Neocaridina denticulata

1. Identification of putative ecdysteroid and
juvenile hormone pathway genes in the shrimp
Neocaridina denticulata
Y.W. Sin et al./General and Comparative Endocrinology xxx (2014) xxx–xxx
Environmental Physiology of Marine Organisms
Van-Tinh Nguyen
201356732
Department: Biomedical Engineering/ Marine Biomedical
Science Laboratory.
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2. The arm of this study
• In this study, the authors identify genes involved in the biosynthesis
and degradation of sesquiterpernoids, and biosynthesis of
ecdysteroids in the newly sequenced cherry shrimp Neocaridina
denticulata.
• This information will provide an understanding of the evolution of
hormonal systems across the Pancrustacea and Arthropoda as a
whole.
Keywords: Crustacean, Ecdysteroid, Juvenile hormone, Methyl farnesoate, Insect, Arthropod
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3. Introduction
SHELLFISH (MOLLUSCS AND CRUSTACEA) j Characteristics of the Groups
http://taxo4254.wikispaces.com/Balanus+Amphitrite
http://en.wikipedia.org/wiki/Neocaridina
http://animaldiversity.ummz.umich.edu/
Neocaridina
The Crustacea is conventionally divided into 6 classes, the Branchiopoda, Maxillopoda,
Ostracoda, Remipedia, Cephatocarida and Malacostraca
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4. Neocaridina denticulata Life Cycle
Nathan J. Kenny et al., Mar. Drugs 2014, 12
They have chosen N. denticulata for further study as it has much potential to become a model for aquacultural,
developmental, ecotoxicological, food safety, genetic, hormonal, physiological and reproductive studies in the
laboratory at pH of 6.5–8.0, and temperature is 22–24°C
a) The lifecycle of N. denticulata
b) The appearance of gravid female compared to a female without eggs. The scale bar represents 5 mm on three adult
shrimp.
c) Some of the colour forms available commercially. (i) Red patched; (ii) punctate red patterning; and (iii) blue.
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5. Sesquiterpenoid pathway and
Ecdysteroid biosymthesic pathway genes
• The steroidogenic CYPs are encoded by the Halloween genes
phantom (CYP306A1), disembodied (CYP302A1), shadow (CYP315A1)
and shade (CYP314A1)
• Each of the Halloween enzymes is believed to mediate one specific
enzymatic step in the biosynthesis of 20-hydroxyecdysone (20E), as
mutations in these genes result in low ecdysteroid levels and
embryonic death.
• Spook (CYP307A1), is believed to mediate an uncharacterized step in
the biosynthesis of 20E, as its mutation will also result in low 20E
production.
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6. Materials and methods
• Gene identification in N. denticulata genome resources.
• Gene sequences were identified in the N. Denticulata genome using TBLASTN
searches.
• Phylogenetic analysis.
• The Bayesian phylogenetic tree showing inter-relationships of a variety of
malacostracan crustacean species, including the position of N. denticulate.
• RNA isolation, RT-PCR, subcloning and sequencing.
• To check if contigs containing different exons of some genes could be merged.
Primers for PCR amplification of identified target genes were designed based on
exonic regions.
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7. Results
• Juvenoid hormone biosynthesis
• Juvenoid hormone degradation
• Juvenoid hormone regulation
• Ecdysteroid production
• Regulators of both juvenoid hormones and ecdysteroids
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8. Juvenoid hormone biosynthesis
Juvenile hormone pathway overview (biosynthesis). Genes
identified in Neocaridina denticulataare shown in boxes with
darker coloration (i.e. JHAMT, JHBP, JHEH, JHE and JHEBP).
In insects, juvenile hormone acid
methyltransferase (JHAMT) plays a major role in
the biosynthesis of juvenile hormones (JHs).
The last two steps of JH biosynthesis differ
depending on the insect group.
In the Lepidoptera, epoxidation by an epoxidase
converts FA to JH III acid and its homologues,
which are subsequently methylated by JHAMT,
whereas in most other insects epoxidation follows
methylation of Farnesoic acid (FA) to Methyl
farnesote (MF) by JHAMT.
JHAMT has only been reported in the
crustacean water flea D. pulex to date, but the
discovery of JHAMT in N. denticulate.
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9. Amino acid sequence identity for JHAMT of
N. denticulata and other arthropods
Figure S1 Amino acid sequence identity for JHAMT
Figure S25 Phylogenetic tree of JHAMT amino acid sequences from N.
denticulata and other arthropods, based on the 50% majority rule tree from the
Bayesian analysis.
Hence, JHAMT is more generally present across
crustaceans and may play an important role in the
biosynthesis of Methyl farnesote (MF) in crustaceans.
11/25/2014 of N. denticulata and other arthropods. 9

