Integrative and Comparative Biology

21 Pages·2015·0.82 MB·English

Integrative and Comparative Biology Advance Access published June 30, 2015
Integrative and Comparative Biology
IntegrativeandComparativeBiology,pp.1–21
doi:10.1093/icb/icv080 Society forIntegrative and Comparative Biology
SYMPOSIUM
Exoskeletons across the Pancrustacea: Comparative Morphology,
Physiology, Biochemistry and Genetics
Robert Roer,1,* Shai Abehsera† and Amir Sagi†
*Department of Biology and Marine Biology, University of NC Wilmington, 601 S. College Road, Wilmington, NC
28403-5915, USA; †Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion
University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel D
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From the symposium ‘‘Linking Insects with Crustacea: Comparative Physiology of the Pancrustacea’’ presented at the lo
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annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2015 at West Palm Beach, Florida. de
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1E-mail: [email protected] fro
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Synopsis The exoskeletons of pancrustaceans, as typified by decapod crustaceans and insects, demonstrate a high degree b.o
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of similarity with respect to histology, ultrastructure, function, and composition. The cuticular envelope in insects and fo
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the outer epicuticle in crustaceans both serve as the primary barrier to permeability of the exoskeleton, preventing loss of jo
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water and ions to the external medium. Prior to and following ecdysis, there is a sequence of expression and synthesis of rn
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different proteins by the cuticular epithelium for incorporation into the pre-exuvial and post-exuvial procuticle of insects ls.o
and the exocuticle and endocuticle of crustaceans. Both exhibit regional differences in cuticular composition, e.g., the arg/
articular (intersegmental) membranes of insects and the arthrodial (joint) membranes of crustaceans. The primary t B
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difference between these cuticles is the ability to mineralize. Crustaceans’ cuticles express a unique suite of proteins n G
that provide for the nucleation and deposition of calcium carbonate. Orthologs of genes discussed in the present review urio
were mined from a recently completed cuticular transcriptome of the crayfish, Cherax quadricarinatus, providing new n U
insights into the nature of these proteins. niv
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Introduction the literature are further discussed in light of a case - A
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One of the defining characteristics of the Arthropoda study of a molt-related transcriptomic library re- nne
cently established for a crayfish (Abehsera et al. L
is the possession of a rigid exoskeleton comprised of ib
2015). This library originates from different exoskel- ra
chitin and protein as its principal organic compo- ry
etal-forming epithelia at four distinctive molt stages o
nents. Being enclosed in such a rigid exoskeleton n
thus is representative of the heavily calcified crusta- Ju
requires that all arthropods must undergo a molt ly
cean cuticle as opposed to the non-mineralized insect 8
or ecdysis in order to metamorphose and grow. , 2
cuticle. 0
Thus, many of the features of the exoskeletons of 15
the Pancrustacea are common across modern taxa
Morphology
and presumably predate the divergence of the
Malacostraca and Hexapoda. Others, such as calcifi- While the morphologies of the decapod and hexapod
cation, are nearly ubiquitous in the non-hexapod exoskeletons are very similar at the level of the light
Pancrustacea and extremely rare in the Hexapoda. microscope (Fig. 1) and electron microscope (Fig. 2),
The similarities are reflected in the morphology of the nomenclatures that describe them, and which are
the cuticular layers, their deposition and sclerotiza- now well-accepted, differ. Both possess an outer layer
tion, and common motifs in their structural proteins. comprised of two distinct regions. These are desig-
Differences are apparent in proteins involved in min- nated as the outer and inner epicuticles in the deca-
eralization and, perhaps, in the timing of biochemi- pods (Compe`re 1995; Dillaman et al. 2013) and the
cal changes in the cuticle following ecdysis. In this envelope and epicuticle in the hexapods (Locke
review the above similarities and differences found in 2001). In the decapods, the outer epicuticle is
(cid:2)TheAuthor2015.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforIntegrativeandComparativeBiology.Allrightsreserved.
For permissions please email: [email protected].
