptible to precipitation in a solution with low ionic strength than vitellin. This study suggests a close relationship between vitellogenin and vitellin in M. rosenbergii in their biochemical characteristics.
Epidermal growth factor receptors (EGFRs) are highly conserved members of the tyrosine kinase rec... more Epidermal growth factor receptors (EGFRs) are highly conserved members of the tyrosine kinase receptor superfamily found in metazoans and plants. In arthropods, EGFRs are vital for the proper development of embryos and of adult limbs, gonads, and eyes as well as affecting body size. In searching for genes involved in the growth and development of our model organism, the decapod crustacean (Macrobrachium rosenbergii), a comprehensive transcript library was established using next-generation sequencing. Using this library, the expression of several genes assigned to the signal transduction pathways mediated by EGFRs was observed, including a transcript encoding M. rosenbergii EGFR (Mr-EGFR), several potential ligands upstream to the receptor, and most of the putative downstream signal transducer genes. The deduced protein encoded by Mr-EGFR, representing the first such receptor reported thus far in crustaceans, shows sequence similarity to other arthropod EGFRs. The M. rosenbergii gene...
Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endoc... more Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endocrine manipulation, causing them to hypertrophy. Transcripts from these glands were used in the construction of an AG cDNA subtractive library. Screening of the library revealed an AG-specific gene, termed the M. rosenbergii insulin-like AG (Mr-IAG) gene. The cDNA of this gene was then cloned and fully sequenced. The cysteine backbone of the predicted mature Mr-IAG peptide (B and A chains) showed high similarity to that of other crustacean AG-specific insulin-like peptides. In vivo silencing of the gene, by injecting the prawns with Mr-IAG double-stranded RNA, temporarily prevented the regeneration of male secondary sexual characteristics, accompanied by a lag in molt and a reduction in growth parameters, which are typically higher in males of the species. In terms of reproductive parameters, silencing of Mr-IAG led to the arrest of testicular spermatogenesis and of spermatophore developm...
Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the... more Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 1: an ovarian stage dependent involvement
Due to over-fishing and deterioration of wild catch, the ever-growing crustacean market is increa... more Due to over-fishing and deterioration of wild catch, the ever-growing crustacean market is increasingly relying on aquaculture, driving the need for better management techniques. Since most cultured crustacean species exhibit dimorphic growth patterns, the culture of monosex populations (either all-male or all-female) is the preferred approach for gaining higher yields, with the ecological benefit of reducing the risk of invasion by the non-reproducing cultured species. As recently exemplified in prawns, silencing a transcript encoding an androgenic gland-specific insulin-like peptide trough RNAi, has enabled significant yield improvement through all-male monosex cultures that are the progeny of sexually reversed genetic males. The procedure is temporal, not using exogenous hormones and non-genetically modifying (non-GMO), thus, marking the first commercialized, RNAi-based, sustainable biotechnology in the entire aquaculture industry. This tool has the potential to revolutionize pra...
Proceedings of the National Academy of Sciences, 2018
Significance Some aquatic animals use reflectors in their eyes either to form images or to increa... more Significance Some aquatic animals use reflectors in their eyes either to form images or to increase photon capture. Guanine is the most widespread molecular component of these reflectors. Here, we show that crystals of isoxanthopterin, a pteridine analog of guanine, form both the image-forming “distal” mirror and the intensity-enhancing tapetum reflector in the compound eyes of some decapod crustaceans. The crystal structure of isoxanthopterin was determined, providing an explanation for why these crystals are so well suited for efficient reflection. Pteridines were previously known only as pigments, and our discovery raises the question of which other organic molecules may be used to form crystals with superior reflective properties either in organisms or in artificial optical devices.
The “eyestalk-androgenic gland (AG)-testis” endocrine axis is involved in male sexual differentia... more The “eyestalk-androgenic gland (AG)-testis” endocrine axis is involved in male sexual differentiation of crustaceans. The insulin-like androgenic gland hormone (IAG), secreted from the AG, plays a central role in this axis, however key factors upstream the IAG are still poorly understood. Here, two crustacean hyperglycemic hormone (CHH) genes (LvCHH1 and LvCHH2) and their putative receptor guanylate cyclase (LvGC) were identified in Litopenaeus vannamei. LvCHH1 and LvCHH2 belonged to CHH subfamily I members and LvGC was a membrane-bound guanylate cyclase. They were all differentially expressed in eyestalks and gonads of males and females. RNA interference (RNAi) of either LvCHH1 or LvCHH2 increased LvIAG expression, while injection of their recombinant protein decreased LvIAG expression, indicating that LvCHH1 and LvCHH2 are inhibitory factors of LvIAG expression. Yeast two-hybrid assay showed that both LvCHH1 and LvCHH2 interacted with LvGC and their RNAi and recombinant protein in...
Since its role in the prevention of osteoporosis in humans was proven some 30 years ago, calcium ... more Since its role in the prevention of osteoporosis in humans was proven some 30 years ago, calcium bioavailability has been the subject of numerous scientific studies. Recent technology allowing the production of a stable amorphous calcium carbonate (ACC) now enables a bioavailability analysis of this unique form of calcium. This study thus compares the solubility and fractional absorption of ACC, ACC with chitosan (ACC-C), and crystalline calcium carbonate (CCC). Solubility was evaluated by dissolving these preparations in dilute phosphoric acid. The results demonstrated that both ACC and ACC-C are more soluble than CCC. Fractional absorption was evaluated by intrinsically labeling calcium carbonate preparations with 45Ca, orally administrated to rats using gelatin capsules. Fractional absorption was determined by evaluating the percentage of the administrated radioactive dose per milliliter that was measured in the serum, calcium absorption in the femur, and whole-body retention ove...
The insulin-like androgenic gland hormone (IAG) plays a key role in male sexual differentiation a... more The insulin-like androgenic gland hormone (IAG) plays a key role in male sexual differentiation and spermatogenesis in crustaceans. The expression of IAG is usually negatively regulated by neuropeptide hormones through the “eyestalk-AG” endocrine axis. However, the underlying mechanism is still largely unknown. In the present study, we performed a comparative transcriptome analysis on the androgenic gland (AG), the main secretory organ of IAG, of L. vannamei before and after unilateral eyestalk ablation. A total of 67 differentially expressed genes (DEGs) were identified, including some putative genes involved in sexual development. Interestingly, several genes related to molting and endocrine processes were found differentially expressed between the two treatments. Further investigation on the expression profiles of these genes and Lv-IAG showed that their expression patterns were closely correlated throughout the molting cycle. In addition, injection of 20-hydroxyecdysone (20E) co...
Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network tha... more Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network that constitutes the scaffold upon which cuticles form. The chitin fibers that form this network are closely associated with specific structural proteins, while the cuticular matrix contains many additional structural, enzymatic and other proteins. We study the crayfish gastrolith as a simple model for the assembly of calcified cuticular structures, with particular focus on the proteins involved in this process. The present study integrates a gastrolith-forming epithelium transcriptomic library with data from mass spectrometry analysis of proteins extracted from the gastrolith matrix to obtain a near-complete picture of gastrolith protein content. Using native protein separation we identified 24 matrix proteins, of which 14 are novel. Further analysis led to discovery of three putative protein complexes, all containing GAP 65 the most abundant gastrolith structural protein. Using immunological methods we further studied the role of GAP 65 in the gastrolith matrix and forming epithelium, as well as in the newly identified protein complexes. We propose that gastrolith matrix construction is a sequential process in which protein complexes are dynamically assembled and disassembled around GAP 65, thus changing their functional properties to perform each step in the construction process. The scientific interest on which this study is based arises from three main features of gastroliths: (1) Gastroliths possess partial analogy to cuticles both in structural and molecular properties, and may be regarded, with the appropriate reservations (see Introduction), as simple models for cuticle assembly. At the same time, gastroliths are terminally assembled during a well-defined period, which can be controlled in the laboratory, making them significantly easier to study than cuticles. (2) Gastroliths, like the crayfish exoskeleton, contain stable amorphous calcium carbonate (ACC) rather than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification).
In the Australian red-claw crayfish Cherax quadricarinatus (von Martens) (Decapoda, Parastacidae)... more In the Australian red-claw crayfish Cherax quadricarinatus (von Martens) (Decapoda, Parastacidae), a gonochoristic species, seven different combinations of intersex individuals (with both male and female genital openings) have been described. However, to date, the genetic basis for this phenomenon has not been investigated. This study was designed to test a simple chromosome-based sex-determination model for C. quadricarinatus that assumes the male to be the homogametic (ZZ) sex. According to our model, intersex individuals that are functionally males are genetically females (WZ). Individual crosses were performed between intersex and female crayfish, with control crosses being performed between normal males and females. The control crosses yielded, in most cases, the expected 1[ratio ]1 sex ratio in the F1 progeny. Crosses between intersex individuals and females yielded a 1[ratio ]3 (male[ratio ]female) sex ratio in most crosses. According to our hypothesis, one-third of the femal...
Purification and characterization of the female-specific protein (vitellogenin) from the hemolymp... more Purification and characterization of the female-specific protein (vitellogenin) from the hemolymph of mature female prawn, Macrobrachium rosenbergii, were the objectives of this study. The comparison of biochemical characteristics between vitellogenin and ovarian vitellin was also conducted. Hemolymph vitellogenin was purified with DEAE, hydroxylapatite, and another DEAE chromatographic column. The specific protein (vitellogenin) was shown in the fractions of chromatographic columns on the basis of ELISA, Western blotting, and immunoprecipitation. A purified vitellogenin was obtained with an apparent molecular weight of 700 kDa as determined by PAGE. The purified vitellogenin was considered as a lipoglycoprotein on the basis of staining data. Three subunits (170, 100, and 89 kDa) in purified vitellogenin and two subunits (100 and 89 kDa) in vitellin were detected with SDS-PAGE. Nondisulfide bonds were found in the binding of polypeptide subunits. Only the 89-kDa subunit was a glycopolypeptide in both vitellogenin and vitellin. The amino acid composition of vitellogenin differed from that of vitellin in a few amino acids. Eight amino acid sequences from the N-terminal end of 89- and 100-kDa subunits were determined and they were identical between vitellogenin and vitellin. Seven amino acid sequence from the N-terminal end of the 170-kDa subunit were also identical to the 100-kDa subunit. Purified vitellogenin was more susceptible to precipitation in a solution with low ionic strength than vitellin. This study suggests a close relationship between vitellogenin and vitellin in M. rosenbergii in their biochemical characteristics.
Epidermal growth factor receptors (EGFRs) are highly conserved members of the tyrosine kinase rec... more Epidermal growth factor receptors (EGFRs) are highly conserved members of the tyrosine kinase receptor superfamily found in metazoans and plants. In arthropods, EGFRs are vital for the proper development of embryos and of adult limbs, gonads, and eyes as well as affecting body size. In searching for genes involved in the growth and development of our model organism, the decapod crustacean (Macrobrachium rosenbergii), a comprehensive transcript library was established using next-generation sequencing. Using this library, the expression of several genes assigned to the signal transduction pathways mediated by EGFRs was observed, including a transcript encoding M. rosenbergii EGFR (Mr-EGFR), several potential ligands upstream to the receptor, and most of the putative downstream signal transducer genes. The deduced protein encoded by Mr-EGFR, representing the first such receptor reported thus far in crustaceans, shows sequence similarity to other arthropod EGFRs. The M. rosenbergii gene...
Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endoc... more Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endocrine manipulation, causing them to hypertrophy. Transcripts from these glands were used in the construction of an AG cDNA subtractive library. Screening of the library revealed an AG-specific gene, termed the M. rosenbergii insulin-like AG (Mr-IAG) gene. The cDNA of this gene was then cloned and fully sequenced. The cysteine backbone of the predicted mature Mr-IAG peptide (B and A chains) showed high similarity to that of other crustacean AG-specific insulin-like peptides. In vivo silencing of the gene, by injecting the prawns with Mr-IAG double-stranded RNA, temporarily prevented the regeneration of male secondary sexual characteristics, accompanied by a lag in molt and a reduction in growth parameters, which are typically higher in males of the species. In terms of reproductive parameters, silencing of Mr-IAG led to the arrest of testicular spermatogenesis and of spermatophore developm...
Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the... more Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 1: an ovarian stage dependent involvement
Due to over-fishing and deterioration of wild catch, the ever-growing crustacean market is increa... more Due to over-fishing and deterioration of wild catch, the ever-growing crustacean market is increasingly relying on aquaculture, driving the need for better management techniques. Since most cultured crustacean species exhibit dimorphic growth patterns, the culture of monosex populations (either all-male or all-female) is the preferred approach for gaining higher yields, with the ecological benefit of reducing the risk of invasion by the non-reproducing cultured species. As recently exemplified in prawns, silencing a transcript encoding an androgenic gland-specific insulin-like peptide trough RNAi, has enabled significant yield improvement through all-male monosex cultures that are the progeny of sexually reversed genetic males. The procedure is temporal, not using exogenous hormones and non-genetically modifying (non-GMO), thus, marking the first commercialized, RNAi-based, sustainable biotechnology in the entire aquaculture industry. This tool has the potential to revolutionize pra...
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Papers by Amir Sagi