Genomic identification of a putative circadian system in the cladoceran crustacean Daphnia pulex

Abstract

Essentially nothing is known about the molecular underpinnings of crustacean circadian clocks. The genome of Daphnia pulex, the only crustacean genome available for public use, provides a unique resource for identifying putative circadian proteins in this species. Here, the Daphnia genome was mined for putative circadian protein genes using Drosophila melanogaster queries. The sequences of core clock (e.g. CLOCK, CYCLE, PERIOD, TIMELESS and CRYPTOCHROME 2), clock input (CRYPTOCHROME 1) and clock output (PIGMENT DISPERSING HORMONE RECEPTOR) proteins were deduced. Structural analyses and alignment of the Daphnia proteins with their Drosophila counterparts revealed extensive sequence conservation, particularly in functional domains. Comparisons of the Daphnia proteins with other sequences showed that they are, in most cases, more similar to homologs from other species, including vertebrates, than they are to those of Drosophila. The presence of both CRYPTOCHROME 1 and 2 in Daphnia suggests the organization of its clock may be more similar to that of the butterfly Danaus plexippus than to that of Drosophila (which possesses CRYPTOCHROME 1 but not CRYPTOCHROME 2). These data represent the first description of a putative circadian system from any crustacean, and provide a foundation for future molecular, anatomical and physiological investigations of circadian signaling in Daphnia.

1. Introduction

Virtually all organisms exhibit physiological and behavioral rhythms oscillating with a period of approximately 24-h. Regardless of species, these physiological/behavioral patterns, commonly referred to as circadian rhythms, are characterized by four basic properties (Allada and Chung, 2010): I. they persist under constant conditions (indicating the presence of a self-sustaining clock), II. the clock-driven activity reoccurs approximately every 24-h, III. the activity pattern is entrained by the solar day, and IV. the period of the activity, while sensitive to changes in environmental conditions, is stable over a wide range of temperatures. In addition, all circadian systems have three functional components (Allada and Chung, 2010): I. a core clock, which is responsible for time keeping, II. input pathways that act to synchronize the clock to the environment, and III. output pathways that transmit the timing information for the control of physiology and behavior (see Table 1 for the proteins in each category). Depending on the system in question, the cellular location of these components may be distinct or contained within a common locus.

Table 1.

Putative Daphnia pulex genes identified via genome mining

Protein	Gene name*	Gene location/length		Homology to query
		Scaffold	Length (nucleotides)	Blast score	E-value
CORE CLOCK PROTEINS1
CASEIN KINASE II α-subunit (CKII α)	dappu-ckII α	17	3693	576	1e-164
CASEIN KINASE II β-subunit (CK II β)	dappu-ckII β	41	2093	410	5e-115
CLOCK (CLK)	dappu-clk	27	5939	407	2e-113
CLOCKWORK ORANGE (CWO)	dappu-cwo†	19	3773	130	3e-30
CYCLE (CYC)	dappu-cyc	1	6473	437	1e-122
DOUBLETIME (DBT)	dappu-dbt	1	2124	550	1e-156
PAR DOMAIN PROTEIN 1e (PDP1e)	dappu-pdp1ε	21	4794	139	3e-33
PERIOD (PER)	dappu-per	47	5860	289	1e-77
PROTEIN PHOSPHATASE 1 (PP1) A	dappu-pp1 a	145	2591	584	3e-167
PROTEIN PHOSPHATASE 1 (PP1) B	dappu-pp1 b	12	1972	566	1e-161
PROTEIN PHOSPHATASE 2A (PP2A) catalytic subunit – MICROTUBULE STAR (MTS)	dappu-mts	13	2099	427	5e-120
PROTEIN PHOSPHATASE 2A (PP2A) regulatory subunit – WIDERBORST (WBT) A	dappu-wbt a	8	6199	746	0.0
PROTEIN PHOSPHATASE 2A (PP2A) regulatory subunit – WIDERBORST (WBT) B	dappu-wbt b	2	4882	614	5e-176
PROTEIN PHOSPHATASE 2A (PP2A) regulatory subunit – TWINS (TWS)	dappu-tws	43	4157	777	0.0
SHAGGY (SGG)	dappu-sgg	76	5712	665	0.0
SUPERNUMERARY LIMBS (SLIMB)	dappu-slimb	169	2880	807	0.0
TIMELESS 1 (TIM1) A	dappu-tim1 a	24	14166	326	8e-89
TIMELESS 1 (TIM1) B	dappu-tim1 b	6	5052	282	2e-75
TIMELESS 1 (TIM1) C	dappu-tim1 c	10	3650	276	1e-73
TIMELESS 1 (TIM1) D	dappu-tim1 d	6	5438	270	5e-72
TIMELESS 1 (TIM1) E	dappu-tim1 e	6	5542	258	3e-68
TIMELESS 1 (TIM1) F	dappu-tim1 f	24	11674	235	2e-61
TIMELESS 1 (TIM1) G	dappu-tim1 g	75	2798	218	3e-56
TIMELESS 1 (TIM1) H	dappu-tim1 h	91	4537	181	5e-45
VRILLE (VRI)	dappu-vri	92	2225	153	5e-37
INPUT PATHWAY PROTEINS1
CRYPTOCHROME (CRY) A – (CRY1)	dappu-cry a≠	40	2706	474	6e-134
CRYPTOCHROME (CRY) B – (CRY2)	dappu-cry bΔ	18	4661	358	7e-99
CRYPTOCHROME (CRY) C	dappu-cry c‡	10	3072	355	5e-98
CRYPTOCHROME (CRY) D – (CRY DASH)	dappu-cry d+	7	2052	129	5e-30
OUTPUT PATHWAY PROTEINS
PIGMENT DISPERSING HORMONE RECEPTOR (PDHR)	dappu-pdhr£	1	4621	362	5e-100

¹It should be noted that the group designations are based on the organization of the Drosophila melanogaster clock, whose proteins were used for querying the Daphnia genome. Unlike Drosophila, which possess only CRY1 (an input pathway protein), D. pulex appears to contain multiple CRY isoforms, including both CRY1 and CRY2 (a core clock protein), though in this table, all are presented under the “INPUT PATHWAY PROTEIN” heading.

^*All genes appear correctly annotated (Genes2010 Annotation) in wFleaBase (http://wfleabase.org/) unless otherwise noted.

^†Gene annotated as “conserved protein” in wFleaBase.

^≠Gene annotated as “dna photolyase” in wFleaBase.

^ΔGene annotated as “phr6-4, cryptochrome-1” in wFleaBase.

^‡Gene annotated as “phr6-4, cryptochrome-1” in wFleaBase.

⁺Gene annotated as “Cryptochrome-1” in wFleaBase.

^£Gene annotated as “class b secretin-like g-protein coupled receptor gprcal2” in wFleaBase.

While a number of species have been the subjects of investigations into the molecular mechanisms underlying the core circadian clock, perhaps the best studied are insects, and in particular, the fruit fly Drosophila melanogaster (Allada and Chung, 2010; Tomioka and Matsumoto, 2010). In Drosophila, work from many laboratories has elucidated several interacting molecular feedback loops, which form the core of a molecular clock. As recently reviewed by Allada and Chung (2010), a heterodimer formed by the CLOCK (CLK) and CYCLE (CYC) proteins binds to E-box elements in the promoter regions of the period (per) and timeless (tim) genes, activating their transcription (typically peaking late in the day). Due to this activation, PERIOD (PER) and TIMELESS (TIM) proteins are produced, accumulate and dimerize in the cytoplasm during the early evening hours, are translocated to the nucleus at approximately midnight, ultimately binding to the CLK/CYC heterodimer. The binding of the PER/TIM heterodimer to CLK/CYC inhibits this complex’s DNA binding to, and hence activation of, the per and tim genes during the late night.

In addition to the core clock proteins CLK, CYC, PER and TIM, a number of others are also involved in the control of the core clock feedback loop of Drosophila (Allada and Chung, 2010). Specifically, PER, TIM and CLK each exhibit rhythmic phosphorylation, with the peak in this state occurring in the late night or early day; PER is phosphorylated by casein kinase Iε (DOUBLETIME [DBT]) and CASEIN KINASE II (CKII), while TIM is phosphorylated by GLYCOGEN SYNTHASE KINASE 3B (SHAGGY [SGG]) and CKII. CLK is phosphorylated by a nuclear complex of PER and DBT. The phosphorylation of PER is known to enhance its repressor activity. In addition, phosphorylated PER and TIM are targets of the phosphatases PROTEIN PHOSPHATASE 2A (PP2A) and PROTEIN PHOSPHATASE 1 (PP1), the former of which is believed to be involved in generation of PER’s phosphorylation rhythm. The peak in phosphorylation of these proteins is known to precede their disappearance, which at least partially involves the ubiquitination of DBT-phosphorylated PER (and its resulting degradation via the ubiquitin-proteasome pathway) by the E3 ubiquitin ligase SUPERNUMERARY LIMBS (SLIMB); the proteolysis of PER removes repression of the CLK/CYC complex allowing for a new cycle of per and tim transcription. TIM is the target for CYPTOCHROME (CRY), a cell autonomous blue-light photoreceptor protein, which triggers its degradation (in the remainder of this paper Drosophila-type CRY is referred to as CRY1 to distinguish it from vertebrate-type CRY or CRY2, which is present both with and without CRY1 in non-drosophalid insects; e.g. Yuan et al., 2007).

In addition to their roles in regulating the PER-TIM feedback loop, the CLK/CYC heterodimer also activates several other interdependent feedback loops that are hypothesized to play roles in setting the phase and amplitude of the Drosophila core clock, as well as its rhythmic output (Allada and Chung, 2010). Specifically, CLK/CYC bind to E-box elements in the promoters of the par domain protein 1 (pdp1) and vrille (vri) genes, activating their transcription. In turn, the PAR DOMAIN PROTEIN 1 (PDP1) and VRILLE (VRI) proteins activate and repress, respectively, the transcription of the clock (clk) and cryptochrome (cry) genes. Because the accumulation of PDP1 is delayed relative to that of VRI, the rhythms of clk and cry activation are antiphase (peaking in early day) to those of per and tim. In addition the CLK/CYC heterodimer also activates transcription of the clockwork orange (cwo) gene. The CLOCKWORK ORANGE (CWO) protein, a basic helix-loop-helix (HLH) repressor, in turn targets the E-box elements of CLK/CYC target genes, repressing their activation.

Interestingly, while the Drosophila circadian system is arguably the best understood in the animal kingdom, it may not be stereotypical, even within insects (e.g. Zhu et al., 2005; Yuan et al., 2007; Zhu et al., 2008). Based on the complement of CRYs present, several models have been proposed for clock systems in insects (Yuan et al., 2007). Specifically, whereas D. melanogaster possesses a single CRY, in many insects, two CRYs have been identified, one similar to that of Drosophila, commonly referred to as dCRY or CRY1, and the other similar to that present in vertebrates, commonly referred to as CRY2; in several insects, only CRY2 has been found. In essentially all systems where it is present, CRY1 is proposed as a photosensitive input to the clock, providing a mechanism for entraining the clock to the solar day (Yuan et al., 2007). In contrast, CRY2 does not appear to play a role in photic entrainment, but rather appears to be a core clock protein, functioning as a repressor of CLK/CYC-mediated transcription (e.g. Zhu et al., 2005; Yuan et al., 2007; Zhu et al., 2008). Thus, in Drosophila, CRY likely functions solely as an input to the clock system, whereas in other insects members of the CRY family appear to serve both as inputs to the clock (CRY1) and as members of the core clock ensemble itself (CRY2); in insects with only CRY2, novel photic entrainment pathways are hypothesized, with CRY2 proposed to function primarily, perhaps solely, as a transcriptional repressor (e.g. Zhu et al., 2005; Yuan et al., 2007; Zhu et al., 2008). Evolutionary studies of CRY gene duplication and loss suggest that the clock system possessing both CRY1 and CRY2 is the most ancestral organization (Yuan et al., 2007).

As in other organisms, many crustaceans are known to display circadian patterns in physiology and behavior. As recently reviewed by Strauss and Dircksen (2010), known/postulated crustacean circadian behaviors include, but are not limited to, locomotion, feeding, moulting, reproduction, hatching/larval release, color change, and diel vertical migration. Interestingly, and despite the rich repertoire of circadian rhythms exhibited by crustaceans, essentially nothing is known about the molecular underpinnings of circadian clocks in these animals. While many laboratories have attempted to molecularly clone crustacean circadian proteins via reverse transcription polymerase chain reaction using degenerate primers, only two putative circadian proteins have thus far been identified and characterized from crustaceans, i.e. a putative homolog CLK from the freshwater prawn Macrobrachium rosenbergii (Yang et al., 2006) and a CRY homolog from the Antarctic krill Euphausia superba (Mazzotta et al., 2010).

The recent sequencing of the genome of the cladoceran crustacean Daphnia pulex provides an alternative avenue for identifying putative crustacean homologs of known insect circadian proteins, namely identification via genome mining; members of the genus Daphnia, like many other planktonic crustaceans, are known to exhibit pronounced diel migratory behaviors (e.g. Lampert, 1989; Loose, 1993; Loose and Dawidowicz, 1994). In the study presented here, we have used such a strategy to predict a large suite of D. pulex proteins that show significant homology to those that form the molecular underpinnings of the D. melanogaster circadian clock. Structural analysis of the identified proteins, which include, among others, putative homologs of PER, TIM, CLK and CYC, revealed that essentially all contain the domains known to be required for function in the fruit fly. Moreover, putative homologs of both CRY1 and CRY2 were identified, suggesting that the clock system of Daphnia is organized more like that proposed for lepidopterans and mosquitoes, than it is to the Drosophila system (Yuan et al., 2007), i.e. CRY1 acting as a circadian photoreceptor to the clock and CRY2 participating in the establishment of the core clock itself. In addition, a protein likely involved in mediating the output signaling of the clock, i.e. a receptor for pigment dispersing hormone, was identified and characterized. Taken collectively, the data presented here represent the first description of a putative circadian system from any crustacean, and provide a foundation for future molecular, anatomical and physiological investigations of circadian signaling in D. pulex and other crustacean species.

2. Materials and methods

2.1. Genome sequencing and gene modeling

For current descriptions of the preparation, sequencing and modeling of the D. pulex genome, readers are referred to http://wfleabase.org/ (Colbourne et al., 2005; 2011), which is maintained by the Indiana University Genome Informatics Laboratory (Indiana University, Bloomington, IN, USA).

2.2. Genome mining

Genome mining was accomplished using BLAST+ 2.2.23 software (downloadable from the National Center for Biotechnology Information, Bethesda, MD, USA; ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/) and the beta-release of the D. pulex Genes 2010 frozen genome assembly (Indiana University Genome Informatics Laboratory, and Center for Genomics and Bioinformatics at Indiana University, Bloomington, IN, USA; http://wfleabase.org/) as described in several earlier publications (Christie et al., 2011; McCoole et al., 2011); D. melanogaster proteins were used to query the genome. For all searches, the BLAST score and BLAST-generated E-value for significant alignment are provided in Table 1. To strengthen our gene identifications, the sequence of the Daphnia protein deduced from each gene was reciprocally blasted against the Drosophila proteins curated in FlyBase (Tweedie et al., 2009); the results of these analyses are shown in Table 2. In addition, each protein was blasted against all non-redundant protein sequences curated at NCBI (excluding Daphnia proteins, obvious partial proteins, synthetic constructs and provisional protein sequences) using the online program protein blast (blastp algorithm used; http://blast.ncbi.nlm.nih.gov/Blast.cgi); the top five hits for each protein are shown in Table 3.

Table 2.

Reciprocal blasting of deduced Daphnia pulex proteins versus known Drosophila melanogaster protein sequences in FlyBase

Daphnia protein	Top named protein hit in FlyBase
	Protein name	FlyBase ID	Homology to query
			Blast score	E-value
CORE CLOCK PROTEINS1
Dappu-CKII α	CKIIalpha-PC	FBpp0070043	566.2	1.06e-161
Dappu-CKII β	CKIIbeta-PE	FBpp0089135	409.1	9.91e-115
Dappu-CLK	CLK-PA	FBpp0076500	397.9	1.64e-110
Dappu-CWO	CWO-PA	FBpp0081723	106.3	7.84e-23
Dappu-CYC	CYC-PA	FBpp0074693	432.6	3.92e-73
Dappu-DBT	DCO-PC (DBT)	FBpp0085106	540.8	5.61e-154
Dappu-PDP1e	PDP1-PD	FBpp0076495	109.8	2.72e-24
Dappu-PER	PER-PA	FBpp0070455	264.6	3.30e-70
Dappu-PP1 A	PP1alpha-96A-PA	FBpp0084026	598.2	2.36e-171
Dappu-PP1 B	FLW-PB (PP1)	FBpp0071382	599.7	8.31e-172
Dappu-PP2A MTS	PP4-19C-PE (MTS)	FBpp0077017	592.8	1.02e-169
Dappu-PP2A WBT A	WDB-PE	FBpp0084579	625.9	1.67e-179
Dappu-PP2A WBT B	PP2A-B′-PJ	FBpp0288759	744.2	0
Dappu-PP2A TWS	TWS-PH	FBpp0081671	755.0	0
Dappu-SGG	SGG-PA	FBpp0070450	658.7	0
Dappu-SLIMB	SLMB-PA (SLIMB)	FBpp0083434	803.1	0
Dappu-TIM1 A	TIM-PJ	FBpp0291971	308.1	2.47e-83
Dappu-TIM1 B	TIM-PJ	FBpp0291971	261.9	1.69e-69
Dappu-TIM1 C	TIM-PJ	FBpp0291971	275.8	9.85e-74
Dappu-TIM1 D	TIM-PC	FBpp0077254	251.1	3.03e-66
Dappu-TIM1 E	TIM-PI	FBpp0291970	255.8	1.10e-67
Dappu-TIM1 F	TIM-PC	FBpp0077254	212.2	2.43e-54
Dappu-TIM1 G	TIM-PI	FBpp0291970	219.2	7.13e-57
Dappu-TIM1 H	TIM-PI	FBpp0291970	193.7	5.21e-49
Dappu-VRI	VRI-PD	FBpp0289297	149.1	8.29e-36
INPUT PATHWAY PROTEINS1
Dappu-CRY A	CRY-PA	FBpp0083150	476.1	5.56e-140
Dappu-CRY B	PHR6-4-PB	FBpp0080935	495.0	2.59e-134
Dappu-CRY C	PHR6-4-PB	FBpp0080935	594.7	5.15e-170
Dappu-CRY D	PHR6-4-PB	FBpp0080935	167.5	2.12e-41
OUTPUT PATHWAY PROTEINS
Dappu-PDHR	PDFR-PA	FBpp0099841	345.5	5.62e-95

Abbreviations: CKII, casein kinase II; CLK, CLOCK; CRY, CRYPTOCHROME; CWO, CLOCKWORK ORANGE; CYC, CYCLE; DBT, DOUBLETIME; MTS, MICROTUBULE STAR; PDFR, PIGMENT DISPERSING FACTOR RECEPTOR; PDHR, PIGMENT DISPERSING HOROMONE RECEPTOR; PDP1ε; PAR DOMAIN PROTEIN 1ε; PER, PERIOD; PHR, PHOTOLYASE; PP1, PROTEIN PHOSPHATASE 1; PP2A, PROTEIN PHOSPHATASE 2A; SGG, SHAGGY; SLIMB, SUPERNUMERARY LIMBS; TIM, TIMELESS; TWS, TWINS; VRI, VRILLE; WBT, WIDERBORST.

¹It should be noted that the group designations are based on the organization of the Drosophila melanogaster clock, whose proteins were used for querying the Daphnia genome. Unlike Drosophila, which possesses only CRY1 (an input pathway protein), D. pulex appears to contain multiple CRY isoforms, including both CRY1 and CRY2 (a core clock protein), though in this table, all are presented under the “INPUT PATHWAY PROTEIN” heading.

Table 3.

blastp analyses of putative Daphnia pulex circadian system proteins vs. all NCBI curated non-redundant protein sequences^*

Daphnia protein	Top five blastp protein hits
	Accession no.	Species	Protein description	Blast score	E-value
CASEIN KINASE II (CKII) – α subunit
Dappu-CKII α	1NA7_A	Homo sapiens	Chain A, Catalytic Subunit of Protein Kinase Ck2	624	9e-177
	EFN84867	Harpegnathos saltator	Casein kinase II subunit alpha	621	6e-176
	EFN62439	Camponotus floridanus	Casein kinase II subunit alpha	620	6e-176
	EGI62250	Acromyrmex echinatior	Casein kinase II subunit alpha	619	2e-175
	O76484	Spodoptera frugiperda	Casein kinase II subunit alpha	605	3e-171
CASEIN KINASE II (CKII) – β subunit
Dappu-CKII β	CAI18393	Homo sapiens	casein kinase 2, beta polypeptide	425	2e-117
	EDL26660	Mus musculus	casein kinase II, beta subunit, isoform CRA_c	424	3e-117
	EDL83523	Rattus norvegicus	casein kinase 2, beta subunit, isoform CRA_b	424	4e-117
	CBK38916	Mytilus galloprovincialis	protein kinase CK2beta regulatory subunit	422	2e-116
	CAJ83806	Xenopus tropicalis	casein kinase 2, beta subunit	421	4e-116
CLOCK (CLK)
Dappu-CLK	BAJ16353	Thermobia domestica	CLOCK	467	5e-129
	AAR14936	Antheraea pernyi	CLOCK	461	2e-127
	EGI62057	Acromyrmex echinatior	Circadian locomoter output cycles protein kaput	444	2e-122
	EFN76178	Hapregnathos saltator	Circadian locomoter output cycles protein kaput	442	1e-121
	EFN61630	Camponotus floridanus	Circadian locomoter output cycles protein kaput	441	3e-121
CLOCKWORK ORANGE (CWO)
Dappu-CWO	AAF54527	Drosophila melanogaster	Clockwork orange	132	3e-28
	AAF24476	Drosophila melanogaster	Sticky ch1	132	3e-28
	EFN67685	Camponotus floridanus	Hairy/enhancer-of-split related with YRPW motif protein 2	131	4e-28
	ACK77653	Drosophila melanogaster	RE11081p	131	5e-28
	EGI62806	Acromyrmex echinatior	Hairy/enhancer-of-split related with YRPW motif protein 1	130	9e-28
CYCLE (CYC)
Dappu-CYC	BAJ16354	Thermobia domestica	CYCLE	612	8e-173
	ABI21880	Lutzomyia longipalpis	Cycle	592	5e-167
	EFN72014	Camponotus floridanus	Aryl hydrocarbon receptor nuclear translocator-like protein 1	567	2e-159
	AAW80970	Xenopus laevis	BMAL1	483	5e-134
	Q6YGZ5	Tyto alba	Aryl hydrocarbon receptor nuclear translocator-like protein 1	474	3e-131
DOUBLETIME (DBT)/CASEIN KINASE Iε
Dappu-DBT	EFN64010	Camponotus floridanus	Casein kinase I isoform epsilon	590	9e-167
	EGI65788	Acromyrmex echinatior	Casein kinase I isoform epsilon	584	6e-165
	Q5ZLL1	Gallus gallus	Casein kinase I isoform epsilon	582	4e-164
	AAF65549	Mesocricetus auratus	casein kinase I epsilon	582	4e-164
	EFB26732	Ailuropoda melanoleuca	PANDA_002512	582	5e-164
PAR DOMAIN PROTEIN 1ε (PDP 1ε)
Dappu-PDP 1ε	EDM05669	Rattus norvegicus	rCG33934	176	5e-42
	AAN12026	Drosophila melanogaster	PAR-domain protein 1, isoform J	175	7e-42
	AAN12025	Drosophila melanogaster	PAR-domain protein 1, isoform D	175	8e-42
	AAN12027	Drosophila melanogaster	PAR-domain protein 1, isoform C	174	2e-41
	EFB19547	Ailuropoda melanoleuca	PANDA_015107	174	2e-41
PERIOD (PER)
Dappu-PER	AAN02439	Blattella germanica	circadian clock protein PERIOD	397	4e-108
	BAG48878	Gryllus bimaculatus	period	370	6e-100
	BAI47546	Modicogryllus siamensis	period clock protein	367	6e-99
	Q25637	Periplaneta americana	Period circadian protein	366	1e-98
	ADO24377	Laupala paranigra	period	353	7e-95
PROTEIN PHOSPHATASE 1 (PP1)
Dappu-PP1 A	EFN69572	Camponotus floridanus	Serine/threonine-protein phosphatase alpha-1 isoform	654	0.0
	ADY40510	Ascaris suum	Serine/threonine-protein phosphatase PP1-beta	643	0.0
	EFA05189	Tribolium castaneum	TcasGA2_TC015321	642	0.0
	ACO15366	Caligus clemensi	Serine/threonine-protein phosphatase PP1-beta	641	0.0
	BAJ85104	Hordeum vulgare	predicted protein	640	0.0
Dappu-PP1 B	CAD61270	Danio rerio	similar to human protein phosphatase 1, catalytic subunit, beta isoform	624	4e-177
	AAM88380	Canis lupus familiaris	protein phosphatase type 1 catalytic subunit delta isoform	622	2e-176
	ABQ18261	Carassius auratus	protein serine/threonine phosphatase-1 catalytic subunit beta isoform	622	2e-176
	EFN76901	Harpegnathos saltator	Serine/threonine-protein phosphatase PP1-beta catalytic subunit	622	2e-176
	BAE38902	Mus musculus	unnamed protein product	622	3e-176
PROTEIN PHOSPHATASE 2A – catalytic subunit MICROTUBULE STAR (MTS)
Dappu-MTS	EFN85419	Harpegnathos saltator	Serine/threonine-protein phosphatase 4 catalytic subunit	605	3e-171
	AAD01262	Takifugu rubripes	serine/threonine phosphatase	601	4e-170
	AAV38551	Homo sapiens	protein phosphatase 4 (formerly X), catalytic subunit	598	2e-169
	ACO10717	Caligus rogercresseyi	Serine/threonine-protein phosphatase 4 catalytic subunit	595	3e-168
	ACO10485	Caligus rogercresseyi	Serine/threonine-protein phosphatase 4 catalytic subunit	595	4e-168
PROTEIN PHOSPHATASE 2A – regulatory subunit WIDERBORST (WBT)
Dappu-WBT A	EFN66909	Camponotus floridanus	Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit epsilon isoform	769	0.0
	EGI66726	Acromyrmex echinatior	Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit alpha isoform	761	0.0
	EFA06323	Tribolium castaneum	TcasGA2_TC009194	747	0.0
	AAN14114	Drosophila melanogaster	widerborst, isoform C	742	0.0
	AAH64358	Homo sapiens	PPP2R5E protein	678	0.0
Dappu-WBT B	EFN69797	Camponotus floridanus	Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform	855	0.0
	EFN83893	Harpegnathos saltator	Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform	848	0.0
	BAG61599	Homo sapiens	unnamed protein product	836	0.0
	AAQ01559	Mus musculus	protein phosphatase 2A B56 delta subunit	830	0.0
	BAF84486	Homo sapiens	unnamed protein product	828	0.0
PROTEIN PHOSPHATASE 2A – regulatory subunit TWINS (TWS)
Dappu-TWS	EFA10095	Tribolium castaneum	TcasGA2_TC012273	823	0.0
	AAN13458	Drosophila melanogaster	twins, isoform E	777	0.0
	AAF54498	Drosophila melanogaster	twins, isoform A	776	0.0
	AAA99871	Drosophila melanogaster	phosphoprotein phosphatase 2A 55 lDa regulatory subunit	775	0.0
	P56932	Rattus norvegicus	Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B delta isoform	761	0.0
SHAGGY (SGG)/GLYCOGEN SYNTHASE KINASE 3B
Dappu-SGG	ABO61882	Rhipicephalus microplus	glycogen synthase kinase	676	0.0
	EFN78376	Harpegnathos saltator	Protein kinase shaggy	676	0.0
	EGI58906	Acromyrmex echinatior	Protein kinase shaggy	673	0.0
	Q5YJC2	Spermophilus citellus	Glycogen synthase kinase-3 beta	670	0.0
	P18266	Rattus norvegicus	Glycogen synthase kinase-3 beta	666	0.0
SUPERNUMERARY LIMBS (SLIMB)/E3 UBIQUITIN LIGASE
Dappu-SLIMB	BAE26547	Mus musculus	unnamed protein product	840	0.0
	EAW49771	Homo sapiens	beta-transducin repeat containing, isoform CRA_b	839	0.0
	EDL94307	Rattus norvegicus	beta-transducin repeat containing	839	0.0
	DAA14816	Bos taurus	beta-tranducin repeat containing protein	838	0.0
	BAG36210	Homo sapiens	unnamed protein product	838	0.0
TIMELESS (TIM)
Dappu-TIM A	AAR15505	Danaus plexippus	timeless	468	3e-129
	BAB85487	Sarcophaga bullata	timeless	344	4e-92
	AAB94890	Drosophila melanogaster	circadian clock protein	344	6e-92
	AAF51098	Drosophila melanogaster	timeless, isoform B	343	7e-92
	AAN10371	Drosophila melanogaster	timeless, isoform D	343	7e-92
Dappu-TIM B	AAF66996	Antheraea pernyi	timeless	394	4e-107
	AAR15505	Danaus plexippus	timeless	383	7e-104
	AAB94930	Drosophila virilis	circadian clock protein	313	1e-82
	ABW71828	Chymomyza costata	timeless	303	1e-79
	EFA04644	Tribolium castaneum	timeless	302	2e-79
Dappu-TIM C	EFA04644	Tribolium castaneum	timeless	397	4e-108
	AAR15505	Danaus plexippus	timeless	371	3e-100
	AAN10371	Drosophila melanogaster	timeless, isoform D	302	1e-79
	P49021	Drosophila melanogaster	Protein timeless	302	1e-79
	AAB94890	Drosophila melanogaster	circadian clock protein	302	2e-79
Dappu-TIM D	EFA04644	Tribolium castaneum	timeless	387	4e-105
	AAF66996	Antheraea pernyi	timeless	386	1e-104
	AAR15505	Danaus plexippus	timeless	382	2e-103
	AAB94890	Drosophila melanogaster	circadian clock protein	293	8e-77
	AAF51098	Drosophila melanogaster	timeless, isoform B	293	8e-77
Dappu-TIM E	ADV36936	Drosophila melanogaster	timeless, isoform I	282	2e-73
	AAB94890	Drosophila melanogaster	circadian clock protein	282	2e-73
	AAF51098	Drosophila melanogaster	timeless, isoform B	282	2e-73
	AAN10372	Drosophila melanogaster	timeless, isoform C	281	3e-73
	AAL13507	Drosophila melanogaster	GH03106p	281	3e-73
Dappu-TIM F	P49021	Drosophila melanogaster	Protein timeless	236	2e-59
	AAN10371	Drosophila melanogaster	timeless, isoform D	236	2e-59
	AAF51098	Drosophila melanogaster	timeless, isoform B	236	2e-59
	AAB94890	Drosophila melanogaster	circadian clock protein	236	2e-59
	AAF51097	Drosophila melanogaster	timeless, isoform K	236	3e-59
Dappu-TIM G	O17482	Drosophila virilis	Protein timeless	236	6e-60
	AAB94930	Drosophila virilis	tim	236	9e-60
	BAB85487	Sarcophaga bullata	timeless	232	2e-58
	AAY40757	Aedes aegypti	TIMELESS	231	3e-58
	AAF51098	Drosophila melanogaster	timeless, isoform B	231	4e-58
Dappu-TIM H	BAJ16356	Gryllus bimaculatus	TIMELESS	216	1e-53
	O17482	Drosophila virilis	Protein timeless	214	6e-53
	AAB94930	Drosophila virilis	tim	213	9e-53
	BAB85471	Sarcophaga crassipalpis	TIMELESS	212	3e-52
	BAB85487	Sarcophaga bullata	timeless	211	7e-52
VRILLE (VRI)
Dappu-VRI	AAT86041	Danaus plexippus	vrille	169	2e-39
	AAS92609	Antheraea pernyi	vrille	166	1e-38
	CAX37108	Acyrthosiphon pisum	vrille	159	1e-36
	CAA72535	Drosophila melanogaster	bZIP transcription factor	155	3e-35
	AAF52237	Drosophila melanogaster	vrille, isoform A	155	3e-35
CRYPTOCHROME (CRY)
Dappu-CRY A	BAF45421	Dianemobius nigrofasciatus	cryptochrome precursor	523	2e-146
	AAK11644	Antheraea pernyi	cryptochrome	516	2e-144
	AAX58599	Danaus plexippus	cryptochrome	511	1e-142
	BAI67362	Bactrocera cucurbitae	cryptochrome	506	5e-141
	BAI67363	Bactrocera cucurbitae	cryptochrome	504	9e-141
Dappu-CRY B	EFR20390	Anopheles darlingi	AND_20159	882	0.0
	ABB29887	Anopheles gambiae	cryptochrome 2	877	0.0
	ABO31112	Bombus impatiens	cryptochrome 2 protein	851	0.0
	EFN85258	Harpegnathos saltator	Cryptochrome-1	850	0.0
	BAG07408	Riptotus pedestris	cryptochrome-m	844	0.0
Dappu-CRY C	AAI66277	Xenopus tropicalis	LOC100144974 protein	669	0.0
	EFR25332	Anopheles darlingi	AND_09443	620	1e-175
	ACN10565	Salmo salar	Cryptochrome-1	612	5e-173
	CAG02357	Tetraodon nigroviridis	unnamed protein product	612	6e-173
	AAL90322	Drosophila melanogaster	RE11660p	595	8e-168
Dappu-CRY D	CBN81995	Dicentrarchus labrax	Cryptochrome DASH	634	1e-179
	Q4KML2	Danio rerio	Cryptochrome DASH	623	2e-176
	AAH98514	Danio rerio	Cryptochrome DASH	619	3e-175
	CAO90052	Microcystis aeruginosa	unnamed protein product	475	8e-132
	P77967	Synechocystis sp.	Cryptochrome DASH	452	5e-125
PIGMENT DISPERSING HORMONE RECEPTOR (PDHR)
Dappu-PDHR	BAH85843	Marsupenaeus japonicus	pigment dispersing hormone receptor	385	8e-105
	ADG46049	Drosophila melanogaster	MIP16931p	380	3e-103
	AAF45788	Drosophila melanogaster	PDF receptor	380	3e-103
	CAB72288	Drosophila melanogaster	EG:BACR25B3.3	347	2e-93
	CAP34509	Caenorhabditis briggsae	CBG_16586	227	7e-71

^*excluding Daphnia proteins, partial proteins, synthetic constructs and provisional protein sequences.

2.3. Analyses of protein structure

Analyses of protein structural motifs were accomplished using the online program SMART (http://smart.embl-heidelberg.de/ [Schultz et al., 1998; Letunic et al., 2009]) and homology to the structural motifs of previously described insect circadian proteins, predominantly ones from D. melanogaster. Alignment of all proteins shown in our figures was done using the online program MAFFT version 6 (http://align.bmr.kyushu-u.ak.jp/mafft/online/server/; [Katoh and Toh, 2008]). Amino acid identity was calculated as number of identical amino acids (denoted by [*]) divided by the total number of amino acids in the longest sequence, while amino acid similarity was calculated as number of identical and similar amino acids (the latter denoted by the [:] and [.] symbols in the protein alignments) divided by the total number of amino acids in longest sequence.

2.4. Figure production

Alignments generated in MAFFT were copied and pasted into Microsoft Word, and the structural domains identified by SMART analyses, colored using this program. For all figures, a common coloring scheme was used to highlight each structural domain: serine/threonine kinase catalytic, red; HLH, green; PAS, light blue; PAC, blue; coiled-coil, pink; orange, yellow; basic region leucine zipper, dark blue; protein phosphatase 2A, dark green; WD40, dark red; FBOX, dark gray; transmembrane, light gray; hormone receptor, black.

3. Results

As stated in Section 1, all known circadian systems are composed of three functional components: a core clock, input pathways that act to synchronize the clock to the environment, and output pathways that transmit timing information. With one exception, the results of genome searches and protein analyses are grouped according their putative role within the theoretical Daphnia circadian system, i.e. core clock proteins, input pathway proteins, or output pathway proteins; the results for CRY are presented under “input pathway proteins”, though, in some species, family members also serve as key components of the core clock as well. Within each of these grouping, data are presented in alphabetical order based on the Drosophila protein name. It should be noted that the Daphnia protein sequences reported in this study are based on the Genes 2010 gene model algorithm, which, in a previous study (Christie et al., 2011), was found to typically have the best fit with the extant D. pulex transcriptome data, at least for peptide precursor protein genes. This said, other gene model algorithms (i.e. JGI, Gnomon, PASA and SNAP) did in some cases predict slightly different protein sequences from those shown here, and readers should take heed of this and treat the sequences presented as theoretical rather than biochemically-confirmed.

3.1. Core clock proteins

3.1.1. CASEIN KINASE II (CKII)

3.1.1.1. CKII α-subunit

A single D. pulex gene (dappu-ckII α) was identified as encoding a putative CKII α-subunit protein via a query using a D. melanogaster CKII α (Accession no. AAN11415; Adams et al., 2000). The Genes 2010 gene model shows dappu-ckII α to be located on Scaffold 17 of the genome, with a predicted length of 3693 nucleotides (Table 1).

Figure 1A shows the alignment of the protein deduced from dappu-ckII α (Dappu-CKII α; 365 amino acids in overall length) with that of the Drosophila query (336 amino acids long).

Figure 1.

Putative Daphnia pulex CASEIN KINASE II (CKII) α- and β-subunit proteins. (A) Alignment of Drosophila melanogaster CKII α-subunit (Drome-CKII α) with D. pulex CKII α-subunit (Dappu-CKII α). (B). Alignment of D. melanogaster CKII β-subunit (Drome-CKII β) with D. pulex CKII β-subunit (Dappu-CKII β). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, serine/threonine kinase catalytic domains predicted by SMART analyses are highlighted in red.

Comparison of the sequence of Dappu-CKII α with that of Drome-CKII α revealed 81.1% amino acid identity/89.6 % amino acid similarity between the two proteins. SMART analyses of Dappu-CKII α and Drome-CKII α identified a single, highly conserved (93.4% identical/97.9% similar) serine/threonine kinase catalytic domain within each protein (Fig. 1A).

Reciprocal blasting of Dappu-CKII α against all proteins curated in FlyBase revealed CKII α (Flybase no. FBpp0070043) as the D. melanogaster protein most similar to the Daphnia query (Table 2). Interestingly, blastp comparison of Dappu-CKII α with all non-redundant protein sequences curated by NCBI revealed the catalytic subunit of human CK II (1NA7_A) to be the most similar protein match (Table 3); the remaining top five blastp hits are all insect CKII α proteins, though Drosophila CKII α is not among them (Table 3).

3.1.1.2. CKII β-subunit

A single D. pulex gene (dappu-ckII β) was identified as encoding a putative CKII β-subunit protein via a query using a D. melanogaster CKII β (AAF48093; Adams et al., 2000). The Genes 2010 gene model shows dappu-ckII β to be located on Scaffold 41 of the genome, with a predicted length of 2093 nucleotides (Table 1).

Figure 1B shows the alignment of the protein deduced from dappu-ckII β (Dappu-CKII β; 221 amino acids in overall length) with that of the Drosophila query (215 amino acids long). Comparison of the sequence of Dappu-CKII β with that of Drome-CKII β revealed 84.2% amino acid identity/94.6 % amino acid similarity between the two proteins. No functional domains were identified in either Dappu-CKII β or Drome-CKII β via SMART analysis.

Reciprocal blasting of Dappu-CKII β against all proteins curated in FlyBase identified CKII β (Flybase No. FBpp0089135) as the D. melanogaster protein most similar to the Daphnia query (Table 2). Interestingly, and similar to the result found for Dappu-CKII α, a human CKII β protein (CAI18393) was found to have the highest homology score when Dappu-CKII β was blasted against all non-redundant protein sequences curated by NCBI (Table 3). Perhaps even more surprising was the finding that none of the remaining top five blastp hits from the NCBI database were from insects. In fact, only one invertebrate protein, a CKII β from the bivalve mollusc Mytilus galloprovincialis (CBK3891), was among these hits (Table 3).

3.1.2. CLOCK (CLK)

A single D. pulex gene (dappu-clk) was identified as encoding a putative CLK protein via a query using a D. melanogaster CLK (AAC62234; Bae et al., 1998). The Genes 2010 gene model shows dappu-clk to be located on Scaffold 27 of the genome, with a predicted length of 5939 nucleotides (Table 1).

Figure 2A shows the alignment of the protein deduced from dappu-clk (Dappu-CLK; 890 amino acids in overall length) with that of the Drosophila query (1027 amino acids long). Comparison of the sequence of Dappu-CLK with that of Drome-CLK revealed 30.8% amino acid identity/57.4% amino acid similarity between the two proteins. SMART analyses of Dappu-CLK and Drome-CLK identified similar, though not identical, sets of structural domains within each protein. Specifically, both Dappu-CLK and Drome-CLK are predicted to contain a single HLH domain, two PAS domains, and a single PAC domain (Fig. 2A). In addition, Drome-CLK is predicted to contain three coiled-coil regions; this motif is absent in Dappu-CLK (Fig. 2A). For those domains that are shared between Dappu-CLK and Drome-CLK, extensive amino acid conservation is evident: HLH, 60.8% identity/96.1 % similarity; PAS1, 43.3 % identity/80.6 % similarity; PAS2, 73.1 % identity/92.5 % similarity; PAC, 70.5 % identity/93.2 % similarity. Essentially no conservation of sequence is seen between the two proteins in any of the coiled-coil regions (Fig. 2A).

Figure 2.

Putative Daphnia pulex CLOCK (CLK) protein. (A) Alignment of Drosophila melanogaster CLK (Drome-CLK) with D. pulex CLK (Dappu-CLK). (B). Alignment of Macrobrachium rosenbergii CLK (Macro-CLK) with Dappu-CLK. In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, helix-loop-helix, PAS, PAC and coiled-coil domains identified by SMART analyses are highlighted in green, light blue, blue, and pink, respectively.

As stated in Section 1, CLK is one of the few circadian proteins for which a putative crustacean family member has been identified, i.e. M. rosenbergii CLK (Yang et al., 2006). Comparison of the sequences of Dappu-CLK and Macro-CLK (AAX44045; Yang et al., 2006), revealed a level of amino acid identity/similarity similar to that seen for Dappu-CLK and Drome-CLK (i.e. 30.4 % identity/56.0 % similarity; Fig. 2B). As for Dappu-CLK and Drome-CLK, SMART analysis of Macro-CLK identified single HLH domain, two PAS domains, and a single PAC domain within this protein (Fig. 2B). In addition, this analysis identified a single coiled-coil region in Macro-CLK (Fig. 2B). Comparison of the HLH, PAS and PAC domains of Macro-CLK and Dappu-CLK revealed slightly higher levels of conservation to those reported above for Drome-CLK and Dappu-CLK (HLH, 62.7% identity/100 % similarity; PAS1, 60.0% identity/85.0% similarity; PAS2, 80.0% identity/95.0% similarity; PAC, 81.8% identity/93.2% similarity). Little sequence conservation is seen between the Macrobrachium and Daphnia CLKs in the coiled-coil region of the former protein.

Reciprocal blasting of Dappu-CLK against the proteins curated in FlyBase identified CLK (Flybase no. FBpp0076500) as the D. melanogaster protein most similar to the Daphnia query (Table 2). Comparison of Dappu-CLK with all non-redundant protein sequences curated by NCBI revealed the top five blastp hits to be insect CLK sequences, with a CLK from the firebrat Thermobia domestica (BAJ16353) showing the highest similarity (Table 3); Drosophila CLK was not among these proteins (Table 3).

3.1.3. CLOCKWORK ORANGE (CWO)

A single D. pulex gene (dappu-cwo) was identified as encoding a putative CWO protein via a query using a D. melanogaster CWO (AAF54527; Adams et al., 2000). The Genes 2010 gene model shows dappu-cwo to be located on Scaffold 19 of the genome, with a predicted length of 3773 nucleotides (Table 1).

Figure 3 shows the alignment of the protein deduced from dappu-cwo (Dappu-CWO; 809 amino acids in overall length) with that of the Drosophila query (698 amino acids long). Comparison of the sequence of Dappu-CWO with that of Drome-CWO revealed 23.9% amino acid identity/51.4% amino acid similarity between the two proteins. SMART analyses of Dappu-CWO and Drome-CWO identified a single HLH and a single orange domain within each protein (Fig. 3); high levels of amino acid conservation were evident when the Daphnia domains were compared to their Drosophila counter parts (HLH, 78.2% identity/92.7% similarity; ORANGE, 37.5% identity/85% similarity).

Figure 3.

Putative Daphnia pulex CLOCKWORK ORANGE (CWO) protein. Alignment of Drosophila melanogaster CWO (Drome-CWO) with D. pulex CWO (Dappu-CWO). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, helix-loop-helix and orange domains identified by SMART analyses are highlighted in green and yellow, respectively.

Reciprocal blasting of Dappu-CWO against all proteins curated in FlyBase identified CWO (Flybase No. FBpp0081723) as the D. melanogaster protein most similar to the Daphnia query. Moreover, blastp comparison of Dappu-CWO with all non-redundant protein sequences curated by NCBI revealed D. melanogaster CWO (AAF54527) as most similar to this protein as well (Table 3); three of the top five blastp hits are D. melanogaster sequences, the remaining two are also insect proteins (Table 3).

3.1.4. CYCLE (CYC)

A single D. pulex gene (dappu-cyc) was identified as encoding a putative CYC protein via a query using a D. melanogaster CYC (AAF49107; Adams et al., 2000). The Genes 2010 gene model shows dappu-cyc to be located on Scaffold 1 of the genome, with a predicted length of 6473 nucleotides (Table 1).

Figure 4 shows the alignment of the protein deduced from dappu-cyc (Dappu-CYC; 654 amino acids in overall length) with that of the Drosophila query (413 amino acids long). Comparison of the sequence of Dappu-CYC with that of Drome-CYC revealed 33.9% amino acid identity/49.7% amino acid similarity between the two proteins. SMART analyses of Dappu- and Drome-CYC identified a single HLH domain, two PAS domains and a single PAC domain in each protein (Fig. 4). Comparisons of the D. pulex domains with those of D. melanogaster show high levels of amino acid conservation in these portions of the proteins: HLH, 71.4 % identity/95.2 % similarity; PAS1, 75.0 % identity/94.1 % similarity; PAS2, 59.7 % identity/91.9 % similarity; PAC, 53.8% identity/94.9 % similarity.

Figure 4.

Putative Daphnia pulex CYCLE (CYC) protein. Alignment of Drosophila melanogaster CYC (Drome-CYC) with D. pulex CYC (Dappu-CYC). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, helix-loop-helix, PAS and PAC domains identified by SMART analyses are highlighted in green, light blue and blue respectively.

Reciprocal blasting of Dappu-CYC against all proteins curated in FlyBase identified CYC (Flybase no. FBpp0074693) as the D. melanogaster protein most similar to the Daphnia query (Table 2). Comparison of Dappu-CYC with all non-redundant protein sequences curated by NCBI identified a CYC from the firebrat T. domestica (BAJ16354) to be the most similar protein match (Table 3); no Drosophila proteins were among the top five blastp hits (Table 3).

3.1.5. DOUBLETIME (DBT)

A single D. pulex gene (dappu-dbt) was identified as encoding a putative DBT protein via a query using a D. melanogaster DBT (AAF57110; Adams et al., 2000). The Genes 2010 gene model shows dappu-dbt to be located on Scaffold 1 of the genome, with a predicted length of 2124 nucleotides (Table 1).

Figure 5 shows the alignment of the protein deduced from dappu-dbt (Dappu-DBT; 409 amino acids in overall length) with that of the Drosophila query (440 amino acids long). Comparisons of the sequence of Dappu-DBT with Drome-DBT revealed 61.4% amino acid identity/78.4% amino acid similarity between the two proteins. SMART analyses of Dappu-DBT and Drome-DBT identified a serine/threonine kinase domain in each protein, the sequences of which were nearly identical, i.e. 82.6% amino acid identity/96.2% amino acid similarity (Fig. 5).

Figure 5.

Putative Daphnia pulex DOUBLETIME (DBT) protein. Alignment of Drosophila melanogaster DBT (Drome-DBT) with D. pulex DBT (Dappu-DBT). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, serine/threonine kinase domains identified by SMART analyses are highlighted in red.

Reciprocal blasting of Dappu-DBT against all proteins curated in FlyBase identified DBT (Flybase no. FBpp0085106) as the most similar D. melanogaster protein to the Daphnia query (Table 2). Comparison of Dappu-DBT with all non-redundant protein sequences curated by NCBI revealed a casein kinase Iε (an alternative name for DBT) from the ant Camponotus floridanus (EFN64010) to be most similar to Daphnia DBT (Table 3); no Drosophila proteins were among the top five blastp hits (Table 3).

3.1.6. PAR DOMAINE PROTEIN 1ε (PDP1ε)

A single D. pulex gene (dappu-pdp1ε) was identified as encoding a putative PDP1ε protein via a query using a D. melanogaster PDP1ε (AAF04509; Lin et al., 1997). The Genes 2010 gene model shows dappu-pdp1ε to be located on Scaffold 21 of the genome, with a predicted length of 4794 nucleotides (Table 1).

Figure 6 shows the alignment of the protein deduced from dappu-pdp1ε (Dappu-PDP1ε; 350 amino acids in overall length) with that of the Drosophila query (351 amino acids long). Comparisons of the sequence of Dappu-PDP1ε with that of Drome-PDP1ε revealed 39.6% amino acid identity/63.5% amino acid similarity between the two proteins. SMART analyses of Dappu-PDP1ε and Drome-PDP1ε identified a single basic region leucine zipper domain in each protein, which were 67.7% identical/81.5% similar in amino acid composition (Fig. 6).

Figure 6.

Putative Daphnia pulex PAR DOMAIN PROTEIN 1ε (PDP1ε) protein. Alignment of Drosophila melanogaster PDP1ε (Drome-PDP1ε) with D. pulex PDP1ε (Dappu-PDP1ε). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, basic region leucine zipper domains identified by SMART analyses are highlighted in dark blue.

Reciprocal blasting of Dappu-PDP1ε against all proteins curated in FlyBase identified PDP1 (Flybase No. FBpp0076495) as the D. melanogaster protein most similar to the Daphnia query (Table 2). Interestingly, comparison of Dappu-PDP1ε with all non-redundant protein sequences curated by NCBI identified a rat protein (EDM05669), likely a PDP1, to be most similar to Dappu-PDP1ε (Table 3), though the next three of the top five blastp hits were D. melanogaster PDP1 isoforms (Table 3).

3.1.7. PERIOD (PER)

A single D. pulex gene (dappu-per) was identified as encoding a putative PER protein via a query using a D. melanogaster PER (AAF45804; Adams et al., 2000). The Genes 2010 gene model shows dappu-per to be located on Scaffold 47 of the genome, with a predicted length of 5860 nucleotides (Table 1).

Figure 7 shows the alignment of the protein deduced from dappu-per (Dappu-PER; 1286 amino acids in overall length) with that of the Drosophila query (1218 amino acids long). Comparisons of the sequence of Dappu-PER with Drome-PER revealed 27.4% amino acid identity/59.6% amino acid similarity between the two proteins. SMART analyses of Dappu-PER and Drome-PER identified two PAS domains in each protein (Fig. 7), both of which showed considerable amino acid conservation between the two proteins: PAS-1, 50.0% amino acid identity/82.3% amino acid similarity; PAS-2, 47.3% amino acid identity/84.2% amino acid similarity. In addition, SMART analysis identified a PAC domain within the Drome-PER (Fig. 7) but not in Dappu-PER. Interestingly, the portion of Dappu-PER that overlaps with the Drosophila PAC domain is 72.7% identical/93.2% similar in amino acid composition to that of the Drosophila protein (Fig. 7).

Figure 7.

Putative Daphnia pulex PERIOD (PER) protein. Alignment of Drosophila melanogaster PER (Drome-PER) with D. pulex PER (Dappu-PER). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, PAS and PAC domains identified by SMART analyses are highlighted in light blue and blue, respectively.

Reciprocal blasting of Dappu-PER against the proteins curated in FlyBase identified PER (Flybase No. FBpp0070455) as the D. melanogaster protein most similar to the Daphnia query (Table 2). Moreover, blastp comparison of Dappu-PER with all non-redundant protein sequences curated by NCBI showed the top five hits to be insect PER proteins (Table 3), with an isoform from the cockroach Blattella germanica (AAN02439; Table 3) exhibiting the highest similarity.

3.1.8. PROTEIN PHOSPHATASE 1 (PP1)

Two D. pulex genes (dappu-pp1 a and dappu-pp1 b) were identified as encoding putative PP1 proteins via a query using a D. melanogaster PP1 (CAA39820; Dombradi et al., 1990). The Genes 2010 gene model shows dappu-pp1 a and dappu-pp1 b to be located on Scaffolds 145 and 12 of the genome, respectively, with lengths of 2591 and 1972 nucleotides (Table 1).

Figure 8A shows the alignment of the protein deduced from dappu-pp1 a (Dappu-PP1 A; 332 amino acids in overall length) with that of the Drosophila query (327 amino acids long). Comparison of the sequences of Dappu-PP1 A with Drome-PP1 revealed 84.0% amino acid identity/90.7% amino acid similarity between the two proteins. Comparison of the sequence of Dappu-PP1 B (325 amino acids in overall length) with that of the Drosophila query with that of Drome-PP1 revealed a similar level of amino acid conservation, i.e. 81.3% identity/89.6% similarity (alignment not shown). Figure 8B shows the alignment of the two Daphnia PP1s with one another. As can be seen from this panel, the two proteins are nearly identical in amino acid sequences (84.3% identity/96.9% similarity in amino acid composition). SMART analyses of Dappu-PP1 A, Dappu-PP1 B, and Drome-PP1 identified a single serine/threonine protein kinase domain in each protein (Fig. 8A–B). The serine/threonine protein kinase domain in each of the Daphnia proteins is nearly identical to that present in their Drosophila counterpart: Dappu-PP1 A vs. Drome-PP1, 95.9% amino acid identity/100% amino acid similarity; Dappu-PP1 B vs. Drome-PP1, 89.6% amino acid identity/99.3% amino acid similarity. Similarly, this domain is highly conserved between the two Daphnia proteins (91.5% identity/98.5% similarity).

Figure 8.

Putative Daphnia pulex PROTEIN PHOSPHATASE 1 (PP1) proteins. (A) Alignment of Drosophila melanogaster PP1 (Drome-PP1) with D. pulex PP1 A (Dappu-PP1 A). (B). Alignment of Dappu-PP1 A and PP1 B. In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, serine/threonine kinase domains identified by SMART analyses are highlighted in red.

Reciprocal blasting of Dappu-PP1 A and B against all proteins curated in FlyBase identified isoforms of PP1 (Flybase nos. FBpp0084026 and FBpp0071382, respectively; Table 2) as the most similar D. melanogaster proteins to the Daphnia queries. Comparison of the Dappu-PP1s with all non-redundant protein sequences curated by NCBI identified a serine/threonine-protein phosphatase alpha-1 isoform (of which PP1 is a family member) from the ant C. floridanus (EFN69572) to possess the highest similarity score to Dappu-PP1 A (Table 3), with a PP1 from the zebra fish Danio rerio (CAD61270) being most similar to Dappu-PP1 B (Table 3); no Drosophila proteins were among the top five blastp hits for either of the Dappu-PP1s (Table 3).

3.1.9. PROTEIN PHOSPHATASE 2A (PP2A)

3.1.9.1. PP2A catalytic subunit – MICROTUBULE STAR (MTS)

A single D. pulex gene (dappu-mtr) was identified as encoding a putative PP2A catalytic subunit protein (MTS) via a query using a D. melanogaster MTS sequence (AAF52567; Adams et al., 2000). The Genes 2010 gene model shows dappu-mts to be located on Scaffold 13 of the genome, with a predicted length of 2099 nucleotides (Table 1).

Figure 9A shows the alignment of the protein deduced from dappu-mts (Dappu-MTS; 308 amino acids in overall length) with that of the Drosophila query (309 amino acids long). Comparisons of the sequence of Dappu-MTS with Drome-MTS revealed 64.4% amino acid identity/90.3% amino acid similarity between the two proteins. SMART analyses of Dappu-MTS and Drome-MTS identified a single protein phosphatase 2A catalytic domain in each protein (Fig. 9A). Comparison of the sequences of these domains revealed 66.9% amino acid identity/92.6% amino acid similarity between these two regions of the proteins (Fig. 9A).

Figure 9.

Putative Daphnia pulex PROTEIN PHOSPHATASE 2A (PP2A) proteins. (A) Alignment of Drosophila melanogaster PP2A catalytic subunit MICROTUBULE STAR (MTS) protein (Drome-MTS) with D. pulex MTS (Dappu-MTS). (B1). Alignment of D. melanogaster PP2A regulatory subunit WIDERBORST (WBT) protein (Drome-WBT) with D. pulex WBT A (Dappu-WBT A). (B2). Alignment of Dappu-WBT A and Dappu-WBT B. (C). Alignment of D. melanogaster PP2A regulatory subunit TWINS (TWS) protein (Drome-WBT) with D. pulex TWS (Dappu-TWS). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, protein phosphatase 2A catalytic, coiled-coil and WD40 domains identified by SMART analyses are highlighted in dark green, pink and dark red, respectively.

Reciprocal blasting of Dappu-MTS against all proteins curated in FlyBase identified MTS (Flybase no. FBpp0077017) as the D. melanogaster protein most similar to the Daphnia query (Table 2). Comparison of Dappu-MTS with all non-redundant protein sequences curated by NCBI revealed a serine/threonine-protein phosphatase 4 catalytic subunit from the ant Harpegnathos saltator (EFN85419) to be most similar to Daphnia MTS (Table 3); no Drosophila proteins were among the top five blastp hits (Table 3).

3.1.9.2. PP2A regulatory subunit

3.1.9.2.1. WIDERBORST (WBT)

Two D. pulex genes (dappu-wbt a and dappu-wbt b) were identified as encoding putative PP2A regulatory subunit proteins (WBTs) via a query using a D. melanogaster WBT sequence (AAF56720; Adams et al., 2000). The Genes 2010 gene model shows dappu-wbt a and b to be located on Scaffolds 8 and 2 of the genome, respectively, with a predicted lengths of 6199 and 4882 nucleotides (Table 1).

Figure 9B1 shows the alignment of the protein deduced from dappu-wbt a (Dappu-WBT A; 481 amino acids in overall length) with that of the Drosophila query (524 amino acids long). Comparisons of the sequence of Dappu-WBT with Drome-WBT revealed 73.7% amino acid identity/85.5% amino acid similarity between the two proteins. Alignment of Dappu-WBT B with Drome-WBT revealed a lower level of amino acid conservation between these two proteins, 52.6% identity/74.1% similarity (alignment not shown). Figure 9B2 shows the alignment of the two Daphnia WBTs; these proteins are 50.5% identical/69.7% similar in amino acid sequence. SMART analyses of Dappu-WBT A identified a single coiled-coil domain; this domain was not predicted by SMART analyses in either Dappu-WBT B or Drome-WBT, though in the former protein this region is 45.5% identical/69.7% similar in amino acid composition to that of Dappu-WBT A and in the latter protein 72.7%identical/97.0% similar to that of Dappu-WBT A (Fig. 9).

Reciprocal blasting of Dappu-WBT A and B against all proteins curated in FlyBase identified PP2A regulatory subunit isoforms (Flybase nos. FBpp0084579 and FBpp0288759, respectively; Table 2) as the most similar D. melanogaster proteins to the Daphnia queries. Comparison of the Dappu-WBTs with all non-redundant protein sequences curated by NCBI identified serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit isoforms from the ant C. floridanus to show the highest similarity scores to each of Dappu-WBTs (Accession nos. EFN66909 and EFN69797, respectively; Table 3); while a Drosophila isoform of WBT was among the top five blastp hits for Dappu-WBT A, none were among the top hits for Dappu-WBT B (Table 3).

3.1.9.2.2. TWINS (TWS)

A single D. pulex gene (dappu-tws) was identified as encoding a putative PP2A regulatory subunit protein (TWS) via a query using a D. melanogaster TWS sequence (AAF54498; Adams et al., 2000). The Genes 2010 gene model shows dappu-tws to be located on Scaffold 43 of the genome, with a predicted length of 4157 nucleotides (Table 1).

Figure 9C shows the alignment of the protein deduced from dappu-tws (Dappu-TWS; 443 amino acids in overall length) with that of the Drosophila query (499 amino acids long). Comparisons of the sequence of Dappu-TWS with Drome-TWS revealed 71.5% amino acid identity/83.6% amino acid similarity between the two proteins. SMART analyses of Dappu-TWS and Drome-TWS identified six and seven WD40 domains in these proteins, respectively (Fig. 9C). