Identification of the molecular components of a Tigriopus californicus (Crustacea, Copepoda) circadian clock.

Comparative Biochemistry and Physiology, Part D 12 (2014) 16–44

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Identification of the molecular components of a Tigriopus californicus (Crustacea, Copepoda) circadian clock Katherine T. Nesbit, Andrew E. Christie ⁎ Békésy Laboratory of Neurobiology, Pacific Biosciences Research Center, University of Hawaii at Manoa, 1993 East-West Road, Honolulu, HI 96822, USA

a r t i c l e

i n f o

Article history: Received 31 July 2014 Received in revised form 15 September 2014 Accepted 15 September 2014 Available online 23 September 2014 Keywords: Bioinformatics Circadian rhythm Transcriptome shotgun assembly

a b s t r a c t Copepods of the genus Tigriopus have been proposed as marine models for investigations of environmental perturbation. One rapidly increasing anthropogenic stressor for intertidal organisms is light pollution. Given the sensitivity of circadian rhythms to exogenous light, the genes/proteins of a Tigriopus circadian pacemaker represent a potential system for investigating the influences of artificial light sources on circadian behavior in an intertidal species. Here, the molecular components of a putative Tigriopus californicus circadian clock were identified using publicly accessible transcriptome data; the recently deduced circadian proteins of the copepod Calanus finmarchicus were used as a reference. Transcripts encoding homologs of all commonly recognized ancestral arthropod core clock proteins were identified (i.e. CLOCK, CRYPTOCHROME 2, CYCLE, PERIOD and TIMELESS), as were ones encoding proteins likely to modulate the core clock (i.e. CASEIN KINASE II, CLOCKWORK ORANGE, DOUBLETIME, PROTEIN PHOSPHATASE 1, PROTEIN PHOSPHATASE 2A, SHAGGY, SUPERNUMERARY LIMBS and VRILLE) or to act as inputs to it (i.e. CRYPTOCHROME 1). PAR DOMAIN PROTEIN 1 was the only circadianassociated protein not identified in Tigriopus; it appears absent in Calanus too. These data represent just the third full set of molecular components for a crustacean circadian pacemaker (Daphnia pulex and C. finmarchicus previously), and only the second obtained from transcribed sequences (C. finmarchicus previously). Given Tigriopus' proposed status as a model for investigating the influences of anthropogenic stressors in the marine environment, these data provide the first suite of gene/protein targets for understanding how light pollution may influence circadian physiology and behavior in an intertidal organism. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Increasing utilization of coastal areas has resulted in an ever-growing number of anthropogenic stressors impacting intertidal and near-shore ecosystems (e.g. Crain et al., 2009; Defeo et al., 2009; Dugan et al., 2011; Kennish et al., 2014). One of the most rapidly growing of these stressors is light pollution (e.g. Longcore and Rich, 2004; Smith, 2009; Davies et al., 2012; Gaston et al., 2012; Davies et al., 2013; Gaston et al., 2013; Bennie et al., 2014; Inger et al., 2014). Within all environments, the presence of artificial light sources can manifest in alterations in the spatial distribution of light, the timing of light vs. darkness, and/or the spectral composition of the light present in a given location (e.g. Gaston et al., 2013). Perturbations such as these can disrupt the natural day/night cycle of an organism exposed to an artificial light source, and as such have the potential to alter the physiological processes and behaviors that would naturally be cued to the solar day (e.g. Moore et al., 2000; Riley et al., 2012; Henn et al., 2014; Perkin et al., 2014). One biological network that is likely to be affected by light pollution in intertidal marine organisms is the circadian system: the interacting complex of genes and ⁎ Corresponding author. Tel.: +1 808 956 5212; fax: +1 808 956 6984. E-mail address: [email protected] (A.E. Christie).

http://dx.doi.org/10.1016/j.cbd.2014.09.002 1744-117X/© 2014 Elsevier Inc. All rights reserved.

proteins that provides the molecular pacemaker for timing physiological and behavioral processes that operate on a ~24-hour cycle and are cued to the solar day. Circadian rhythms have been documented in essentially all living organisms (e.g. Dvornyk et al., 2003; Hut and Beersma, 2011; Loudon, 2012; McClung, 2013). While there is some variation in the components that comprise their pacemakers, much of the molecular machinery responsible for the generation of circadian clocks appears to be broadly conserved across phylogeny (e.g. Pegoraro and Tauber, 2011; Tarrant and Reitzel, 2013). While many model organisms have been used to study the molecular basis of circadian rhythmicity, for example the plant Arabidopsis thaliana (e.g. Yerushalmi et al., 2011) and a variety of rodent species (e.g. Mohawk et al., 2012), the fruit fly Drosophila melanogaster, a member of the Arthropoda, has arguably the most thoroughly characterized circadian system (e.g. Williams and Sehgal, 2001; Stanewsky, 2003; Allada and Chung, 2010; Tomioka and Matsumoto, 2010; Hardin, 2011). In Drosophila, as in all organisms, the molecular machinery that comprises the circadian clock system can be divided into several functional subunits, namely the core clock, clock input pathways, and clock output pathways (Allada and Chung, 2010). The core clock, as its name implies, is responsible for time keeping (for a detailed review see Allada and

K.T. Nesbit, A.E. Christie / Comparative Biochemistry and Physiology, Part D 12 (2014) 16–44

Chung (2010)). In greatly simplified terms, the D. melanogaster core clock is initiated through the binding of a protein heterodimer consisting of CLOCK (CLK) and CYCLE (CYC) to E-box elements located in the promoter regions of the period (per) and timeless (tim) genes. This interaction, which typically occurs late in the day, activates the transcription of the per and tim, and results in the subsequent production of PERIOD (PER) and TIMELESS (TIM) proteins. PER and TIM accumulate within the neurons that form the cellular locus of the circadian clock and dimerize within the cytoplasm of these cells, an event that typically occurs during the early evening hours. At approximately midnight, the PER/TIM heterodimer is translocated from the cytoplasm to the nucleus, allowing for its binding to the CLK/CYC heterodimer, inhibiting the latter complex's activation of the per and tim genes late at night. The core clock of D. melanogaster is modulated by a number of genes/ proteins (for a detailed review see Allada and Chung (2010)). For example, kinases, e.g. DOUBLETIME (DBT) and SHAGGY (SGG), are responsible for phosphorylating a number of the core clock proteins, with ligases, including SUPERNUMERARY LIMBS (SLIMB), subsequently responsible for the degradation of the phosphorylated substrates. Moreover, the CLK/CYC heterodimer regulates other feedback loops that are involved in modulating the core clock's phase and amplitude, as well as its rhythmic output (for a detailed review see Allada and Chung (2010)), e.g. it is responsible for the transcriptional activation of the par domain protein 1 (pdp1) and vrille (vri) genes, whose protein products activate and repress the clk and cryptochrome 1 (cry1) genes, respectively; CRYPTOCHROME 1 (CRY1) protein, a blue light photoreceptor, acts as an input pathway protein, influencing the core clock by promoting the degradation of TIM. While Drosophila is clearly not a marine species, and thus not a potential model for investigating the influence of light pollution on intertidal circadian oscillators, many other members of the Arthropoda are, in particular members of the Crustacea. Many crustaceans, including ones inhabiting the intertidal zone, are known to exhibit robust circadian rhythms in physiology and behavior. For example, locomotion, feeding, molting, reproduction, hatching, larval release, color change, and vertical migration within the water column are all likely to be under circadian control in at least some crustaceans (e.g. de la Iglesia and Hsu, 2010; Farca Luna et al., 2010; Strauss and Dircksen, 2010). Interestingly, and despite the clear phenomenological evidence for circadian behavior in member of the Crustacea, little work has focused on identifying the molecular machinery responsible for establishing circadian rhythms in these animals. In fact, prior to 2011, just two crustacean circadian proteins had been identified and characterized: a homolog of CLK from the giant river prawn Macrobrachium rosenbergii (Yang et al., 2006) and a CRY from the Antarctic krill Euphausia superba (Mazzotta et al., 2010). In 2011, the first, and thus far only, genome for a crustacean was released, namely that of the cladoceran Daphnia pulex (Colbourne et al., 2011). This resource was instrumental in transforming our knowledge of crustacean circadian biology, as it, in conjunction with known sequences from Drosophila, allowed for the identification of the first complete set of clock genes/proteins from a member of the Crustacea (Tilden et al., 2011). Interestingly, and unlike Drosophila, the Daphnia circadian system includes the CRYPTOCHROME 2 gene/protein (cry2/ CRY2), which has been lost in Drosophila but is present in what are typically considered more ancestral arthropod circadian systems, e.g. ones similar to that described for the monarch butterfly Danaus plexippus (Yuan et al., 2007). In ancestral arthropod circadian systems, CRY2, which is photo-insensitive, appears to be member of the core clock, inhibiting transcription mediated by the CLOCK/CYCLE heterodimer (Yuan et al., 2007). The identification of the molecular components of the Daphnia circadian clock was followed rapidly by the identification of a circadian system from the copepod crustacean Calanus finmarchicus (Christie et al., 2013a). Here, the deduced Drosophila proteins were used as a template to mine a de novo assembled Calanus transcriptome (Lenz et al., 2014)

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for homologous transcripts/proteins. In addition, the molecular machinery that likely functions to produce the hormonal outputs from the Calanus clock system, e.g. peptide precursor proteins (Christie et al., 2013b) and amine and diffusible gas transmitter biosynthetic enzymes (Christie et al., 2014a, 2014b), were also characterized via in silico analyses. Like the circadian system of Daphnia, the putative C. finmarchicus circadian pacemaker also include CRY2, suggesting that the circadian clocks of many crustaceans may be of the ancestral type, more similar to that of Danaus than to the derived one present in Drosophila. While the molecular components of circadian systems have now been identified from two crustaceans, the animals from which they are known are not tractable ones for investigating the influence of anthropogenic stressors on marine intertidal species; D. pulex is a freshwater crustacean, and C. finmarchicus, while marine, is an offshore, deep water pelagic animal. In contrast, copepods of the genus Tigriopus, e.g. Tigriopus californicus, have been proposed as models for marine environmental genomics (e.g. Raisuddin et al., 2007); T. californicus, a species native to the west coast of North America, is an inhabitant of the high intertidal/supralittoral zones (e.g. Altermatt et al., 2012). In the study presented here, the publicly accessible transcriptome shotgun assembly (TSA) dataset for T. californicus was mined for transcripts encoding homologs of a variety of circadian proteins; known sequences from C. finmarchicus (Christie et al., 2013a) were used as the reference set. As the data that follow will show, transcripts encoding homologs of each of the commonly recognized ancestral arthropod core clock proteins were identified (i.e. CLK, CRY2, CYC, PER and TIM), as were most of those encoding proteins likely to modulate the core clock or act as inputs to this system. These data represent only the third “complete” set of molecular components for a crustacean circadian pacemaker, and are the only full set known from an intertidal marine crustacean. As T. californicus is one species that has been proposed as a model for investigating the influences of anthropogenic stressors in the intertidal marine environment, these data provide, for the first time, a set of gene/ protein targets for understanding how light pollution may influence circadian physiology and behavior in an intertidal marine organism. 2. Materials and methods 2.1. Transcriptome mining To identify Tigriopus transcripts encoding putative circadian protein homologs, known circadian sequences from C. finmarchicus (Christie et al., 2013a) were used as the input queries for BLAST searches of the publicly accessible T. californicus TSA data; this method is a wellvetted strategy that has been used previously for the identification of transcripts/genes encoding a variety of proteins, both from this and other crustacean species (Christie et al., 2008; Gard et al., 2009; Ma et al., 2009; Christie et al., 2010; Ma et al., 2010; Christie et al., 2011; McCoole et al., 2011; Tilden et al., 2011; McCoole et al., 2012a,b; Christie et al., 2013a,b,c; Christie, 2014a, 2014b, 2014c, 2014d, 2014e, 2014f; Christie et al., 2014a,b; Lenz et al., 2014). Specifically, the database of the online program tblastn (National Center for Biotechnology Information, Bethesda, MD; http://blast.ncbi.nlm.nih.gov/Blast.cgi) was set to “Transcriptome Shotgun Assembly (TSA)” and restricted to sequence data from “Tigriopus californicus (taxid:6832)”. A known C. finmarchicus protein (Christie et al., 2013a) was input into tblastn as the query sequence and the program was run using its default parameters. All hits returned by tblastn were subsequently translated using the “Translate” tool of ExPASy (http://web.expasy.org/translate/). Hits that were ultimately deemed to be positive (via sequence homology to known circadian protein isoforms and structural analysis; see Section 2.2) are shown in Table 1. This table also provides the lengths of the identified transcripts and their deduced proteins, the completeness of the deduced protein sequences (i.e. full-length or partial), as well as the BLAST-generated maximum score and E-value for significant alignment of each of the proteins deduced from the identified