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Fig. 1 Epifluorescent light micrographs of the exoskeletons of the dorsal carapace of the blue crab, Callinectes sapidus (left), and the n
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pronotumofthefieldcricket,Grylluspennsylvanicus(right).Thecuticlewasfixedinalcoholicformalinandstainedwithacridineorange. a
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Notethatinbothtissues,theacridineorangedifferentiatestheexocuticle(Ex)fromtheendocuticle(En).Theepicuticle(visibleinthe d
crab’s exoskeleton, arrowhead) is autofluorescent. The image of the blue crab’s cuticle modified from Marlowe and Dillaman (1995) fro
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with permission from the publisher. h
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bilaminate, displaying an outer surface coat and an (Andersen 2010). The secretion and the initiation fo
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inner cuticulin layer that has five distinguishable of sclerotization appear to be under the control of jo
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sublayers. The hexapod envelope is trilaminate in the hormone bursicon in both taxa (Luo et al. 2005; rna
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appearance (Locke 2001). In both taxa, the outer Wilcockson and Webster 2008); however, the mech- .o
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regions lack chitin and are composed largely of anism of transport of compounds into the pre- a/
lipid and protein. These outer regions are important exuvial layers is unknown. t B
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as barriers to permeability (see Hadley [1994] for Unlike most hexapods, decapods commonly im- G
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review), and as protection from abrasion and infec- pregnate the largest part of their epicuticles, exocu- rio
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tion. The establishment of the permeability barrier ticles, and endocuticles with calcium carbonate, U
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relative to the molt cycle is discussed below. either in the form of calcite or amorphous calcium iv
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The exocuticle and endocuticle of the decapods carbonate (Dillaman et al. [2013] for review). Even rsity
are collectively designated as the procuticle in hexa- in the heavily mineralized decapods, certain cuticu- - A
pods (Fig. 3) (Roer and Dillaman 1984; Locke 2001). larized regions remain uncalcified, notably the ra
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In both taxa they have an organic matrix that is arthrodial membranes at the joints of appendages, e L
principally formed from chitin and protein microfi- the lining of the branchial cavity, the gills, and por- ibra
bers that are laid down in parallel sheets. Each sheet tions of the foregut and hindgut. It is also vital that ry
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is slightly offset in its orientation from the one above the pre-exuvial layers that are destined to mineralize J
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it, forming a helicoidal arrangement first described do not do so until after ecdysis. The control over ly
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by Bouligand (1965, 1972) for the decapod exoskel- which areas mineralize and when that occurs resides , 2
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eton. In cross-section, this arrangement gives rise to in the composition of the cuticular proteins (CPs) 5
the lamellate appearance of these cuticular layers, and glycoproteins, and in their postecdysial alter-
such that each lamella represents a rotation of 1808 ations, as is discussed below (Roer and Dillaman
in the orientation of the fibers (Fig. 4). The exocu- 1984, 1993; Shafer et al. 2006; Dillaman et al.
ticle of decapods and the outer portion of the pro- [2013] for review).
cuticle in hexapods (along with the epicuticles and There are a few examples of hexapods in which
envelope) are deposited prior to ecdysis and are, regions of the cuticle mineralize, at least during some
therefore, called pre-exuvial layers. Subsequent to ec- developmental stages. While not strictly an example
dysis, both the pre-exuvial exocuticle and the procu- of calcification, the tips of ovipositors of the parasitic
ticle are hardened by quinone crosslinking or wasps Gabunia sp. (Ichneumonidae: Cryptinae) from
sclerotization. This process entails the active secre- Uganda and the cosmopolitan Heterospilus prosopidis
tion of acyldopamines into the cuticle from the un- (Braconidae: Doryctinae) display high concentrations
derlying epithelium and the conversion of these both of calcium and of manganese in their cuticles
compounds to quinones by phenyloxidase (Quicke et al. 2004). The face fly, Musca autumnalis
ExoskeletonsacrossthePancrustacea 3
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Fig. 2 Scanning electron micrographs of the exoskeletons of the pronotum of the field cricket, Gryllus pennsylvanicus (A, C) and the .o
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dorsalcarapaceofthebluecrab,Callinectessapidus(B,D).Notethatbothtissuesdisplaylamellae(A,B)thatarecomprisedofparallel fo
sheets of chitin–protein fibrils whose orientation changes between layers (C, D). Pore canals (PC) are evident throughout the rdjo
exocuticles and endocuticles in the crab (D), but are not apparent in those of the insect (C). urn
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and house fly, M. domestica (Diptera: Muscidae), the decapod exoskeleton, thereby forming pore ca- a/
produce a calcified puparium (Fraenkel and Hsiao nals. In the dorsal carapace of the green crab, t B
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1967; Gilby and McKellar 1976; Grodowitz et al. Carcinus maenas, the diameter of the typical pore G
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1987). The closest homolog to the mineralized deca- canal is approximately 0.4mm and there are nearly rio
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pod exoskeleton is found in certain members of the 950,000 pore canals/mm2 of epithelial surface (Roer U
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Coleoptera. Within the family Tenebrionidae, the 1980). Pore canals are not universally present in the iv
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subfamily Phrenapetinae contains a number of cuticle of decapods; they are conspicuously absent in rsity
genera in which the cuticle is impregnated with cal- the cuticle of gills, for example (Dickson et al. 1991). - A
cium carbonate (Leschen and Cutler 1994) (Fig. 5). While pore canals do occur in hexapods, they are not ran
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Although some analysis of the amino-acid composi- always apparent, even in the pronotum (Fig. 2), a e L
tion of the organic matrix of the puparium was per- region that would correspond to the dorsal carapace ibra
formed (Bodnaryk 1972), no comparison of the of decapods (Fig. 2). Decapods’ pore canals are likely ry o
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proteins expressed in the mineralized hexapod cuticle involved in the post-ecdysial mineralization of the J
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to those of the decapods has been made. pre-exuvial layers (Roer 1980), and probably contrib- ly 8
Beneath the decapod endocuticle lies the membra- ute to the transport of enzymes and aclydopamines , 2
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nous layer. This layer resembles the endocuticle in its as well. Their role in the hexapods and the mode of 5
lamellate structure, but remains uncalcified. There is transport in regions that lack pore canals needs to be
no similar structure described in the literature on investigated.
hexapods. It may be that the function of the mem-
branous layer is to limit the extent of mineralization
Resorption and deposition during the
of the innermost endocuticle. As such, it would not
molt cycle
be expected to be deposited in most hexapods. An
investigation of whether or not a membranous layer The dynamics of cuticular resorption and deposition
is present in the calcified cuticles of beetles would in relation to the molt cycle of decapods and hexa-
provide further evidence of its potential role in this pods have been extensively reviewed (Roer and
regard. Dillaman 1993; Moussian 2010). There are a
The epithelium that underlies the cuticle bears nu- number of phenomena that emerge when comparing
merous microvilli that extend up through the exocu- these two taxa, however, that bear further scrutiny
ticle and into the inner epicuticle in most regions of and future research.
4 R.Roeretal.
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Fig. 3 Schematic representation of the pancrustacean exoskeletal structure and its changes throughout the molt cycle. The nomen- - A
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claturefor theintermoltcuticleofdecapodsisshowninA,whilethatforhexapodsisshowninF.Theouterepicuticleofthedecapod n
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correspondstotheenvelopeinthehexapods,andthedecapod’sinnerepicuticleiscalledtheepicuticleinthehexapods.Thedecapods’ L
exocuticle and endocuticle are the procuticle in the hexapods. The onset of pre-molt in both taxa is markedbythe separation of the ibra
epithelial layer from the overlying old cuticle (B). During late pre-molt, the pre-exuvial layers (the new epicuticle and exocuticle in ry
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decapods;theenvelope,epicuticle,andpre-exuvial procuticleinhexapods)aredeposited beneaththeoldcuticle(C).Ecdysis(D)and n J
post-moltdepositionoftheendocuticle(decapods)andpost-exuvialprocuticle(hexapods)continues(E,F)untiltheexoskeletonisfully uly
formed in intermolt (A). Modified from Dillaman et al. (2013). 8, 2
0
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During premolt, there is the simultaneous enzy- suggested that the envelope becomes impermeable
matic digestion of the old exoskeleton (or puparium as soon as it is fully synthesized by the epithelium.
in hexapods) and deposition of the pre-exuvial layers If that is the case, the envelope would isolate the
of the new exoskeleton (Fig. 3). Degradation of the newly synthesized chitin–protein matrix from the
old cuticle is accomplished by the secretion of chit- molting space and the enzymes that act on the old
inases, chitobiases, and proteases into the molting exoskeleton. However, an impermeable envelope
space. This poses a potential problem since the com- would prevent the resorption of the products of
ponents of the new and old cuticle are chemically the breakdown of the old cuticle through the new
identical and, therefore, the newly synthesized cuticle cuticle and epithelium into the hemolymph. Indeed,
might be susceptible to degradation by the enzymes Cornell and Pan (1983) and Yarema et al. (2000)
digesting the old exoskeleton. provided evidence that the molting fluid containing
It has been proposed that the new cuticle in hexa- breakdown products (e.g., glutamate) are not ab-
pods is protected by the envelope. Locke (2001) sorbed across the integument, but passed either
ExoskeletonsacrossthePancrustacea 5
mineral deposition in Callinectes does not appear
before 2h postmolt (Dillaman et al. 2005).
Thus, in the decapods and in at least some regions
of the hexapods, the new cuticle may be exposed to
the enzymes that are actively digesting the old cuti-
cle. Data from the literature on hexapods suggest
that protection of the new cuticular chitin–protein
fibrils may be afforded by a highly conserved protein
(Knickkopf) that is incorporated into the newly syn-
thesized cuticle by the underlying epithelium
(Chaudhari et al. 2011). The Knickkopf protein was
initially implicated in cuticular synthesis in
Drosophila melanogaster, where knk deletion mutants
D
exhibited cuticular defects (Ostrowski et al. 2002). o
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First, Chaudhari et al. (2011) established that, in n
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fact, chitinases co-located with chitin in the newly ad
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deposited cuticle of the red flour beetle, Tribolium fro
castaneum. This observation suggested that the enve- m
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lope may not provide protection to the new cuticle ttp
from the degradative enzymes in the molting fluid/ ://ic
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space. They then identified a T. castaneum ortholog .o
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(TcKnk) of the D. melanogaster gene, and determined ford
that dsRNA-mediated knockdown of TcKnk resulted jo
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Fig. 4 The changing orientation of chitin–protein fibrils in the in lethal defects in molting in all stages of larval, rna
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cuticle. A 1808 rotation in the orientation of the layers (L) pro- pupal, and adult development. Quantitative chemical .o
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duces a lamella within the cuticle. From Bouligand (1972) with analyses and immunohistochemistry showed that a/
permission from the publisher. RNAi of TcKnk resulted in near-total loss of cuticu- t Be
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lar chitin in the newly secreted cuticle. The loss of G
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chitin was due to the action of secreted chitinases, as rio
anteriorly or posteriorly and ingested via the mouth n
simultaneous knockdown of two chitinases genes U
or anus. However, Lensky et al. (1970), using Buffalo n
(TcCht-5 and TcCht-10) restored the chitin to con- iv
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Babladcokmdenye,ofdeCmecornosptiraatwedasthreastormbeodltinthgrofulugihd pinitsthine tlorocallliezveeslsw(iCthhacuhdithinariinetthael.n2e0w11ly).sFyinntahlelys,izTedcKcnukticcloe-, rsity - A
the underlying new envelope. The pits were associ- but is absent from the old cuticle that is being ran
ated with tonofibrillar attachments and became im- degraded. ne L
permeable to dye after the moth emerged from the Knickkopf protein orthologs were subsequently ibra
puparium. found to be present in most taxa with chitin in ry o
In the decapods, it is clear that the pre-exuvial n
their extracellular matrices, except fungi, and includ- J
cuticle is permeable and is the site of resorption, at ing one non-hexapod pancrustacean, Daphnia pulex. uly
least of Ca2þ that is released from the old cuticle Here we report the first evidence of a knickkopf pro- 8, 2
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(Roer 1980). Williams et al. (2009) demonstrated 1
tein ortholog in the Malacostraca. In the transcrip- 5
that the pre-exuvial cuticle of the blue crab,
tomic library of the crayfish (Cherax quadricarinatus)
Callinectes sapidus, remained permeable to water
that we studied, a knickkopf ortholog showing a high
and p-nitrophenylphosphate (used as a tracer) until
similarity to known hexapod knickkopf proteins was
just after molting (Fig. 6). Permeability decreased found, and named Cq knickkopf protein
markedly within the first 15 min postmolt, and was (KR025533). It is interesting to note that the expres-
entirely impermeable by 1h after ecdysis. The change sion pattern throughout the molt cycle of this knick-
in permeability was associated with an alteration in kopf ortholog (Fig. 8) is similar to the previously
the structure of the outer epicuticle (Fig. 7), and described expression pattern of genes related to
transmission electron microscopy, using La3þ as a chitin metabolism (Abehsera et al. 2015). In the cu-
marker, showed that the outer epicuticle was ticle-forming epithelium the expression pattern is
indeed the barrier to permeability. The change in molt-independent, being expressed through the
permeability precedes the initiation of postmolt min- entire molt cycle with no differences. In the
eralization in the epicuticle. The first evidence of gastrolith-forming epithelium the expression pattern
6 R.Roeretal.
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Fbirgio.n5idSbceanentliensg. eTlheectrcounticmleicorfogTrraibpohlisumancdonefnuesurgmy-(dAis)pesrhsoivwesannoalyesvisideonfcXe-roafym(EinDeAraXliz)astpioenc,trcaonoffirtmheedcubtyicaleslacokf towfoasCpaecpieesakofintetnhee- ://icb
EDAX spectrum (B, full scale¼800 counts). The cuticle of Zypoetes epieroides (C) shows a dense cross-section characteristic of .ox
mineralized cuticle, confirmed by a pronounced Ca peak in the EDAX spectrum (D, full scale¼10,000 counts). From Leschen and ford
Cutler (1994); the labels for the Ca peaks have been enhanced. jo
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Fig. 7 Transmissionelectronmicrographs oftheepicuticle ofthe L
dorsal carapace of the blue crab, Callinectes sapidus. Note the ibra
change in the appearance of the outer epicuticle (arrows) be- ry
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tweenanewly moltedcrab (0m)and one2hafter ecdysis (2h). n
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The outerepicuticle changes from an amorphous morphology to uly
atrilaminatestructure.Thechangescorrespondtotheformation 8, 2
of a permeability barrier in this layer. Modified from Williams 0
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(2000).
Fig. 6 Change in cuticular permeability between late pre-molt
(stages D –D ) and times immediately following ecdysis in the
2 4
blue crab, Callinectes sapidus. Permeability was assessed in vitro
using p-nitrophenol as a marker. Note the pronounced decrease
exoskeleton, referred to as apolysis. It is generally
in cuticular permeability upon ecdysis. From Williams et al.
(2009) with permission from the publisher. agreed that the formation of the new envelope and
epicuticle in the hexapods and the epicuticle in the
decapods occurs by self-assembly after components
is molt-related, being highly expressed during pre- are secretedfrom vesiclesof the underlying epithelium
molt. The search for knickkopf orthologs among (Leopold et al. 1992; Compe`re 1995; Locke 1998).
other taxa of the Pancrustacea should be a focus of While some question remains regarding the role of
future investigation. epithelial plaques, electron-dense regions at the apex
Deposition of the new cuticle during premolt fol- of epithelial microvilli, in the organization of the epi-
lows the separation of the epithelium from the old cuticular layers. The absence of plaques in regions of
ExoskeletonsacrossthePancrustacea 7
Fig.8 Normalizedreadcountofcqknickkopfproteintranscript.Normalizedreadcountfromthecuticle-formingepithelium(left)and
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thegastrolith-formingepithelium(right).TheX-axisrepresentsthefourmoltstages:inter-molt,earlypre-molt,latepre-molt,andpost- o
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molt. Letters represent statistical groups that are significantly different (P50.05). Error bars represent standard error. n
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the cuticle in the green crab, C. maenas, and the fact form a highly ordered assembly in an extracellular m
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that full assembly of the outer epicuticle occurs at environment could have significant fundamental bi- ttp
some distance from the epithelium preclude direct ological, as well as commercial, implications. ://ic
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organization of the layers by the epithelial microvilli Both in decapods and hexapods, a surface layer is .o
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in decapods (Compe`re 1995). deposited outside of the envelope or outer epicuticle. fo
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Following the formation of the epicuticle, the ep- This is the wax layer in insects (Locke 1998) and is jo
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ithelial cells begin to deposit the pre-exuvial layers of referred to as a surface coat in decapods (Compe`re rna
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the procuticle (hexapods) and exocuticle (decapods). 1995). Both appear to be formed by secretions of .o
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In both cases, the components of the chitin–protein specialized cells or glands that communicate with a/
fibrils are secreted at the surface of epithelial micro- the epicuticle via ducts or canals. While the wax t B
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villi and are associated with the microvillar plaques layer is secreted postmolt, the surface coat of G
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(Leopold et al. 1992; Locke 1998; Dillaman et al. Carcinus is formed prior to ecdysis, during late rio
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2013) (Fig. 9). In Drosophila, Moussian (2012) stage D , after the pre-exuvial exocuticle has been U
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showed that these plaques are located on the apical completed (Compe`re 1995). The role of the wax iv
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ridges of epithelial undulae (rather than on micro- layer in waterproofing and protection is well- rsity
villi). The plaques are associated with membrane- established (Hadley 1994), but the function of deca- - A
bound chitin synthase, and he hypothesized that pods’ surface coat is as yet unknown. ran
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the chitin fibrils are secreted from the peaks of the e L
undulae, while the CPs are secreted in the valleys ib
Comparison of cuticular structural ra
(Moussian 2012). Moussian also ascribed a role of ry
proteins o
the undulae in orienting the fibrils. Locke (1998) also n J
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hypothesized that the apical microvilli controlled the Numerous studies have identified and characterized ly
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orientation of the chitin–protein fibrils as they were CPs both from hexapods (Andersen 1988b, 2000; He , 2
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being deposited. However, micrographs both from et al. 2007; Charles 2010; Dittmer et al. 2012; Willis 15
decapods (Callinectes, Greenaway et al. 1995; et al. 2012; Noh et al. 2014) and from decapods
Dillaman et al. 2013) and hexapods (Anthonomus, (Andersen 1988a, 1999; Endo et al. 2000, 2004;
Leopold et al. 1992) show a pronounced assembly Watanabe et al. 2000, 2006; Inoue et al. 2001,
or polymerization zone in which the secreted mate- 2003, 2004; Wynn and Shafer 2005; Shafer et al.
rial is clearly unorganized at a distance from the 2006; Faircloth and Shafer 2007; Kuballa and Elizur
apical surface of the epithelial cells (Fig. 9). These 2008; Kuballa et al. 2011; Ma et al. 2013; Suzuki
data suggest that the chitin–protein fibrils self- et al. 2013; Tom et al. 2014; including a mini-
assemble and self-orient. Data from a number of review of known gastrolith proteins, Glazer and
researchers using material from insect cuticle have Sagi 2012). However, only a few of these have deter-
demonstrated that chitin fibrils will self-assemble mined the expression patterns of these proteins rel-
into a helicoidal arrangement (see Neville [1998] ative to the molt (Togawa et al. 2008) and to the
for review). This is an important area for future re- type of cuticle in which they are expressed. In a
search. The ability of complex macromolecules to recently studied transcriptomic library of cuticular
8 R.Roeretal.
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Fig. 9 Transmission electron micrographs of the epithelial–cuticular interfaces of insects (A, C) and crabs (B, D) during cuticular ://ic
deposition. (A) Pre-exuvial deposition of the fifth larval procuticle of Calpodes ethlius showing apical microvilli depositing elements b
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of the lamellar cuticle (Lc) presumably supplied by coated vesicles (cv). From Locke (1998) with permission from the publisher. xfo
(B) Pre-exuvial deposition of the exocuticle of Callinectes sapidus showing similar apical microvilli at the site of deposition along with rd
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cytoplasmic extensions (pore canals, arrows) and abundant microtubules within the epithelial cells (arrowheads). From Dillaman et al. u
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(2013). (C) Higher magnification of a pre-exuvial adult insect (Anthonomus grandis) showing apical microvilli secreting cuticular com- a
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ponents (arrowhead) into the assembly zone (AZ). Cytoplasmic extensions (pore canals, PC) are also evident. From Leopold et al. .o
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(1992) with permission from the publisher. (D) Post-molt deposition of the endocuticle in Callinectes sapidus showing the polymeri- a/
zation (¼assembly) zone (pz) above the apical epithelial membrane and associated vesicles (v). From Greenaway et al. (1995) with t B
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permission from the publisher. n G
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elements of the crayfish C. quadricarinatus, the ex- cuticle and pre-molt versus post-molt expression iv
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pression pattern through the molt cycle of each of (Table 1). There are no proteins that have been iden- rsity
the above genes can be visualized, thereby providing tified for insects that are exclusively expressed during - A
a resourceful tool (Abehsera et al. 2015) that is used post-molt in soft cuticle. However, three proteins are ra
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throughout the present review to identify and evalu- expressed in pre-molt hard cuticle and eight in soft e L
ate crustaceans’ orthologs. cuticle; 20 proteins are expressed post-molt in hard ibra
In order to fully understand the structural and cuticle. For the decapods, seven proteins are ex- ry
o
n
functional similarities and differences between hexa- pressed prior to ecdysis, four in arthrodial cuticle, J
u
pod and decapod CPs, it is important to know if and three in calcifying cuticle. Post-molt, seven are ly
8
they are expressed prior to ecdysis and, therefore, expressed in arthrodial cuticle and nine in calcifying , 2
0
1
incorporated into the pre-exuvial exoskeleton, or cuticle. Some of these (as discussed further below) 5
post-ecdysis and thus be components of the post- are expressed both pre-molt and post-molt, and one
exuvial procuticle of insects or the endocuticle of both in arthrodial and calcifying cuticle. In addition,
malacostracans. It is also important to know if the orthologs of each protein were searched in the re-
proteins are expressed in hard (heavily-sclerotized cently established molt-related transcriptomic library
and/or mineralized) or soft (flexible) cuticle. While originating from the hard exoskeleton-forming epi-
a number of studies satisfy a subset of these criteria thelia of a decapod (Abehsera et al. 2015). The cri-
(e.g., Togawa et al. 2008; Charles 2010; Tom et al. teria utilized for ortholog discovery are listed in
2014), this section focuses on those CPs for which Supplementary Table S1. The temporal expression
these expression patterns are known, and for which was examined for each of the orthologs found. A
there is enough sequence known to give an indica- higher degree of conservation was found among pro-
tion of function. teins originating from the soft cuticle both for hexa-
The proteins have been sorted into eight bins, four pods and decapods compared with our studied
each for hexapods and decapods: hard versus soft decapod (Table 1). These findings suggest that the
ExoskeletonsacrossthePancrustacea 9
Degreeofhomology Low Low continued)
(
dricarinatusmeetal.2015) Homologexpression Postmolt Postmolt
Cheraxquatranscripto(Abehsera Homologexistence None None None None None None None None None None Exists Exists None None None None None None None None
2) 2) 2) 2) 2) 2)
1 1 1 1 1 1
Citation Nohetal.(2014) Nohetal.(2014) Dittmeretal.(20 Dittmeretal.(20 Dittmeretal.(20 Dittmeretal.(20 Dittmeretal.(20 Dittmeretal.(20 Andersen(2000) Andersen(2000) Andersen(2000) Andersen(2000) Andersen(2000) Andersen(2000) Andersen(2000) Andersen(2000) Andersen(2000) Andersen(2000) Andersen(2000) Andersen(2000) Down
onnumber 13987 13988 14462 08228 11338 15908 loaded from
Accessi GLEAN GLEAN GLEAN GLEAN GLEAN GLEAN P82122 P82118 P82119 P82120 P82121 P82168 P82169 P82170 P82171 P82165 P82166 P82167 http://ic
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podaandDecapoda Postmolt ProteinFunction TcasCPR33—RR-2Chitin-bindingstructural TcasCPR34—RR-2Chitin-bindingstructural Glu,Arg,HisrichUnknown—lowcomplexity SimilartoTmACP17Unknown—Glycine-rich TcasCPR101—RR-2Chitin-bindingstructural TcasCPR87—RR-2Chitin-bindingstructural BcNCP3.8 BcNCP14.6—Postmolt-18 BcNCP14.9—RR-3,Chitin-bindingPostmolt-18structural BcNCP15.0—RR-3,Chitin-bindingPostmolt-18structural BcNCP21.1—RR-2Chitin-bindingstructural LmNCP4.9 LmNCP5.1 LmNCP6.4 LmNCP9.5 LmNCP18.7—Postmolt-18 LmNCP19.8—RR-2Chitin-bindingstructural LmNCP21.3 at Ben Gurion Universityrdjournals.org/
exa - A
moltstageamongtheH AccessionFunctionnumber Chitin-bindingXP_967633structural Chitin-bindingXP_971678structural ranne Library on July 8, 20
d 15
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a
s
e 2 2
ci R- R-
e R R
p — —
s
NAby Premolt Protein TcCPR18 TcCPR27
R
m
P m
xpressionofC Species Triboliumcastaneu (redflourbeetle) Blaberuscraniifer (cockroach) Locustamigratoria (locust)
E e
1 oda uticl
e p c
Tabl Hexa Hard
10 R.Roeretal.
Degreeofhomology Low Low Low Medium High Low Low High Low Low High continued)
(
dricarinatusmeetal.2015) Homologexpression Premolt Premolt Premolt Premolt PremoltandPostmolt Premolt Premolt Premolt Premolt ostmoltP Postmolt
Cheraxquatranscripto(Abehsera Homologexistence None None Exists Exists Exists Exists None Exists Exists Exists Exists Exists Exists Exists None
6)
Citation Accessionnumber VectorBase:Vanninietal.(2014)AGAP008446 VectorBase:Vanninietal.(2014)AGAP008447 Dittmeretal.(2012) Dittmeretal.(2012) Dittmeretal.(2012) Dittmeretal.(2012) Dittmeretal.(2012) Dittmeretal.(2012) Dittmeretal.(2012) Dittmeretal.(2012) AB103035Inoueetal.(2003) AB167814Inoueetal.(2004) Endoetal.(2004)AB114444/AB114445 AB049147Ikeyaetal.(2001) AB194409Watanabeetal.(200 http://icDownloaded from
d d d d b
Function Structural/insecticideresistance Structural/insecticideresistance Calcification-associatepeptide Calcification-associatepeptide Calcification-associateprotein Calcification-associateprotein Unknownfunction—endocuticular a.oxfordjournals.org/
t B
e
n
G
Postmolt AccessionFunctionnumberProtein EpicuticularVectorBase:AgamCPLCG3structuralAGAP004690 AgamCPLCG4 Chitin-bindingGLEAN03363structural Chitin-bindingGLEAN13127structural Chitin-bindingGLEAN03830structural Chitin-bindingGLEAN03831structural Pro-resilinGLEAN03362 CuticularproteinGLEAN11349analogoustoperitrophin CuticularproteinGLEAN00316analogoustoperitrophin Chitin-bindingGLEAN13137structural CAP-1—RR CAP-2—RR DD4—RR-like(Crustocalcin) DD5 DD1 urion University - Aranne Library on July 8, 20
1
Premolt Protein AgamCPF3 TcasCPR17—RR-2 TcasCPR81—RR-1 TcasCPR22—RR-1 TcasCPR23—RR-1 TcasCPR103—RR-2 TcasCPAP3-E—CPAPtype3 TcasCPAP1-C—CPAPtype1 TcasCPR69—RR-1 5
ontinued Species Anophelesgambiae (mosquito) Triboliumcastaneum (redflourbeetle) Procambarusclarkii (crayfish) Penaeusjaponicus (shrimp)
e1C Cuticle poda cuticle
Tabl Soft Deca Hard