10. Expression of JHAMT in N. denticulate embryos,
juveniles and adults
Figure S3 Amino acid sequence identity for juvenile
hormone (JH) binding protein (JHBP) of N. denticulata
and other arthropods, which has been proposed to be
the cytoplasmic receptor of JH in insects.
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Expression of JHAMT in N. denticulate embryos, juveniles and adults.
Expression of JHAMT in embryonic, juvenile and adult N. denticulatasamples as
noted.
JHAMT in the sesquiterpenoid biosynthetic pathway of crustaceans and insects indicates the JH biosynthetic pathway found in
insects may have already been present in the Pancrustacean as shown.
The JHAMT gene in embryonic, juvenile and adult samples, showing at least weak transcription is ubiquitous across the body of
this species, albeit very weak in posterior regions of juveniles and adults.
This suggests that JHAMT may be active around the body.

11. Juvenile hormone pathway overview
(degradation). Genes identified in Neocaridina
denticulata are shown in boxes with darker
coloration (i.e. JHAMT, JHBP, JHEH, JHE and
JHEBP).
Juvenoid hormone degradation
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12. Amino acid sequence identity for JHE/
JHEBP/JHEH
Figure S2 Amino acid sequence identity for JHE of N. denticulata and
other arthropods.
Figure S4 Amino acid sequence identity for JHEBP of N. denticulata and
other arthropods.
Figure S5 Amino acid sequence identity for JHEH of N. denticulata and other
arthropods.
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13. Juvenoid hormone regulation
Figure S6 Amino acid sequence identity for type A allatostatin of N.
denticulata and other arthropods.
Allatostatins (ASTs) are well known neuropeptides that inhibit
the biosynthesis of juvenile hormones (JH) by the corpora allata
(CA) in insects, and several allatostatin-like peptides were
identified in the N. denticulate genome.
In insects, there are three major types of ASTs: A-type, muscle-inhibiting
peptide (MIP) type, and C-type.
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14. Juvenoid hormone regulation
Figure S9 Amino acid sequence identity for allatostatin receptors of N. denticulata and
other arthropods.
Figure S7 Amino acid sequence identity for muscle-inhibiting peptide (MIP) of N.
denticulata and other arthropods.
The MIP identified in N. denticulate also possesses amino acid sequence with
tryptophan in the second and ninth positions.
Figure S8 Amino acid sequence identity for type C allatostatin of N. denticulata and
other arthropods.
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15. Ecdysteroid production
Ecdysteroid synthesis
The steroidogenic CYP enzymes mediating steps in the conversion of cholesterol to
ecdysone in the crustacean molting gland (Y-organ), and the conversion of ecdysone to
20-hydroxyecdysone (20E) in the peripheral tissues.
The terminal hydroxylations at carbons are catalyzed by enzymes encoded by Spook,
Phantom, Disembodied, Shadow, and Shade.
A central tenet of insect biology is that ecdysteroids mediate larval-larval molts in the
presence of the sesquiterpenoid, JH.
In the absence of JH, by contrast, ecdysteroid induces a larval-pupal transition.
Ecdysteroid 20E regulates insect development and reproduction.
Molting is triggered by ecdysteroid, which is secreted from a pair of Y-organs. The Y-organ
is normally degenerates after puberty, and no subsequent molts occur.
Teruaki Nakatsuji, et al., General and Comparative Endocrinology,135,3,2004,358-364
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16. Transductory cascade of Prothoracicotropic Hormone (PTTH)
interaction with a prothoracic gland cell. S6*, multiple
phosphorylated form.
Halloween genes encode P450 enzymes that mediate steroid
hormone biosynthesis in Drosophila melanogaster
Halloween gene disembodied, plays a critical role in
ecdysteroidogenesis
Molecular and Cellular Endocrinology, Volume 215, Issues 1–2, 2004, 1 - 10
Abbreviated scheme of ecdysteroidogenesis.
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17. Ecdysteroid pathway genes
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18. Amino acid sequence identity for
spook/phantom
Figure S18 Amino acid sequence identity for spook
(CYP307A1) of N. denticulata and other arthropods.
Figure S19 Amino acid sequence identity for phantom
(CYP306A1) of N. denticulata and other arthropods.
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19. Figure S20 Amino acid sequence identity for disembodied
(CYP302A1) of N. denticulata and other arthropods.
11/25/2014 Figure S21 Amino acid sequence identity for shadow 19
(CYP315A1) of N. denticulata and other arthropods.
Amino acid sequence identity for
disembodied/shadow

20. Figure S22 Amino acid sequence identity for Cytochrome P450-18a1
(CYP18A1) of N. denticulata and other arthropods.
Figure S24 Phylogenetic tree of CYP (cytochrome P450) amino acid sequences from N.
denticulata and other arthropods, based on the 50% majority rule tree from the Bayesian
analysis. The substitution model selected was Blosum, with 99.7% posterior 20
probability.
Bayesian posterior probabilities above 50% are shown above the branches.
Amino acid sequence identity for Cytochrome
P450-8A1/ Cytochrome P450

21. Regulators of both juvenoid hormones and
ecdysteroids
Figure S10 Amino acid sequence identity for
Methoprene-tolerant (Met) of N. denticulata and
other arthropods.
Met can also interact with Ecdysone receptor
(EcR) and retinoid X receptor (RXR) in their
binding to ecdysone response elements, so 20E could
drive molting and metamorphosis.
The action of ecdysteroids is mediated through a
heterodimer of two members of the nuclear receptor
family, the EcR and Ultraspiracle (Usp).
The USP protein is an ortholog of the RXR.
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22. Regulators of both juvenoid hormones and
ecdysteroids
Figure S11 Amino acid sequence identity for EcR of N. denticulata and other
arthropods.
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23. Regulators of both juvenoid hormones and
ecdysteroids
Figure S12 Amino acid sequence identity for RXR of N. denticulata and other arthropods.
Figure S14 Amino acid sequence identity for Broad-complex of N.
denticulata and other arthropods.
Broad expression eventually disappears during the early pupal stage
coinciding with the onset of adult cuticular synthesis. The role of Broad
was further defined by applying sufficient JH to pupae to elicit the
formation of a second pupal cuticle.
Broad’s presence in the cell appears to inhibit larval cuticular
synthesis, while permitting pupal cuticular synthesis to proceed.
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24. Regulators of both juvenoid hormones and
ecdysteroids
Figure S15 Amino acid sequence identity for Chd64 of N. denticulata and other arthropods.
Chd64 and FKBP39 could mediate the action of MF-Met on the MF response element
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25. Regulators of both juvenoid hormones and
ecdysteroids
Figure S16 Amino acid sequence identity for FKBP39 of N. denticulata and FKBP39, FKBP46
and FK506 of other arthropods.
Figure S17 Amino acid
sequence identity for
CHH/MIH/GIH of N.
denticulata and other
arthropods. They are
functions for potential
regulator of MF.
Figure S23 Phylogenetic tree of Crustacean hyperglycemic hormone/molt-inhibiting hormone/gonad-inhibiting
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hormone (CHH/MIH/GIH) amino acid sequences from N. denticulata and other arthropods.

26. Conclusions
• This study has been suggested that the complete juvenile hormone (JH) and
ecdysteroid pathways were present in the insect-crustacean common ancestor,
and these regulatory systems could possibly also date back to the arthropod
ancestor.
• However, many questions remain, could the JH and ecdysteroid systems have
evolved concurrently with the emergence of the exoskeleton in arthropod
radiation?
• How are these different pathways components regulated in vivo in different
arthropods?
• The investigations of their regulation and functions in different arthropods,
including the crustaceans, would be of immense importance for research on
aquaculture, development, endocrinology.
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27. Thank you for
your attention
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