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Table 1 Putative Tigriopus californicus circadian protein-encoding transcripts identified via in silico transcriptome mining. Protein family

Transcript/protein identifications

tblasn search statistics Transcript lengtha

Deduced protein lengthb

Deduced protein typec

BLAST score

E-value

Core clock transcripts/proteins CLK JW523144 JW503893 JW523143 CRY2 JW504321 JW523592 JV190442 CYC JW523145 JW503894 PER JW535312 JV196344 JV190453 JW509073 TIM JW538330 JW511710 JV197201 JW511708 JV191316

614 887 444 2862 2813 933 3679 3023 4826 4380 4386 4545 5461 2202 2358 3416 1875

68 295 148 748 752 331 693 672 1460 1460 1462 1411 1438 529 786 970 625

N I I F F I F C F F F N N N I C C

104 283 138 855 854 466 560 557 511 512 512 511 673 199 652 671 425

5e−25 3e−90 7e−38 0.0 0.0 1e−157 0.0 0.0 6e−155 3e−156 4e−156 1e−155 0.0 2e−52 0.0 0.0 3e−133

Core clock-associated transcripts/proteins CKII-α JV195708 JV189813 JW518769 JW522448 CKII-β JV195743 JV189848 JW522449 JW503280 CWO JW506198 JW525737 DBT JV195325 JV189421 JW522445 JW503277 JW522447 JV198427 JW503279

1110 1110 1562 2264 663 663 1522 1525 2751 722 1197 1197 2180 2172 3717 1890 1217

370 370 370 370 221 221 221 221 626 217 399 399 399 399 663 630 405

F F F F F F F F F N F F F F F N I

639 639 641 640 384 384 375 375 207 204 575 575 575 575 529 528 504

0.0 0.0 0.0 0.0 2e−135 2e−135 5e−128 5e−128 5e−57 1e−60 0.0 0.0 0.0 0.0 6e−178 0.0 1e−178

1020 1821 2014 441 1002 1002 2331 2657 945 945 1473 1716 3107 1425 1425 3930 4274 1383 762 2828 2899 1347 1347 2711 1335 2786 1413 1866 2873 2865 1869 1345 397

340 340 340 147 334 334 334 306 315 315 315 315 616 475 475 501 501 461 254 449 449 449 449 445 445 471 471 622 622 622 623 297 99

F F F C F F F F F F F F F C C F F N N F F F F F F F F F F F F F N

659 659 659 290 592 592 592 577 612 612 612 612 884 737 734 603 603 595 296 834 832 835 832 750 752 749 749 839 838 838 818 130 125

0.0 0.0 0.0 1e−97 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2e−95 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7e−35 8e−36

2037

553

F

248

8e−76

Transcript accession number

PDP1 PP1

PP2A-MTS

PP2A-WDB

PP2A-TWS

SGG

SLIMB

VRI

JV189862 JW537406 JW510909 JV195757 JV195507 JV189609 JW510910 JW537407 JV195775 JV189880 JW510908 JW537405 JW510900 JV191225 JV197112 JW537398 JW510901 JV189438 JV195342 JW536418 JW510059 JV196234 JV190342 JW537477 JV196574 JW519106 JV190685 JV192661 JW505360 JW524779 JV198524 JW538477 JW511832

Clock input pathway transcripts/proteins CRY1 JW523594


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Table 1 (continued) Protein family

Transcript/protein identifications Transcript accession number

Clock input pathway transcripts/proteins JW504323

tblasn search statistics Transcript lengtha

Deduced protein lengthb

1440

443

Deduced protein typec

BLAST score

E-value

N

244

2e−76

Abbreviations: CLK, CLOCK; CRY2, CRYPTOCHROME 2; CYC, CYCLE; PER, PERIOD; TIM, TIMELESS; CKII-α, CASEIN KINASE II α-subunit; CKII-β, CASEIN KINASE II β-subunit; CWO, CLOCKWORK ORANGE; DBT, DOUBLETIME; PDP1, PAR DOMAIN PROTEIN 1; PP1, PROTEIN PHOSPHATASE 1; PP2A-MTS, PROTEIN PHOSPHATASE 2A-MICROTUBULE STAR; PP2A-WDB, PROTEIN PHOSPHATASE 2A-WIDERBORST; PP2A-TWS, PROTEIN PHOSPHATASE 2A-TWINS; SGG, SHAGGY; SLIMB, SUPERNUMERARY LIMBS; VRI, VRILLE; CRY1, CRYPTOCHROME 1. a Length in nucleotides. b Length in amino acids. c Deduced protein type: F, full-length; N, amino-terminal partial; I, internal protein fragment; C, carboxyl-terminal partial.

transcripts to its original query sequence. All searches of the Tigriopus database were conducted on or before July 25, 2014. 2.2. Analyses of protein conservation and structure

protocols employed previously for the characterization of a number of protein types (McCoole et al., 2011, 2012a, 2012b; Christie et al., 2013b,c, 2014a, 2014b), including circadian ones (Tilden et al., 2011; Christie et al., 2013a).

To help confirm the identity of the putative T. californicus circadian proteins deduced from the transcripts identified here, analyses of protein sequence and structural motif conservation were conducted using

2.2.1. Sequence similarity To determine the proteins most similar to each of the T. californicus circadian sequences identified in this study, each of the deduced

Table 2 blastp analyses of putative Tigriopus californicus (Tigca) circadian system proteins vs. all annotated Drosophila melanogaster proteins curated in FlyBasea and all non-redundant arthropod proteins curated in GenBankb. Query

FlyBase searches

GenBank searches

FlyBase no.

Species

BLAST score

FBpp0076500 FBpp0306710 ABA62409 ABA62409 FBpp0074693 FBpp0074693 FBpp0304590 FBpp0304590 FBpp0304590 FBpp0304590 FBpp0305455

D. melanogaster D. melanogaster Danaus plexippus D. plexippus D. melanogaster D. melanogaster D. melanogaster D. melanogaster D. melanogaster D. melanogaster D. melanogaster

82 243 810 809 392 390 319 319 323 319 516

Clock-associated proteins Tigca- CKII-α FBpp0070041 Tigca- CKII-β FBpp0305406 Tigca-CWO-A FBpp0081723 Tigca-CWO-B FBpp0081723 Tigca-DBT-I FBpp0306615 Tigca-DBT-IIA FBpp0306615 Tigca-DBT-IIB FBpp0306615 Tigca-DBT-IIC FBpp0306615 Tigca-PP1-I FBpp0306442 Tigca-PP1-IIA FBpp0305497 Tigca-PP1-IIB FBpp0071381 Tigca-MTS FBpp0310063 Tigca-WDB-IA FBpp0288759 Tigca-WDB-IB FBpp0082978 Tigca-WDB-IIA FBpp0084575 Tigca-WDB-IIA FBpp0084575 Tigca-TWS-A FBpp0311386 Tigca-TWS-B FBpp0311386 Tigca-SGG-A FBpp0070450 Tigca-SGG-B FBpp0070450 Tigca-SLIMB-A FBpp0306059 Tigca-SLIMB-B FBpp0303082 Tigca-SLIMB-C FBpp0303082 Tigca-VRI FBpp0309715

D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D.

Clock input pathway proteins Tigca-CRY1-A AAX58599 Tigca-CRY1-B AAX58599

D. plexippus D. plexippus

Core clock proteins Tigca-CLK-NT Tigca-CLK-IF Tigca-CRY2-A Tigca-CRY2-B Tigca-CYC-A Tigca-CYC-B Tigca-PER-A Tigca-PER-B Tigca-PER-C Tigca-PER-D Tigca-TIM

melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster melanogaster

E-value

Accession no.

Species

Blast score

E-value

1e−16 2e−64 0.0 0.0 1e−108 3e−108 1e−86 3e−86 2e−87 2e−86 1e−145

AGD94516 BAJ16353 BAG07408 BAG07408 ABI21880 ABI21880 AGA01525 AGA01525 AGA01525 AGA01525 AAY40757

Solenopsis invicta Thermobia domestica Riptortus pedestris R. pedestris Lutzomyia longipalpis L. longipalpis Rhyparobia maderae R. maderae R. maderae R. maderae Aedes aegypti

105 268 830 831 532 531 498 495 495 495 705

4e−26 1e−83 0.0 0.0 5e−179 6e−179 2e−149 3e−149 3e−149 2e−149 0.0

574 423 107 104 528 494 493 469 618 602 574 609 777 686 776 377 780 780 659 656 801 802 782 123

9e−164 1e−118 6e−23 9e−23 5e−150 2e−139 4e−139 3e−132 3e−177 3e−172 8e−164 2e−174 0.0 0.0 0.0 5e−105 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4e−28

KDR22024 EFX77371 KDR16323 KDR16323 AGV28719 XP_008196766 XP_008196766 XP_972641 ACO15366 XP_008192788 EFA02685 ENN77718 XP_008195946 XP_006559896 XP_004924522 XP_002435135 AFK24473 AFK24473 XP_008208522 XP_008543312 KDR19729 KDR19729 KDR19729 EFA11543

Zootermopsis nevadensis Daphnia pulex Z. nevadensis Z. nevadensis Eurydice pulchra Tribolium castaneum T. castaneum T. castaneum Caligus clemensi T. castaneum T. castaneum Dendroctonus ponderosae T. castaneum Apis mellifera Bombyx mori Ixodes scapularis Scylla paramamosain Scylla paramamosain Nasonia vitripennis Microplitis demolitor Z. nevadensis Z. nevadensis Z. nevadensis T. castaneum

632 445 180 179 560 526 525 501 646 626 586 627 836 700 827 439 828 828 696 692 844 845 823 132

0.0 2e−158 2e−47 1e−50 0.0 4e−180 3e−180 3e−174 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2e−152 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6e−34

518 431

8e−179 4e−146

BAF45421 BAF45421

Dianemobius nigrofasciatus D. nigrofasciatus

561 456

0.0 2e−154

Abbreviations: CLK, CLOCK; CRY2, CRYPTOCHROME 2; CYC, CYCLE; PER, PERIOD; TIM, TIMELESS; CKII-α, CASEIN KINASE II α-subunit; CKII-β, CASEIN KINASE II β-subunit; CWO, CLOCKWORK ORANGE; DBT, DOUBLETIME; PDP1ε, PAR DOMAIN PROTEIN 1ε; PP1, PROTEIN PHOSPHATASE 1; MTS, PROTEIN PHOSPHATASE 2A-MICROTUBULE STAR; WDB, PROTEIN PHOSPHATASE 2A-WIDERBORST; TWS, PROTEIN PHOSPHATASE 2A-TWINS; SGG, SHAGGY; SLIMB, SUPERNUMERARY LIMBS; VRI, VRILLE; CRY1, CRYPTOCHROME 1. a As D. melanogaster possesses CRY1 but not CRY2, the D. plexippus dataset was used for both CRY searches as Danaus possesses both CRYs, and they are well characterized in this species. b Excluding T. californicus proteins, synthetic constructs and provisional protein sequences.

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isoforms was used to query the annotated D. melanogaster proteins dataset present in FlyBase (version FB2014_04; St. Pierre et al., 2014), as well as those present in the non-redundant arthropod protein dataset (taxid:6656) curated in GenBank (excluding T. californicus proteins, synthetic constructs, and provisional protein sequences). The Drosophila dataset of FlyBase were chosen for comparison to the Tigriopus sequences, as it is one of the largest, most complete, and thoroughly characterized arthropod protein databases extant; the non-redundant arthropod protein dataset was chosen as it allows for a broad species comparison. The BLAST algorithm blastp (Altschul et al., 1997) was used for both the FlyBase and non-redundant protein analyses. Table 2 summarizes the results of both of these database searches. To determine amino acid identity/similarity between proteins (and structural motifs [see Section 2.2.2]), the sequences in question were aligned using MAFFT version 7 (http://align.bmr.kyushu-u.ak.jp/mafft/ online/server/; Katoh et al., 2002; Katoh and Toh, 2008), and amino acid identity/similarity was subsequently determined using the alignment output. Specifically, percent identity was calculated as the number of identical amino acids (denoted by “*” in the MAFFT output) divided by the total number of residues in the longest sequence (× 100). Amino acid similarity was calculated as the number of identical and similar amino acids (the latter denoted by the “:” and “.” symbols in the protein alignment) divided by the total number of residues in longest sequence (× 100). In the MAFFT output “:” indicates that one of the following strong groups is fully conserved: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY or FYW (Kazutaka Katoh, personal communication). The “.” symbol in MAFFT indicates full conservation of one of the following weaker groups: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM or HFY (Kazutaka Katoh, personal communication). 2.2.2. Structural motif analysis Protein structural motifs were analyzed using the online program SMART (http://smart.embl-heidelberg.de/; Schultz et al., 1998; Letunic et al., 2009); the default parameters of the program were used for all analyses. In a previous study (Christie et al., 2013a), SMART was used to determine the structural motifs present in C. finmarchicus circadian proteins. A common highlighting scheme has been used to denote functional domains in all figures: basic region leucine zipper, pink; casein kinase II regulatory subunit domain, black; coiled coil region, gray; D domain of beta-TrCP, blue; FBOX, dark blue; helix–loop–helix, yellow; orange domain, red; PAC, dark green; PAS, light green; protein phosphatase 2A catalytic domain, dark red; serine/threonine protein kinase catalytic domain, purple; WD40, teal. 3. Results Circadian clock systems can be divided into three functional subunits: the clock, input pathways to the clock, and output pathways from the clock (e.g. Allada and Chung, 2010). The clock group itself can also be divided into two subsystems, namely the genes/proteins that form the core clock and those that contribute to its modulation, i.e. clock-associated proteins. Here, the results have been subdivided into core clock transcripts/proteins, clock-associated transcripts/proteins and clock input pathway transcripts/proteins; within each of these sections the data are presented in alphabetical order based on transcript/protein family name (using the Drosophila nomenclature). Output pathway transcripts/proteins putatively used by the T. californicus circadian system, i.e. peptide hormone precursors, were the subject of a previous study (Christie, 2014b). It is important to note that all of the proteins reported here are theoretical, having been predicted from computationally assembled transcripts. Thus, care must be taken to not assume that any are actually produced by T. californicus until they have been verified by molecular cloning and/or biochemical analysis.

3.1. Core clock proteins 3.1.1. CLOCK (CLK) Using the sequence of the amino (N)-terminal partial C. finmarchicus CLK (Christie et al., 2013a) deduced from GAXK01092177 (Lenz et al., 2014) as the query, three T. californicus transcripts (Accession Nos. JW523144, JW503893, JW523143) were identified as encoding putative CLK homologs (Table 1). Translation of JW523144 revealed a 68 amino acid, N-terminal partial protein (Tigca-CLK-NT; Fig. 1). Translation of JW503893 and JW523143 revealed each to encode a putative internal protein fragment, with that from the former being 295 amino acids long and that from the latter, 148 amino acid in length. The partial protein deduced from JW523143 is identical to that derived from JW503893, with the exception that it is carboxyl (C)-terminally extended by six amino acids. Thus, a 301 amino acid, putative internal protein fragment was achieved by combining these two partial sequences (Tigca-CLK-IF; Fig. 1); there is no region of overlap between TigcaCLK-NT and Tigca-CLK-IF (Fig. 1). Alignment of Tigca-CLK-NT and Tigca-CLK-IF with that of the extant portion of Calfi-CLK is shown in Fig. 1. As can be seen from these alignments, both of the extant portions of the putative Tigriopus CLK show significant sequence homology to their respective portions of the Calanus sequence. Specifically, Tigca-CLK-NT is 67.4% identical/75.2% similar in amino acid composition to its corresponding portion of CalfiCLK (Fig. 1). Similarly, Tigca-CLK-IF shows 44.0% amino acid identity/ 64.7% similarity to its corresponding portion of the C. finmarchicus protein (Fig. 1). In addition to amino acid similarity, the extant portions of TigcaCLK show similar functional domains to those previously described for Calfi-CLK (Christie et al., 2013a). Via SMART analyses, a single helix– loop–helix domain was identified in both Tigca-CLK-NT and in the extant portion of the Calanus protein (highlighted in yellow in Fig. 1). Similarly, one PAS and one PAC domain were predicted by SMART in Tigca-CLK-IF (highlighted in light green and dark green, respectively in Fig. 1), with the same domains predicted for the corresponding region of Calfi-CLK (Christie et al., 2013a). The missing portion of the Tigriopus protein (red dashes in the sequence shown in Fig. 1) is likely to contain an additional PAS domain, as one was predicted previously by SMART for this portion of the Calanus CLK (Christie et al., 2013a). For the functional domains that are shared between the Tigriopus and Calanus proteins, more extensive amino acid conservation was noted than was reported for the proteins as a whole, at least in their regions of overlap (Fig. 1), e.g. the PAC domain is 72.7% identical/95.4% similar in amino acid composition in the two proteins. Taken collectively, the sequence and structural motif conservation seen between the two Tigriopus partial proteins and CalfiCLK suggest that Tigca-CLK-NT and Tigca-CLK-IF are true portions of an authentic CLOCK family member. To further confirm that the two partial Tigriopus proteins are fragments of a true CLK, each was used as the input query for blastp searches of the annotated Drosophila protein database present in FlyBase and the non-redundant arthropod protein database curated in GenBank. For Tigca-CLK-NT, the most similar D. melanogaster protein was identified as CLOCK, isoform A (FlyBase ID. No. FBpp0076500 [Table 2]), while the most similar arthropod protein was identified as a partial CLOCK from the fire ant Solenopsis invicta (Accession No. AGD94516; Ingram et al., 2012 [Table 2]). For Tigca-CLK-IF, the top FlyBase hit was CLOCK, isoform H (FlyBase ID. No. FBpp0306710 [Table 2]), with the most similar arthropod sequence being an isoform of CLOCK from the firebrat Thermobia domestica (Accession No. BAJ16353; Kamae et al., 2010 [Table 2]). These BLAST results also provide strong support for the two Tigriopus partial proteins being portions of a CLOCK family member. 3.1.2. CRYPTOCHROME 2 (CRY2) Using the sequence of C. finmarchicus CRY2 (Christie et al., 2013a) deduced from GAXK01199676 (Lenz et al., 2014) as a query, three transcripts (Accession Nos. JW504321, JW523592 and JV190442) were identified as encoding putative CRY2 homologs (Table 1). Translation


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Fig. 1. CLOCK (CLK) protein. Alignment of Calanus finmarchicus CLK (Calfi-CLK) with two partial Tigriopus californicus CLK proteins, one an amino-terminal partial sequence (Tigca-CLK-NT) and the other an internal protein fragment (Tigca-CLK-IF). In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, helix–loop–helix, PAS, and PAC domains identified by SMART analyses are highlighted in yellow, light green, and dark green, respectively. The red dashes in the Tigriopus protein show an intervening region that is missing but is predicted to exist between Tigca-CLK-NT and Tigca-CLKIF. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of the first two transcripts revealed each to encode a full-length protein, with that deduced from JW504321 being 748 amino acids long (TigcaCRY2-A; Fig. 2) and that deduced from JW523592 752 amino acids in length (Tigca-CRY2-B; Fig. 2A). Translation of JV190442 revealed it to encode an internal fragment of Tigca-CRY2-A (underlined in the sequence of the full-length protein shown in Fig. 2A). Alignment of Tigca-CRY2-A and CRY2-B shows that the two proteins are nearly identical in amino acid composition (i.e. 98.1% identity/98.9% similarity), with the greatest variation occurring at the C-termini of the proteins, where the latter is extended relative to the former by six glutamine residues (Fig. 2A). The alignment of Calfi-CRY2 and Tigca-CRY2-A is shown in Fig. 2B. As can be seen from this alignment, these two proteins share 55.7% identity/73.9% similarity in amino acid composition. Alignment of the Calanus protein with Tigca-CRY2-B revealed nearly identical sequence conservation to that seen for Tigca-CRY2-A, with these proteins sharing 55.7% identity/72.7% similarity in amino acid sequence (alignment not shown). While no functional domains were identified by SMART in either of the Tigriopus CRY2s or in the Calanus protein (Christie et al., 2013a), the extensive sequence conservation seen between each of the Tigriopus proteins and Calfi-CRY2 is supportive of Tigca-CRY2-A and Tigca-CRY2-B being authentic isoforms of CRYPTOCHTOME 2. To increase our confidence in the assignment of the Tigriopus proteins just described to the CRY2 family, each sequence was used to query the non-redundant arthropod protein database of GenBank for the most similar protein sequences. For both Tigca-CRY2-A and TigcaCRY2-B, cryptochrome-m (an alternative designation for CRY2) from the bean bug Riptortus pedestris (Accession No. BAG07408; Ikeno et al., 2008) was returned as the top arthropod protein hit (Table 2). As CRY2 is not present in Drosophila (Yuan et al., 2007), a search of the monarch butterfly D. plexippus database was substituted for the FlyBase one; both CRY2 and CRY1 are present in Danaus, and have been well characterized in this species (Yuan et al., 2007). For both Tigca-CRY2-A and Tigca-CRY2-B, D. plexippus CRYPTOCHROME 2 (Accession No. ABA62409; Zhu et al., 2005) was returned as the top Danaus hit (Table 2). These BLAST results also support the

two Tigriopus proteins being members of the CRYPTOCHROME 2 family. 3.1.3. CYCLE (CYC) Using the sequence of C. finmarchicus CYC (Christie et al., 2013a) deduced from GAXK01131751 (Lenz et al., 2014) as a query, two transcripts (Accession Nos. JW523145 and JW503894) were identified as encoding putative CYC homologs (Table 1). Translation of JW523145 revealed a 693 amino acid, full-length protein (Tigca-CYC-A; Fig. 3); a 672 amino acid, C-terminal partial protein was deduced from JW503894 (Tigca-CYC-B; Fig. 3A). Alignment of Tigca-CYC-A and Tigca-CYC-B shows them to be essentially identical in sequence, at least over the extant portion of the latter (Fig. 3A). In fact, in their region of overlap, the two proteins differ at just 4 residues, all conservative amino acid substitutions (Fig. 3A). Fig. 3B shows the alignment of Calfi-CYC and Tigca-CYC-A. As can be seen from this alignment, extensive amino acid conservation (51.8% identity/78.9% similarity) is present between these two proteins; the extant portion of Tigca-CYC-B is 52.6% identical/79.0% similar to its corresponding portion of the Calanus protein (alignment not shown). In addition to their sequence conservation, extensive conservation of protein functional motifs is evident between the putative Tigriopus CYC isoforms and Calfi-CYC (Fig. 3). Specifically, both Tigca-CYC-A and TigcaCYC-B are predicted by SMART to possess the same complement of one helix–loop–helix (highlighted yellow in Fig. 3), two PAS (highlighted light green in Fig. 3) and one PAC domains (highlighted dark green in Fig. 3); the same domain complement was predicted previously by SMART for Calfi-CYC (Christie et al., 2013a). The domains predicted by SMART are similarly placed and highly conserved in amino acid composition between the Tigriopus and Calanus proteins (Fig. 3). Taken collectively, the sequence and structural conservation seen between the Tigriopus isoforms of CYC and their Calanus counterpart support the inclusion of Tigca-CYC-A and Tigca-CYC-B in the CYCLE family. To further vet the assignment of the Tigriopus proteins just described as true CYCLEs, Tigca-CYC-A and Tigca-CYC-B were each used as queries

22


in blastp searches of the annotated Drosophila proteins curated in FlyBase and the non-redundant arthropod proteins present in GenBank. With respect to the FlyBase searches, CYCLE (FlyBase ID. No. FBpp0074693) was returned as the most similar D. melanogaster protein hit for each of the Tigriopus CYCs (Table 2), with the two searches of the non-redundant arthropod protein database returning an isoform of CYCLE from the sand fly Lutzomyia longipalpis (Accession No. ABI21880; Meireles-Filho et al., 2006) as the most similar protein to Tigca-CYC-A and Tigca-CYC-B (Table 2). Taken collectively, these BLAST results also support the inclusion of the two Tigriopus proteins as isoforms of CYCLE. 3.1.4. PERIOD (PER) Using the sequence of C. finmarchicus PER (Christie et al., 2013a) deduced from GAXK01127710 (Lenz et al., 2014) as a query, four transcripts (Accession Nos. JW535312, JV196344, JV190453 and JW509073) were identified as encoding putative PER homologs (Table 1). Translation of JW535312 revealed a 1460 amino acid, full-length protein (Tigca-PERA; Fig. 4); a putative full-length protein of identical size was predicted from JV196344 (Tigca-PER-B; Fig. 4A). The protein predicted from JV190453 is 1462 amino acids long and also is likely a full-length sequence (Tigca-PER-C; Fig. 4A); JW509073 encodes a 1411 amino acid, N-terminal partial protein (Tigca-PER-D; Fig. 4A). Alignment of the four putative Tigriopus PERs shows them to be essentially identical in amino acid composition (N 99% identity in all pairwise comparisons), at least over the extant sequence of Tigca-PER-D (Fig. 4A). Specifically, TigcaPER-A differs from Tigca-PER-B at just one residue, a conservative substitution (Fig. 4A), while it differs from Tigca-PER-C at five positions, which includes the two amino acid insert present in the latter isoform but absent

in the former (Fig. 4A). Tigca-PER-B differs from Tigca-PER-C at six positions, again these include the two-residue inset present in the latter protein but absent in Tigca-PER-B (Fig. 4A). Tigca-PER-D, like Tigca-PER-C, possess the two amino acid insert that is absent in Tigca-PER-A and Tigca-PER-B, but possesses one substituted residues relative to TigcaPER-C, which establishes it as a distinct isoform (Fig. 4A). Moderate sequence conservation is present between each of the Tigriopus PER isoforms and the Calanus protein used to identify the transcripts encoding them. Fig. 4B shows the alignment of Tigca-PER-A with the C. finmarchicus query sequence. Here, 34.3% amino acid identity/ 66.6% amino acid similarity is present between the two PERs. Identical degrees of sequence conservation, or at least nearly so, are present between Calfi-PER-I and the other two full-length T. californicus PERs (i.e., Tigca-PER-B, 34.3% identity/66.6% similarity; Tigca-PER-C, 34.2% identity/66.6% similarity [alignments not shown]). Moreover, each of the Tigriopus PERs possesses the same complement of functional domains previously identified by SMART analysis in Calfi-PER-I (Christie et al., 2013a), i.e. two PAS and one PAC domains (highlighted in light green and dark green, respectively, in Fig. 4), all of which are similarly located in the Calanus and T. californicus proteins (Fig. 4). Taken collectively, the sequence and structural motif conservation seen between the Tigriopus proteins and Calfi-PER-I support the former group's inclusion as PERIOD family members. As a further confirmation of the Tigriopus proteins just described being members of the PER family, each was used to query the extant annotated Drosophila proteins in FlyBase and the non-redundant arthropod proteins in GenBank for the most similar protein sequences. For each of the Tigriopus PERs, PERIOD, isoform B (FlyBase ID. No.

A)

Fig. 2. CRYPTOCHROME 2 (CRY2) protein. (A) Alignment of Tigriopus californicus CRY2 isoform A (Tigca-CRY2-A) and isoform B (Tigca-CRY2-B). (B) Alignment of Calanus finmarchicus CRY2 (Calfi-CRY2) and Tigca-CRY2-A. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences.


23

B)

Fig. 2 (continued).

FBpp0304590), was returned as the top FlyBase BLAST hit (Table 2). Likewise, PERIOD from the cockroach Rhyparobia maderae (Accession No. AGA01525; Werckenthin et al., 2012) was returned as the top hit for each of the T. californicus proteins when searches of the nonredundant arthropod database were conducted (Table 2). Taken collectively, these BLAST results too support the four Tigriopus proteins being true PERIOD isoforms. 3.1.5. TIMELESS (TIM) Using the sequence of C. finmarchicus TIM (Christie et al., 2013a) deduced from GAXK01195225 (Lenz et al., 2014) as a query, five transcripts (Accession Nos. JW538330, JW511710, JV197201, JW511708 and JV191316) were identified as encoding putative TIM homologs (Table 1). Translation of each of these transcripts revealed a partial protein, with those derived from JW538330 and JW511710 being Nterminal partial sequences, that derived from JV197201 an internal fragment, and those derived from JW511708 and JV191316 C-terminal partial proteins. Alignment of the protein deduced from JV197201 with that derived from JW538330 showed the former to be identical in sequence to a portion of the latter. Similarly, JV191316 encodes a protein that is identical to a portion of the sequence deduced from JW511708. Given a 925 amino acid region of overlap between the protein deduced from JW538330 and the protein predicted from JW511708, a 1483 amino acid, full-length sequence was obtained (for convenience of later

discussion this chimeric protein was named Tigca-TIM). The sequence of Tigca-TIM shown in Fig. 5 represents the entire sequence of the Nterminal partial protein deduced from JW538330, with the final 45 residues being from the C-terminal partial protein deduced from JW511708. While a continuous sequence is shown, alignment of the various partial proteins with the full-length chimera did reveal nine variable residues. Specifically, threonine for asparagine, valine for aspartic acid, serine for glycine, asparagine for serine, and asparagine for serine substitutions at positions 140, 332, 380, 437 and 450, respectively, were noted between the partial protein deduced from JW511710 and sequence presented as Tigca-TIM. Similarly, glutamic acid for aspartic acid, proline for alanine, lysine for glutamic acid and asparagine for serine substitutions at positions 1127, 1273, 1275 and 1331, respectively, were identified in the comparison of the partial protein derived from JW511708/JV191316 and the full-length Tigriopus sequence presented in Fig. 5. These variant residues strongly support the existence of at least two isoforms of TIM being present in T. californicus. However, it is not possible to fully characterize either of these putative full-length TIMs from the Tigriopus TSA data at this time. Alignment of Calfi-TIM and Tigca-TIM revealed moderate amino acid conservation between the two proteins (Fig. 5). Specifically, the two sequences exhibit 36.6% identity/65.0% similarity in amino acid composition. Prior analysis of the sequence of Calfi-TIM using SMART failed to identify any functional motifs within the protein (Christie et al., 2013a).

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A)

B)


Analysis of Tigca-TIM by SMART also failed to identify any functional domains within this protein. Regardless, the sequence conservation seen between Calfi-TIM and the Tigriopus protein identified here supports the inclusion of Tigca-TIM as a member of the TIMELESS family. To help confirm that the Tigriopus protein described above is truly a TIM homolog, the full-length sequence shown in Fig. 5 was used to query the annotated Drosophila proteins of FlyBase and the nonredundant arthropod protein database in GenBank for similar sequences. The search of FlyBase returned TIMELESS, isoform Q (FlyBase ID. No. FBpp0305455), as the most similar D. melanogaster protein to Tigca-TIM (Table 2). Similarly, TIMELESS from the mosquito Aedes aegypti (Accession No. AAY40757; Gentile et al., 2006) was identified as the most similar non-redundant arthropod sequence to the Tigriopus protein (Table 2). Collectively, these data also support our assignment of Tigca-TIM as an authentic member of the TIMELESS family. 3.2. Clock-associated proteins 3.2.1. CASEIN KINASE II (CKII) 3.2.1.1. CKII α-subunit. Using the sequence of C. finmarchicus CKII-α (Christie et al., 2013a) deduced from GAXK01065631 (Lenz et al., 2014) as a query, four transcripts (Accession Nos. JV195708, JV189813, JW518769 and JW522448) were identified as encoding putative CKII-α homologs (Table 1). Translation of these transcripts revealed each to encode an identical, 370 amino acid, full-length protein (Tigca- CKII-α; Fig. 6A). Alignment of Calfi-CKII-α and Tigca-CKII-α (Fig. 6A) revealed the two proteins to be nearly identical in amino acid composition, i.e. they are 87.6% identical/97.0% similar in amino acid sequence. Previously, a single serine/threonine protein kinase catalytic domain (highlighted in purple in Fig. 6A) was identified in the Calanus protein using SMART (Christie et al., 2013a); the same domain, with an essentially identical amino acid sequence, is predicted by this program in Tigca-CKII-α (Fig. 6A). The high degree of sequence and structural motif conservation seen between Calfi-CKII-α and the Tigriopus protein is strongly supportive of Tigca-CKII-α being a true member of the CASEIN KINASE II αSUBUNIT family. To further confirm our assignment of Tigca-CKII-α as a member of the CKII-α family, the amino acid sequence of this protein was used to query both the annotated D. melanogaster proteins of FlyBase and the non-redundant arthropod proteins in GenBank for the most similar sequences. For the FlyBase search, CASEIN KINASE II α, isoform A (FlyBase ID. No. FBpp0070041), was identified as the top Drosophila protein hit (Table 2), while for the search of the non-redundant arthopod proteins, CASEIN KINASE II subunit α from the termite Zootermopsis nevadensis (Accession No. KDR22024; Terrapon et al., 2014) was found to be the most similar sequence (Table 2). These BLAST data also support TigcaCKII-α being an authentic CASEIN KINASE II α-SUBUNIT. 3.2.1.2. CKII β-subunit. Using the sequence of C. finmarchicus CKII-β (Christie et al., 2013a) deduced from GAXK01165930 (Lenz et al., 2014) as a query, four transcripts (Accession Nos. JV195743, JV189848, JW522449 and JW503280) were identified as encoding putative CKII-β homologs (Table 1). Translation of these transcripts revealed each to encode an identical 221 amino acid, full-length protein (TigcaCKII-β; Fig. 6B). Fig. 6B shows the alignment of the Calanus query and Tigca-CKII-β; the two proteins share extensive sequence conservation, being 94.1% identical/96.4% similar in amino acid composition. Analyses by SMART identified a single casein kinase II regulatory subunit domain in both

25

Calfi-CKII-β (Christie et al., 2013a) and Tigca-CKII-β (highlighted in black in Fig. 6B); this domain is identically located and essentially identical in amino acid sequence in the two proteins (they differ at just a single residue). The high degree of sequence and structural motif conservation seen between the Calanus and Tigriopus proteins strongly supports Tigca-CKII-β being a CASEIN KINASE II β-SUBUNIT family member. To further confirm our assignment of Tigca-CKII-β as a member of the CKII-β family, the amino acid sequence of this protein was used to query both the Drosophila proteins annotated in FlyBase and the non-redundant arthropod proteins present in GenBank for the most similar sequences. The FlyBase search returned CASEIN KINASE II β, isoform K (FlyBase ID. No. FBpp0305406), as the top protein hit (Table 2). For the search of the non-redundant arthropod proteins, DAPPUDRAFT_305830 from D. pulex (Accession No. EFX77371; Colbourne et al., 2011) was identified as the most similar protein to the query (Table 2); this Daphnia protein is identical in sequence to a previously described Daphnia isoform of CKII-β (Tilden et al., 2011). These data provide additional support for TigcaCKII-β being a true member of the CASEIN KINASE II β-SUBUNIT family. 3.2.2. CLOCKWORK ORANGE (CWO) Using the sequence of C. finmarchicus CWO (Christie et al., 2013a) deduced from GAXK01116566 (Lenz et al., 2014) as a query, two transcripts (Accession Nos. JW506198 and JW525737) were identified as encoding putative CWO homologs (Table 1). Translation of JW506198 revealed a 626 amino acid, full-length protein (Tigca-CWO-A; Fig. 7), while translation of JW525737 revealed a 217 amino acid, N-terminal partial sequence (Tigca-CWO-B; Fig. 7A). Alignment of the two deduced proteins shows them to be nearly identical in amino acid composition (i.e. 96.8% identity/99.5% similarity), at least over the extant portion of Tigca-CWO-B (Fig. 7A). Alignment of Calfi-CWO and Tigca-CWO-A is shown in Fig. 7B. From this alignment, it is clear that only moderate amino acid conservation (32.4% identity/56.3% similarity) is present between these two proteins; the extant portion of Tigca-CWO-B is 34.4% identical/51.3% similar to the corresponding portion of the Calanus protein (alignment not shown). Despite limited sequence conservation, both Calfi-CWO (Christie et al., 2013a) and the two Tigriopus proteins are predicted by SMART to possess one helix–loop–helix and one orange domain (highlighted in yellow and red, respectively, in Fig. 7). The placement of these two domains within the three proteins is nearly identical, and within these regions, much higher levels of amino acid conservation are evident, e.g. the helix–loop–helix domain of Calfi-CWO and that of Tigca-CWO-A show 91.0% identity/98.2% similarity, while their orange domains are 72.3% identical/95.7% similar (Fig. 7B). Taken collectively, the sequence and structural conservation seen between the Tigriopus CWOs and Calfi-CWO support the inclusion of Tigca-CWO-A and Tigca-CWO-B in the CLOCKWORK ORANGE family. To further confirm that the Tigriopus proteins just described are true members of the CWO family, Tigca-CWO-A and Tigca-CWO-B were each used as queries in blastp searches of the annotated Drosophila proteins curated in FlyBase and the non-redundant arthropod proteins present in GenBank. For both of the Tigriopus proteins, CLOCKWORK ORANGE, isoform A (FlyBase ID. No. FBpp0081723), was returned as the top FlyBase hit (Table 2). For the non-redundant arthropod protein searches, hairy/ enhancer-of-split related with YRPW motif protein 2 (a member of the same transcription factor family as CWO) from the termite Z. nevadensis (Accession No. KDR16323; Terrapon et al., 2014) was returned as the protein most similar to each of the Tigriopus CWOs (Table 2). These BLAST results also support Tigca-CWO-A and Tigca-CWO-B being authentic members of the CLOCKWORK ORANGE family.

Fig. 3. CYCLE (CYC) protein. (A) Alignment of Tigriopus californicus CYC isoform A (Tigca-CYC-A) and isoform B (Tigca-CYC-B). (B) Alignment of Calanus finmarchicus CYC (Calfi-CYC) and Tigca-CYC-A. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, helix–loop–helix, PAS, and PAC domains identified by SMART analyses are highlighted in yellow, light green, and dark green, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

26


3.2.3. DOUBLETIME (DBT) Using the sequence of C. finmarchicus DBT-I (Christie et al., 2013a) deduced from GAXK01100537 (Lenz et al., 2014) as a query, seven transcripts (Accession Nos. JV195325, JV189421, JW522445, JW503277, JW522447, JV198427 and JW503279) were identified as encoding putative DBT homologs (Table 1). Translation of the first four of these transcripts revealed each to encode an identical, 399 amino acid, fulllength protein (Tigca-DBT-I; Fig. 8A and B). Translation of JW522447 revealed a 663 amino acid, full-length protein (Tigca-DBT-IIA; Fig. 8A and C). Translation of JV198427 revealed a 630 amino acid, N-terminal partial protein (Tigca-DBT-IIB; sequence not shown), which is identical, at

least over its extant sequence, to Tigca-DBT-IIA, with the exception of its final three amino acid, which are TIQ in Tigca-DBT-IIB vs. QYN in Tigca-DBT-IIA. JW503279 encodes a 405 amino acid internal protein fragment (Tigca-DBT-IIC; sequence not shown) that is also nearly identical to Tigca-DBT-IIA in their region of overlap; two substituted residues are present in the shared region, i.e. serine for threonine and valine for glycine at positions 516 and 529, respectively, of the fulllength protein (alignment not shown). Alignment of Tigca-DBT-I and Tigca-DBT-IIA shows the latter protein to be both N- and C-terminally extended relative to the former, and as a result, the amino acid sequence conservation seen between the proteins as a whole is relatively modest

A)

Fig. 4. PERIOD (PER) protein. (A) Alighment of Tigriopus californicus (Tigca) PER isoform A (Tigca-PER-A), isoform B (Tigca-PER-B), isoform C (Tigca-PER-C) and isoform D (Tigca-PER-D). (B) Alignment of Calanus finmarchicus PER I (Calfi-PER-I) with Tigca-PER-A. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure PAS and PAC domains identified via SMART analyses are highlighted in green and dark green, respectively. In the sequence of Calfi-PER-I, the dark green lettering indicates a potential PAC domain that was not reported by SMART due to its being below the threshold cutoff for positive identification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


27

Fig. 4 (continued).

(37.6% identity/48.1% similarity), though the majority of their region of overlap is highly conserved (Fig. 8A). Alignment of Calfi-DBT-I with Tigca-DBT-I revealed extensive sequence conservation between the two proteins, with 68.7% identity/ 75.7% similarity in amino acid composition present between the two proteins (Fig. 8B). Alignment of the Calanus query with Tigca-DBT-IIA revealed much more modest sequence conservation, i.e. only 36.8% identity/43.3% similarity (alignment not shown). However, alignment of this Tigriopus protein with a second DBT isoform from Calanus, i.e. Calfi-DBT-II (Christie et al., 2013a), revealed a much greater level of amino acid conservation (Fig. 8C), specifically 52.5% identity/73.3% similarity, and suggests that the Tigca-DBT-IIs are likely homologs of this

Calanus variant rather than of Calfi-DBT-I itself. A single serine/threonine protein kinase catalytic domain was predicted by SMART for each of the Tigriopus full-length DBTs (Fig. 8); this domain was identified previously by SMART in both Calfi-DBT-I (Fig. 8B) and Calfi-DBT-II (Fig. 8C) as well (Christie et al., 2013a). Taken collectively, the sequence and structural motif conservation seen between the two Calanus DBTs and the full set of Tigriopus proteins strongly support the latter's inclusion as true DOUBLETIME family members. To increase our confidence that the Tigriopus proteins just described are members of the DBT family, each was used to query the annotated Drosophila proteins of FlyBase and the non-redundant arthropod proteins present in GenBank for the most similar protein sequences. For

28


B)

Fig. 4 (continued).


29

Fig. 5. TIMELESS (TIM) protein. Alignment of Calanus finmarchicus TIM (Calfi-TIM) with a chimeric, full-length TIM derived from a number of Tigriopus californicus partial proteins (TigcaTIM). In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences.

30


Tigca-DBT-I, the top FlyBase hit was discs overgrown (a synonym for DOUBLETIME), isoform D (FlyBase ID. No. FBpp0306615; Table 2), while the non-redundant arthropod protein search returned casein kinase 1 epsilon (another DOUBLETIME synonym) from the isopod Eurydice pulchra (Accession No. AGV28719; Zhang et al., 2013) as the top BLAST hit (Table 2). For each of the Tigriopus DBT-II isoforms, the top FlyBase hits was also discs overgrown, isoform D (Table 2). For the top non-redundant arthropod searches, the top protein hit for Tigca-DBTIIA and Tigca-DBT-IIB was casein kinase I isoform delta-A isoform X1 from the beetle Tribolium castaneum (Accession No. XP_008196766; Kim et al., 2010), while for Tigca-DBT-IIC the top hit was casein kinase I isoform delta-A isoform X2 (Accession No. XP_972641; Kim et al., 2010) also from T. castaneum (Table 2). The results of these BLAST analyses also support the inclusion of the collection of Tigriopus DBTs reported here as authentic members of the DOUBLETIME family. 3.2.4. PAR DOMAIN PROTEIN 1 (PDP1) A previous investigation failed to identify any transcripts encoding PDP1 homologs in the copepod C. finmarchicus (Christie et al., 2013a). However, using the sequence of a D. melanogaster PDP1 family member (Accession no. AAF04509; Lin et al., 1997), a putative PDP1 was identified from the cladoaceran crustacean D. pulex via analysis of its genome (Tilden et al., 2011). Using the Drosophila protein as the query, the extant Tigriopus TSA data were searched for PDP1-encoding transcripts; none were found (Table 1). Similarly, no PDP1-encoding sequences were identified when Dappu-PDP1 was used to search the T. californicus TSA dataset

(Table 1). Thus, it is an open question as to whether or not members of the Copepoda possess PDP1 genes/proteins. 3.2.5. PROTEIN PHOSPHATASE 1 (PP1) Using the sequence of C. finmarchicus PP1 (Christie et al., 2013a) deduced from GAXK01066774 (Lenz et al., 2014) as a query, eight transcripts (Accession Nos. JV189862, JW537406, JW510909, JV195757, JV195507, JV189609, JW510910 and JW537407) were identified as encoding putative PP1 homologs (Table 1). Translation of JV189862, JW537406 and JW510909 revealed each to encode an identical, 340 amino acid full-length protein (Tigca-PP1-I; Fig. 9A and B), while translation of JV195757 revealed a 147 amino acid, C-terminal partial protein that is identical in sequence to the C-terminus of Tigca-PP1-I. Translation of JV195507, JV189609 and JW510910 revealed each to encode an identical, 334 amino acid, full-length protein (Tigca-PP1-IIA; Fig. 9A and C); Tigca-PP1-II-A is distinct from Tigca-PP1-I, though the two proteins do share a high level of sequence conservation, i.e. they are 83.8% identical/93.2% similar in amino acid composition (Fig. 9A). JW537407 also encodes a full-length protein (Tigca-PP1-IIB), which appears to be a truncated (306 amino acid) version of Tigca-PP1-IIA, i.e. the sequence of Tigca-PP1-IIB starts with the second methionine present in TigcaPP1-IIA (alignment not shown). Alignment of Tigca-PP1-I with the Calanus query used for its identification is shown in Fig. 9B. These two proteins show high degrees of amino acid conservation, being 93.5% identical/98.2% similar in amino acid sequence. Tigca-PP1-IIA also shows considerable sequence

A)

B)

Fig. 6. CASEIN KINASE II α (CKII-α) and β (CKII-β) subunit proteins. (A) CKII-α. Alignment of the Calanus finmarchicus CKII-α (Calfi-CKII-α) and Tigriopus californicus CKII-α (Tigca-CKII-α). (B) CKII-β. Alignment of Calfi-CKII-β and Tigca-CKII-β. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, serine/threonine protein kinase catalytic and casein kinase II regulatory subunit domains identified via SMART analyses are highlighted in purple and black, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


31

A)

B)

Fig. 7. CLOCKWORK ORANGE (CWO) protein. (A) Alignment of Tigriopus californicus CWO isoform A (Tigca-CWO-A) and isoform B (Tigca-CWO-B). (B) Alignment of Calanus finmarchicus CWO (Calfi-CWO) and Tigca-CWO-A. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, the helix–loop–helix and orange domains predicted by SMART analyses are highlighted in yellow and red, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

32


conservation with Calfi-PP1, though less than was seen in the comparison of Tigca-PP1-I and Calfi-PP1, i.e. 84.2% amino acid identity/93.0% amino acid similarity. Analysis by SMART revealed each of the Tigriopus PP1s (including Tigca-PP1-IIB), to possess a single protein phosphatase 2A catalytic domain (highlighted in dark red in Fig. 9), a domain previously identified by SMART in Calfi-PP1 (Christie et al., 2013a). The placement of the protein phosphatase 2A catalytic domain in each of the Tigriopus PP1s, as well as in Calfi-PP1, is identical, with little sequence variation seen in this region within or between species (Fig. 9). The extensive sequence and structural motif conservation seen between the Tigriopus PP1s and Calfi-PP1 support the inclusion of the set of

T. californicus proteins described here as true members of the PROTEIN PHOSPHATASE 1 family. To further confirm the assignment of the Tigriopus proteins just described to the PP1 family, each was used to query the annotated Drosophila proteins curated in FlyBase and the non-redundant arthropod proteins present in GenBank for the most similar sequences. For Tigca-PP1-I, protein phosphatase 1 alpha at 96A, isoform B (FlyBase ID. No. FBpp0306442), was returned as the top FlyBase hit (Table 2), while serine/threonine-protein phosphatase PP1-beta from the copepod Caligus clemensi (Accession No. ACO15366; Yasuik, von Schalburg, Cooper, Leong, Jones and Koop, unpublished direct GenBank submission) was returned

A)

B)

Fig. 8. DOUBLETIME (DBT) protein. (A) Alignment of Tigriopus californicus DBT-I (Calfi-DBT-I) and DBT-II, isoform A (Calfi-DBT-IIA). (B) Alignment of Calanus finmarchicus DBT-I (CalfiDBT-I) and Tigca-DBT-I. (C) Alignment of Calfi-DBT-II and Tigca-DBT-IIA. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, serine/threonine protein kinase catalytic domains predicted by SMART analyses are highlighted in purple. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


33

C)

Fig. 8 (continued).

as the most similar non-redundant arthropod protein in GenBank (Table 2). For both Tigca-PP1-IIA and Tigca-PP1-IIB, an isoform of flapwing (a member of the PP1 family) was returned as the top FlyBase hit (Table 2), specifically isoform C (FlyBase ID. No. FBpp0305497) for Tigca-PP1-IIA and isoform A (FlyBase ID. No. FBpp0071381) for TigcaPP1-IIB. For the GenBank non-redundant arthropod protein searches, serine/threonine-protein phosphatase PP1-beta catalytic subunit isoform X1 from the beetle T. castaneum (Accession No. XP_008192788; Kim et al., 2010) was returned as the protein most similar to TigcaPP1-IIA (Table 2), with protein TcasGA2_TC008406 (a PP1 protein) from T. castaneum (Accession No. EFA02685; Richards et al., 2008) returned as the protein most similar to Tigca-PP1-IIB (Table 2). These BLAST results also support the inclusion of Tigca-PP1-I, Tigca-PP1-IIA and Tigca-PP1-IIB as true members of the PROTEIN PHOSPHATASE 1 family.

in Fig. 10) was identified by SMART in Tigca-MTS; this domain was identified previously by this program in the Calanus protein (Christie et al., 2013a). The sequence and structural motif conservation seen between the Tigriopus protein and Calfi-MTS-I is strongly supportive of Tigca-MTS being a member of the MICROTUBULE STAR family. To further strengthen our assignment of Tigca-MTS to the MTS family, its amino acid sequence was used to query both the annotated Drosophila proteins of FlyBase and the non-redundant arthropod proteins in GenBank for the most similar sequences. For the FlyBase search, MICROTUBLE STAR, isoform C (FlyBase ID. No. FBpp0310063), was identified as the top D. melanogaster protein hit (Table 2), while the non-redundant arthropod protein search returned protein YQE_05789 (a putative PP2A catalytic subunit) from the beetle Dendroctonus ponderosae (Accession No. ENN77718; Keeling et al., 2013) as the top BLAST hit (Table 2). These data too support the inclusion of Tigca-MTS in the MICROTUBULE STAR family.

3.2.6. PROTEIN PHOSPHATASE 2A (PP2A) 3.2.6.1. PP2A catalytic subunit — MICROTUBULE STAR (MTS). Using the sequence of C. finmarchicus MTS-I (Christie et al., 2013a) deduced from GAXK01175489 (Lenz et al., 2014) as a query, four transcripts (Accession Nos. JV195775, JV189880, JW510908 and JW537405) were identified as encoding putative MTS homologs (Table 1). Translation of these transcripts revealed each to encode an identical, 315 amino acid, fulllength protein (Tigca-MTS; Fig. 10). Alignment of Calfi-MTS-I and Tigca-MTS (Fig. 10) revealed the two proteins to possess nearly identical amino acid sequences, i.e. they share 94.3% identity/98.7% similarity in amino acid composition. A single protein phosphatase 2A catalytic domain (highlighted in dark red

3.2.6.2. PP2A regulatory subunit 3.2.6.2.1. WIDERBORST (WDB). Using the sequence of C. finmarchicus WDB-I (Christie et al., 2013a) deduced from GAXK01125267 (Lenz et al., 2014) as a query, seven transcripts (Accession Nos. JW510900, JV191225, JV197112, JW537398, JW510901, JV189438 and JV195342) were identified as encoding putative WDB homologs (Table 1). Translation of JW510900, revealed a 616 amino acid, full-length protein (TigcaWDB-IA; Fig. 11A-B). Translation of JV191225 and JV197112 revealed each to encode a 475 amino acid, C-terminal partial protein. The partial sequence derived from JV191225 is identical to the C-terminal 475 residues of Tigca-WDB-IA. The partial protein deduced from JV197112 (Tigca-WDB-IB; sequence not shown) differs from Tigca-WDB-IA's

34


A)

B)

C)

Fig. 9. PROTEIN PHOSPHATAE 1 (PP1) protein. (A) Alignment of Tigriopus californicus PP1-I (Tigca-PP1-I) and PP1-II isoform A (Tigca-PP1-IIA). (B) Alignment of Calanus finmarchicus PP1 (Calfi-PP1) and Tigca-PP1-I. (C) Alignment of Calfi-PP1 and Tigca-PP1-IIA. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, protein phosphatase 2A catalytic domains predicted by SMART analyses are highlighted in dark red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

C-terminus at 3 positions (residue 144, glycine for glutamic acid; residue 531, aspartic acid for glutamic acid; residue 573, alanine for threonine). Translation of JW537398 and JW510901 revealed each to encode an identical, 501 amino acid, full-length protein (Tigca-WDB-IIA; Fig. 11A and C); Tigca-WDB-IIA is distinct from Tigca-WDB-IA, with the two proteins sharing just 50.5% identity/70.1% similarity in amino acid composition (Fig. 11A). JV189438 encodes a 461 amino acid, Nterminal partial protein that is identical in sequence to the corresponding region of Tigca-WDB-IIA (sequence not shown), while JV195342 encode a 254 amino acid, N-terminal partial protein (Tigca-WDB-IIB; sequence not shown) that differs from the corresponding portion of

Tigca-WDB-IIA at just one residue (a serine for valine substitution at position 253). Alignment of Calfi-WDB-I and Tigca-WDB-IA is shown in Fig. 11B. As can be seen from this alignment, the proteins show a high degree of sequence conservation, being 70.9% identical/87.2% similar in amino acid composition. Tigca-WDB-IIA shows less sequence conservation with Calfi-WDB-I than does Tigca-WDB-IA, i.e. 54.3% identity/76.0% similarity (alignment not shown). However, comparison of the sequence of TigcaWDB-IIA with a different Calanus WDB isoform (Fig. 11C), i.e. CalfiWDB-II (Christie et al., 2013a), shows much greater amino acid conservation, here 87.6% identity/92.0% similarity. Based on this comparison, it


35

Fig. 10. MICROTUBULE STAR (MTS) protein. Alignment of the Calanus finmarchicus MTS-I (Calfi-MTS-I) and Tigriopus californicus MTS (Tigca-MTS). In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, protein phosphatase 2A catalytic domains predicted by SMART analyses are highlighted in dark red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

is likely that Tigca-WDB-IIA is a homolog of Calfi-WDB-II rather than of Calfi-WDB-I. Analysis by SMART revealed Tigca-WDB-IA to possess a single coiled coil region (highlighted gray in Fig. 11A and B); this domain was not identified in Tigca-WDB-IIA, nor was it predicted by SMART in either Calfi-WDB-I or Calfi-WDB-II (Christie et al., 2013a). While there was a difference in the structural domains of Tigca-WDBIA vs. the other proteins, the sequence conservation seen between it, Tigca-WDB-IIA, and the Calanus WDBs supports the inclusion of the Tigriopus sequences described here as members of the WIDERBORST family. To strengthen our confidence that the Tigriopus proteins just described are true members of the WDB family, each was used to query the annotated Drosophila proteins present in FlyBase and the nonredundant arthropod proteins curated in GenBank for the most similar sequences. For Tigca-WDB-IA, protein phosphatase 2A-B′, isoform J (FlyBase ID. No. FBpp0288759), was returned as the top FlyBase hit (Table 2), while serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform isoform X2 from the beetle T. castaneum (Accession No. XP_008195946; Kim et al., 2010) was returned as the most similar non-redundant arthropod protein in GenBank (Table 2); protein phosphatase 2A-B′, isoform B (FlyBase ID. No. FBpp0082978), and serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform-like isoform X3 from the honey bee Apis mellifera (Accession No. XP_006559896; Honeybee Genome Sequencing Consortium, 2006) were returned as the top protein hits in the searches of FlyBase and GenBank, respectively, using Tigca-WDB-IB as the input query (Table 2). For both Tigca-WDB-IIA and Tigca-WDB-IIB, WIDERBORST, isoform F, (FlyBase ID. No. FBpp0084575), was returned as the top FlyBase hit (Table 2). Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit epsilon isoform-like isoform X1 from the silk moth Bombyx mori (Accession No. XP_004924522; International Silkworm Genome Consortium, 2008) was returned as the top GenBank non-redundant arthropod protein hit for Tigca-WDB-IIA (Table 2), with protein phosphatase 2A B56 regulatory subunit gamma 3 from the tick Ixodes scapularis (Accession No. XP_002435135; Nene, unpublished direct GenBank submission) returned as the top GenBank hit for Tigca-WDB-IIB (Table 2). These BLAST results also support the Tigriopus WDBs being authentic members of WIDERBORST family. 3.2.6.2.2. TWINS (TWS). Using the sequence of C. finmarchicus TWS (Christie et al., 2013a) deduced from GAXK01019902 (Lenz et al., 2014) as a query, four transcripts (Accession Nos. JW536418, JW510059, JV196234 and JV190342) were identified as encoding putative TWS homologs (Table 1). Translation of these transcripts revealed each to encode a 449 amino acid, full-length protein. The sequences deduced from JW536418 and JW510059 are identical (Tigca-TWS-A; Fig. 12), with that encoded by both JV196234 and JV190342 (Tigca-TWS-B; sequence

not shown) differing at just one residue, an asparagine at position 224 in Tigca-TWS-A and a serine at this location in Tigca-TWS-B. Alignment of Tigca-TWS-A with the Calanus query used for its identification revealed the two proteins to be nearly identical in sequence (Fig. 12), i.e. they share 90.2% identity/97.8% similarity in amino acid composition; alignment of Calfi-TWS and Tigca-TWS-B showed an identical level of sequence conservation (alignment not shown). Seven WD40 domains were identified by SMART in both Tigca-TWS-A (highlighted in teal in Fig. 12) and Tigca-TWS-B; seven WD40 domains were also predicted by SMART analysis in Calfi-TWS (Christie et al., 2013a). With the exception of the sixth WD40 domain, the locations and amino acid sequences of these structural motifs are essentially identically conserved between Tigriopus proteins and Calfi-TWS (Fig. 12); the sixth WD40 domain in Calanus is N-terminally extended relative to those of Tigca-TWS-A and Tigca-TWS-B. Taken collectively, the sequence and structural motif conservation seen between the Tigriopus proteins and Calfi-TWS support Tigca-TWS-A and Tigca-TWS-B being true members of the TWINS family. To add further confidence to our assignment of Tigca-TWS-A and Tigca-TWS-B to the TWS family, the amino acid sequence of each protein was used to query both the annotated Drosophila proteins curated in FlyBase and the non-redundant arthropod proteins present in GenBank for the most similar sequences. For both Tigca-TWS-A and Tigca-TWS-B, the FlyBase searches returned TWINS, isoform I (FlyBase ID. No. FBpp0311386), as the top D. melanogaster protein hit (Table 2). Likewise, protein phosphatase 2A regulatory subunit B from the crab Scylla paramamosain (Accession No. AFK24473; Liu and Ye, unpublished direct GenBank submission) was identified as the most similar nonredundant arthropod protein to both Tigca-TWS-A and Tigca-TWS-B (Table 2). These BLAST results also support Tigca-TWS-A and TigcaTWS-B being authentic members of the TWINS family. 3.2.7. SHAGGY (SGG) Using the sequence of C. finmarchicus SGG (Christie et al., 2013a) deduced from GAXK01013351 (Lenz et al., 2014) as a query, four transcripts (Accession Nos. JW537477, JV196574, JW519106 and JV190685) were identified as encoding putative SGG homologs (Table 1). Translation of JW537477 and JV196574 revealed each to encode an identical, 445 amino acid, full-length protein (Tigca-SGG-A; Fig. 13). Translation of JW519106 and JV190685 revealed each to encode a 471 amino acid, full-length protein; here too, the deduced sequences are identical to one another (Tigca-SGG-B; Fig. 13A). Alignment of Tigca-SGG-A and TigcaSGG-B is shown in Fig. 13A. As can be seen from this panel, both proteins show extensive sequence conservation (94.0% identity/94.4% similarity), with the only significant difference being that Tigca-SGG-B is N-terminally extended relative to Tigca-SGG-A by 26 amino acids

36


(Fig. 13A); there are also two conservative amino acid substitutions within their region of overlap (Fig. 13A). Fig. 13B shows the alignment of Calfi-SGG with Tigca-SGG-A. Here, with the exceptions of three small areas of insertion, the Tigriopus and

Calanus proteins are nearly identical in sequence (i.e. 84.0% identity/ 90.5% similarity in amino acid composition). Tigca-SGG-B is also very similar in sequence to Calfi-SGG, though its N-terminal extension makes the percentage values lower than was seen for the comparison

A)

B)

Fig. 11. WIDERBORST (WDB) protein. (A) Alignment of Tigriopus californicus WDB-I isoform A (Tigca-WDB-IA) and WBD-II isoform A (Tigca-WDB-IIA). (B) Alignment of Calanus finmarchicus WDB-I and Tigca-WDB-IA. (C) Alignment of Calfi-WDB-II and Tigca-WDB-IIA. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, a single coiled coil region predicted by SMART analysis in Tigca-WDB-IA is highlighted in gray.


37

C)

Fig. 11 (continued).

between Tigca-SGG-A and the Calanus protein (i.e. 79.6% identity/85.5% similarity in amino acid composition). Analysis by SMART identified a single serine/threonine protein kinase catalytic domain in each of the Tigriopus sequences (highlighted in purple in Fig. 13), a domain that is also present in Calfi-SGG (Christie et al., 2013a). The sequence and structural motif conservation seen between the Tigriopus SGGs and Calfi-SGG strongly support the inclusion of Tigca-SGG-A and TigcaSGG-B as members of the SHAGGY family. To further confirm our assignment of Tigriopus proteins just described to the SHAGGY family, the amino acid sequence of each protein was used to query the annotated D. melanogaster proteins curated in FlyBase and the non-redundant arthropod proteins present in GenBank for the most similar sequences. For both Tigca-SGG-A and Tigca-SGG-B, SHAGGY, isoform A (FlyBase ID. No. FBpp0070450), was returned as the top Drosophila protein hit (Table 2). For the non-redundant arthropod peptide searches, glycogen synthase kinase-3 beta isoform X4 (a member

of the same protein family as SGG) from the wasp Nasonia vitripennis (Accession No. XP_008208522; Nasonia Genome Working Group, 2010) was identified as the most similar protein to Tigca-SGG-A (Table 2), while glycogen synthase kinase-3 beta isoform X3 from the wasp Microplitis demolitor (Accession No. XP_008543312; unpublished direct GenBank submission) was identified as the most similar sequence to Tigca-SGG-B (Table 2). The results of these BLAST searches also support Tigca-SGG-A and Tigca-SGG-B being authentic members of the SHAGGY family. 3.2.8. SUPERNUMERARY LIMBS (SLIMB) Using the sequence of C. finmarchicus SLIMB (Christie et al., 2013a) deduced from GAXK01020113 (Lenz et al., 2014) as a query, four transcripts (Accession Nos. JV192661, JW505360, JW524779 and JV198524) were identified as encoding putative SLIMB homologs (Table 1). Translation of JV192661 and JW505360 revealed each to encode an identical,

Fig. 12. TWINS (TWS) protein. Alignment of Calanus finmarchicus TWS (Calfi-TWS) and Tigriopus californicus TWS isoform A (Tigca-TWS-A). In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, WD40 domains predicted by SMART analyses are highlighted in teal. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

38


A)

B)

Fig. 13. SHAGGY (SGG) protein. (A) Alignment of Tigriopus californicus SGG isoform A (Tigca-SGG-A) and isoform B (Tigca-SGG-B). (B) Alignment of Calanus finmarchicus SGG (Calfi-SGG) and Tigca-SGG-A. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, serine/threonine protein kinase catalytic domains predicted by SMART analyses are highlighted in purple. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

622 amino acid, full-length protein (Tigca-SLIMB-A; Fig. 14). JW524779 also encodes a 622 amino acid, full-length protein (Tigca-SLIMB-B; Fig. 14A); this protein differs from Tigca-SLIMB-A at four residues, three of which are conservative amino acid substitutions (Fig. 14A). Translation of JV198524 revealed a 623 amino acid full-length protein (Tigca-SLIMB-C; Fig. 14A). This protein shows extensive sequence conservation with Tigca-SLIMB-A and Tigca-SLIMB-B, differing from the former at 11 positions and the latter at seven residues, in addition to the extra amino acid (Fig. 14A). The alignment of Calfi-SLIMB with Tigca-SLIMB-A is shown in Fig. 14B. As can be seen from this alignment, the two proteins share extensive sequence conservation (i.e. 69.1% identity/79.6% similarity in

amino acid composition). Tigca-SLIMB-B and Tigca-SLIMB-C show nearly identical levels of structural conservation with the Calanus protein, sharing 68.8%/80.1% and 67.7%/79.1% amino acid identity/similarity with Calfi-SLIMB, respectively (alignments not shown). Analysis of the Tigriopus SLIMBs by SMART identified one D domain of beta-TrCP (highlighted in blue in Fig. 14), one FBOX domain (highlighted in dark blue in Fig. 14) and seven WD40 repeats within each protein (highlighted in teal in Fig. 14); this same set of domains was described previously for Calfi-SLIMB (Christie et al., 2013a). The locations and amino acid sequences of all of the domains identified by SMART are highly conserved both among the Tigriopus SLIMBs (Fig. 14A) and between them and Calfi-SLIMB (Fig. 14B). The sequence and structural motif conservation

Fig. 14. SUPERNUMERARY LIMBS (SLIMB) protein. (A) Alignment of Tigriopus californicus SLIMB isoform A (Tigca-SLIMB-A), isoform B (Tigca-SLIMB-B) and isoform C (Tigca-SLIMB-C). (B) Alignment of Calanus finmarchicus SLIMB (Calfi-SLIMB) and Tigca-SLIMB-A. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, D domain of beta-TrCP, FBOX, WD40 domains predicted by SMART analyses are highlighted in blue, dark blue and teal, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


A)

B)

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Fig. 15. VRILLE (VRI) protein. Alignment of Calanus finmarchicus VRI (Calfi-VRI) and Tigriopus californicus VRI (Tigca-VRI). In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences. In this figure, basic region leucine zipper domains predicted by SMART analyses are highlighted in pink. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

seen between the Tigriopus SLIMBs and Calfi-SLIMB strongly support Tigca-SLIMB-A, Tigca-SLIMB-B and Tigca-SLIMB-C being authentic members of the SUPERNUMERARY LIMBS family. As a further confirmation of our assignment of the T. californicus proteins just described to the SLIMB family, the amino acid sequence of each protein was used to query the Drosophila proteins curated in FlyBase and the non-redundant arthropod proteins in GenBank for the most similar sequences. For Tigca-SLIMB-A, SUPERNUMERARY LIMBS, isoform C (FlyBase ID. No. FBpp0306059), was returned as the top Drosophila protein hit (Table 2), while for both Tigca-SLIMB-B and Tigca-SLIMB-C, SUPERNUMERARY LIMBS, isoform B (FlyBase ID. No. FBpp0303082), was identified as the top FlyBase hit (Table 2). For the non-redundant arthropod peptide searches, F-box/WD repeat-containing protein 1A (a member of the same protein family as SLIMB) from the termite Z. nevadensis (Accession No. KDR19729; Terrapon et al., 2014) was identified as the most similar protein to each of the Tigriopus sequences (Table 2). These BLAST results also support Tigca-SLIMB-A, Tigca-SLIMB-B and TigcaSLIMB-C being true members of the SUPERNUMERARY LIMBS family. 3.2.9. VRILLE (VRI) Using the sequence of C. finmarchicus VRI (Christie et al., 2013a) deduced from GAXK01130166 (Lenz et al., 2014) as a query, two transcripts (Accession Nos. JW538477 and JW511832) were identified as encoding putative VRI homologs (Table 1). Translation of JW538477 revealed a 297 amino acid, full-length protein (Tigca-VRI; Fig. 15); JW511832 encodes a 99 amino acid, N-terminal partial protein that is identical in sequence to the N-terminus of Tigca-VRI. Fig. 15 shows the alignment of Calfi-VRI with the Tigriopus protein. The two proteins share a modest amount of sequence conservation, being 23.3% identical/45.6% similar in amino acid composition. Analysis of Tigca-VRI by SMART identified one basic region leucine zipper (highlighted in pink in Fig. 15); this domain was also predicted by SMART for Calfi-VRI (Christie et al., 2013a). The location and amino acid sequence of the basic region leucine zipper is highly conserved between Tigca-VRI and Calfi-SLIMB (Fig. 15). The sequence and structural motif conservation seen between the Tigriopus and Calanus proteins support Tigca-VRI being a true member of the VRILLE family. To further confirm our assignment of the Tigriopus protein just described to the VRI family, it was used to query the Drosophila proteins

present in FlyBase and the non-redundant arthropod proteins present in GenBank for the most similar sequences. VRILLE, isoform E (FlyBase ID. No. FBpp0309715), was identified as the top FlyBase hit (Table 2), while VRILLE from the beetle T. castaneum (Accession No. EFA11543; Richards et al., 2008) was returned as the top arthropod protein hit for the GenBank query (Table 2). These BLAST results too support TigcaVRI being an authentic member of the VRILLE family. 3.3. Clock input proteins 3.3.1. CRYPTOCHROME 1 (CRY1) Using the sequence of an N-terminal partial C. finmarchicus CRY1 (Christie et al., 2013a) deduced from the expressed sequence tag GR410803 (Lenz et al., 2012) as a query, two transcripts (Accession Nos. JW523594 and JW504323) were identified as encoding putative CRY1 homologs (Table 1). Translation of JW523594 revealed a 553 amino acid, full-length protein (Tigca-CRY1-A; Fig. 16). Translation of JW504323 revealed a 443 amino acid, N-terminal partial protein (Tigca-CRY1-B; Fig. 16A) that differs from the corresponding portion of Tigca-CRY1-A at seven positions (Fig. 16A). Alignment Tigca-CRY1-A with the Calanus partial sequence used for its identification is shown in Fig. 16B. As can be seen from this alignment, there is considerable sequence homology present between the two proteins (Fig. 16B), at least over the extant sequence of Calfi-CRY1 (i.e. 42.2% identity/73.1% similarity in amino acid composition). Alignment of the partial Calanus protein and the extant portion of Tigca-CRY1-B also revealed extensive sequence conservation in their region of overlap, i.e. 42.2% identity/72.4% similarity (alignment not shown). No functional domains were predicted by SMART for either Tigca-CRY1-A or Tigca-CRY1-B, nor were any domains predicted for the partial Calanus protein by this program in a previous study (Christie et al., 2013a). Regardless, the sequence conservation seen between the Tigriopus and Calanus proteins suggests Tigca-CRY1-A and Tigca-CRY1-B are true members of the CRYPTOCHROME 1 family. To further increase our confidence in the assignment of the Tigriopus proteins just described to the CRY1 family, each sequence was used to query the arthropod non-redundant protein database of GenBank for the most similar sequence. CRYPTOCHROME precursor from the cricket


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A)

B)

Fig. 16. CRYPTOCHROME 1 (CRY1) protein. (A) Alignment of Tigriopus californicus CRY1 isoform A (Tigca-CRY1-A) and isoform B (Tigca-CRY1-B). (B) Alignment of a partial Calanus finmarchicus CRY1 (Calfi-CRY1) and Tigca-CRY1-A. In the line immediately below each sequence grouping, “*” indicates identical amino acid residues, while “:” and “.” denote amino acids that are similar in structure between sequences.

Dianemobius nigrofasciatus (Accession No. BAF45421; Izawa, Tufail and Takeda, unpublished direct GenBank submission) was returned as the most similar arthropod protein sequence to both Tigca-CRY1-A and Tigca-CRY1-B (Table 2). As for CRY2 (see Section 3.1.2), the monarch butterfly D. plexippus database was probed using each Tigriopus protein as the query rather than FlyBase, as both CRY2 and CRY1 are present in Danaus, but only the latter is present in D. melanogaster (Yuan et al., 2007). Here, D. plexippus CRYPTOCHROME 1 (Accession No. AAX58599; Zhu et al., 2005) was returned as the top BLAST hit for each of the Tigriopus proteins (Table 2). These BLAST results also support the

inclusion of Tigca-CRY1-A and Tigca-CRY1-B in the CRYPTOCHROME 1 family. 4. Discussion 4.1. In silico transcriptome mining as a rapid means of protein identification in crustaceans The molecular cascades that underlie circadian rhythms consist of complex, interacting networks of genes and proteins (e.g. Allada and

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Chung, 2010). Significant effort, resources and much manpower were used to identify and characterized the first of these systems, e.g. the circadian clock of fruit fly D. melanogaster. While the elucidation of the Drosophila clock provided a template for identifying and characterizing the molecular components of circadian systems in other species, particularly other arthropods, until recently the identification of the full complement of homologous genes/proteins was achieved for very few species. This lack of sequence information was due, in no small part, to the fact that the targeted molecular cloning of each of the putative network elements from a species in a gene-by-gene fashion was an arduous and expensive task. For example, prior to 201 l, just two circadian proteins had been identified from members of the Crustacea (Yang et al., 2006; Mazzotta et al., 2010). With the advent of high-throughput nucleotide sequencing, and the development of the computer-based tools for assembling and analyzing the resultant data, it has now become possible, and relatively inexpensive, to produce a full transcriptome, or in some cases a genome, for a species, including ones from crustaceans (e.g. Colbourne et al., 2011; Lenz et al., 2014). When such a dataset is available, and a reference set of genes/proteins from a closely related animal is extant, it is quite easy to identify targets of interest via in silico means. In fact, using this strategy, it is often possible to identify the full set of genes/proteins responsible for a complex signaling system on a timescale of weeks to months, rather than the years likely required to achieve this goal via targeted molecular cloning. For crustaceans, the power of the in silico approach to protein discovery is highlighted by the recent characterizations of complete circadian networks from the cladoceran D. pulex (Tilden et al., 2011) and the copepod C. finmarchicus (Christie et al., 2013a), and here, the circadian clock of T. californicus; the molecular components of these circadian systems were each identified and characterized in approximately one month's time. While the discoveries of these circadian networks highlight the power of this method, the use of in silico genome/transcriptome mining for gene/protein discovery is broadly applicable to the identification/characterization of essentially any gene/protein of interest (e.g. Christie et al., 2008; Gard et al., 2009; Ma et al., 2009; Christie et al., 2010; Ma et al., 2010; Christie et al., 2011; McCoole et al., 2011, 2012a, 2012b; Christie et al., 2013b,c; Christie, 2014a, 2014b, 2014c, 2014d, 2014e, 2014f; Christie et al., 2014a,b; Lenz et al., 2014). While still quite new to the field of crustacean biology, in silico genome/transcriptome mining would appear to be one of the most powerful tools for protein discovery in these animals, and as datasets are generated and publicly deposited for a larger number of crustaceans, which is now happening at a rapidly increasing pace, broad comparative studies of the evolution of gene/protein families and/or complete signaling systems should soon become possible. 4.2. Identification of a putative circadian signaling system in Tigriopus californicus In the study presented here, the recently characterized circadian network of the copepod C. finmarchicus (Christie et al., 2013a) was used as a reference to mine the extant T. californicus TSA data for transcripts encoding homologous proteins. The identified sequences were subsequently translated and their deduced proteins subjected to an identification workflow that included amino acid sequence comparisons and structural motif analysis. Via this vetting process, a complete circadian signaling system was identified for Tigriopus. The identified transcripts/ proteins include all of the components putatively necessary for the establishment of a core circadian clock (i.e. CLOCK, CRYPTOCHROME 2, CYCLE, PERIOD and TIMELESS), the vast majority of those likely involved in the modulation of the core clock (i.e. CASEIN KINASE II, CLOCKWORK ORANGE, DOUBLETIME, PROTEIN PHOSPHATASE 1, PROTEIN PHOSPHATASE 2A, SHAGGY, SUPERNUMERARY LIMBS and VRILLE), as well as a well known input pathway protein to circadian systems (i.e. CRYPTOCHROME 1). The identification of these transcripts/proteins represents just the third

full set of circadian molecular machinery described from a crustacean (those from D. pulex and C. finmarchicus being described previously [Christie et al., 2013a; Tilden et al., 2011]), and just the second obtained via the mining of transcribed sequences (the Daphnia circadian system was predicted via genomic analyses rather than from transcriptomic data [Tilden et al., 2011]). These data are also the only full set of circadian transcripts/proteins thus far identified from an intertidal species. For the most part, the circadian proteins deduced from the Tigriopus transcriptome appear to be full-length sequences. In fact, only for CLOCK and TIMELESS was no full-length protein identified. For most of the circadian-related molecules, multiple distinct proteins were discovered. Specifically, for several protein families, there appear to be multiple genes present in T. californicus, i.e. it is likely that there are two DOUBLETIME, two PROTEIN PHOSPHATASE 1, and two WIDERBORST genes in this species. Likewise, multiple alleles appear to be present for most of the proteins deduced here, as isoforms differing from one another by one or a small set of residues was commonly noted, e.g. the four T. californicus isoforms of PERIOD. Finally, there is also possible evidence for alternative splicing in the proteins deduced in this study, i.e. Tigca-PP1-IIB appears to be a truncated version of Tigca-PP1-IIA. How these different gene/protein variants may manifest themselves in the functioning of the Tigriopus clock, if they in fact do, remains unknown, though circadian protein variants/mutations in other species can clearly have a profound impact on the output of a clock, e.g. the presence of different alleles of TIMELESS in Drosophila can result in circadian rhythms being longer or shorter than the stereotypical 24-hour cycling pattern (Rothenfluh et al., 2000). 4.3. Is PAR DOMAIN PROTEIN 1 truly absent in copepods? Much of the molecular machinery required for the establishment of circadian cycling is highly conserved across phylogeny (e.g. Pegoraro and Tauber, 2011; Tarrant and Reitzel, 2013). This said, there is still variation between organisms in the specific roles played by different circadian molecules, the patterns in which they interact, and even their presence/absence in a given species. Members of the Insecta are a prime example of this variability, with multiple clock models having been proposed for different members of this taxon. For example, three distinct models based on the presence/absence of the CRYPTOCHROMES have been postulated to exist in insects (Yuan et al., 2007). In the first model, typically thought of as the ancestral-type insect clock, both CRY1 and CRY2 are present, but function differently, with the former acting as a photosensitive input pathway protein and the latter a photo-insensitive transcriptional repressor within the core clock itself. In the Drosophila-type clock, only CRY1 is present, serving here as an input pathway protein. In the third model (extant, for example, in beetles), only CRY2 is present, with this protein functioning as a transcriptional repressor within the core clock. Interestingly, in the bee A. mellifera, it appears that, in addition to CRY1, TIMELESS has also been lost (Rubin et al., 2006), though a fully functional circadian system is still present in this species. Thus, a wide variety of variant circadian systems can exist, even within relatively closely related animals. While we identified a large number of sequences encoding circadian proteins in Tigriopus, none encoding PAR DOMAIN PROTEIN 1 was identified. In Drosophila, PDP1 is involved in the transcriptional activation of the clk gene (e.g. Allada and Chung, 2010). Whether or not this transcript/protein is truly absent in Tigriopus remains an open question. While it is possible that PDP1 is absent in T. californicus, it is also possible that its transcript was simply not among those currently present in the publicly accessible transcriptome for this species. Interestingly, and seemingly in support of the former hypothesis, no PDP1encoding transcript was found when the Calanus transcriptome was analyzed for its circadian components (Christie et al., 2013a). Thus, while a member of this protein family is likely present in Daphnia (Tilden et al., 2011), it may have been lost, or may have been modified significantly in members of the Copepoda. Clearly additional investigation will be


needed to clarify this issue, but if PDP1 has been lost in copepods, it will be interesting to see how this manifests in the molecular functioning of circadian system in members of this crustacean order. 4.4. The circadian system of Tigriopus — a potential model for assessing the impacts of anthropogenic light pollution As mentioned in the Introduction, increasing utilization of coastal areas has resulted in a dramatic increase in anthropogenic stressors for intertidal and near-shore marine communities worldwide (e.g. Crain et al., 2009; Defeo et al., 2009; Dugan et al., 2011; Kennish et al., 2014), including pollution by artificial lighting (e.g. Longcore and Rich, 2004; Smith, 2009; Davies et al., 2012; Gaston et al., 2012; Davies et al., 2013; Gaston et al., 2013; Bennie et al., 2014; Inger et al., 2014). It is highly likely that this increase in anthropogenic light is disrupting the natural day/night cycles of organisms that exist in the intertidal zone, though, as of present, there is no good model species in which to assess the impact of this stressor. The proposal of copepods of the genus Tigriopus as models for environmental genomics in the intertidal zone (e.g. Raisuddin et al., 2007) opens an opportunity for such an assessment, and the identification of the transcripts/proteins putatively involved in the establishment of the circadian pacemaker of T. californicus provides the first targets for such analyses. Clearly much work will be needed before such an assessment can be undertaken. For example, all of the transcripts/proteins reported here are predicted and must be confirmed via molecular cloning or through biochemical means. Likewise, a base line for how the various genes and protein interact in Tigriopus must be established, as must their actual circadian cycling. Nonetheless, the data presented here provide a significant foundation for initiating such experiments. Acknowledgments Drs. Ann Castelfranco, Daniel Hartline and Petra Lenz, as well as members of the Castelfranco, Christie, Hartline and Lenz laboratories, especially Tiana Fontanilla and Vittoria Roncalli, are thanked for their helpful discussions concerning this study. Financial support for this study was provided by the Cades Foundation (Honolulu, Hawaii) and through the University of Hawaii at Manoa's Undergraduate Research Opportunities Program. References Allada, R., Chung, B.Y., 2010. Circadian organization of behavior and physiology in Drosophila. Annu. Rev. Physiol. 72, 605–624. Altermatt, F., Bieger, A., Morgan, S.G., 2012. Habitat characteristics and metapopulations dynamics of the copepod Tigriopus californicus. Mar. Ecol. Prog. Ser. 468, 85–93. Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bennie, J., Davies, T.W., Duffy, J.P., Inger, R., Gaston, K.J., 2014. Contrasting trends in light pollution across Europe based on satellite observed night time lights. Sci. Rep. 4, 3789. Christie, A.E., 2014a. Prediction of the first neuropeptides from a member of the Remipedia (Arthropoda, Crustacea). Gen. Comp. Endocrinol. 201, 74–86. Christie, A.E., 2014b. Prediction of the peptidomes of Tigriopus californicus and Lepeophtheirus salmonis (Copepoda, Crustacea). Gen. Comp. Endocrinol. 201, 87–106. Christie, A.E., 2014c. Peptide discovery in the ectoparasitic crustacean Argulus siamensis: identification of the first neuropeptides from a member of the Branchiura. Gen. Comp. Endocrinol. 204, 114–125. Christie, A.E., 2014d. In silico characterization of the peptidome of the sea louse Caligus rogercresseyi (Crustacea, Copepoda). Gen. Comp. Endocrinol. 204, 248–260. Christie, A.E., 2014e. Expansion of the Litopenaeus vannamei and Penaeus monodon peptidomes using transcriptome shotgun assembly sequence data. Gen. Comp. Endocrinol. 206, 235–254. Christie, A.E., 2014f. Identification of the first neuropeptides from the Amphipoda (Arthropoda, Crustacea). Gen. Comp. Endocrinol. 206, 96–110. Christie, A.E., Cashman, C.R., Brennan, H.R., Ma, M., Sousa, G.L., Li, L., Stemmler, E.A., Dickinson, P.S., 2008. Identification of putative crustacean neuropeptides using in silico analyses of publicly accessible expressed sequence tags. Gen. Comp. Endocrinol. 156, 246–264. Christie, A.E., Durkin, C.S., Hartline, N., Ohno, P., Lenz, P.H., 2010. Bioinformatic analyses of the publicly accessible crustacean expressed sequence tags (ESTs) reveal numerous

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Prediction of the peptidomes of Tigriopus californicus and Lepeophtheirus salmonis (Copepoda, Crustacea).

Molecular components of the circadian clock in mammals.

Molecular Phylogeny and Revision of Copepod Orders (Crustacea: Copepoda).

Circadian oscillators in the mouse brain: molecular clock components in the neocortex and cerebellar cortex.

Historical biogeography of the neotropical Diaptomidae (Crustacea: Copepoda).

Identification and validation of cryptochrome inhibitors that modulate the molecular circadian clock.

Identification of small-molecule modulators of the circadian clock.

Methods to study molecular mechanisms of the Neurospora circadian clock.

Molecular Mechanisms Regulating Temperature Compensation of the Circadian Clock.

Morphological and molecular affinities of two East Asian species of Stenhelia (Crustacea, Copepoda, Harpacticoida).

Chromosome-Wide Impacts on the Expression of Incompatibilities in Hybrids of Tigriopus californicus.

The Molecular Circadian Clock and Alcohol-Induced Liver Injury.

The Circadian Clock Gene Period1 Connects the Molecular Clock to Neural Activity in the Suprachiasmatic Nucleus.

Fertilization envelope in diapause eggs of Pontella mediterranea (Crustacea, Copepoda).

Acclimation and adaptation to common marine pollutants in the copepod Tigriopus californicus.

Sex-specific rejection in mate-guarding pair formation in the intertidal copepod, Tigriopus californicus.

Pharmacological control of circadian clock. Pharmacological manipulation of the circadian clock: from animal to human studies.

Circadian clock components RORα and Bmal1 mediate the anti-proliferative effect of MLN4924 in osteosarcoma cells.

Identification of a Circadian Clock in the Inferior Colliculus and Its Dysregulation by Noise Exposure.

Diversity of the free-living marine and freshwater Copepoda (Crustacea) in Costa Rica: a review.

Neurodegeneration and the Circadian Clock.

Physiology. Partitioning the circadian clock.

A new species of Metacyclops from a hyporheic habitat in North Vietnam (Crustacea, Copepoda, Cyclopidae).

The plant circadian clock looks like a traditional Japanese clock rather than a modern Western clock.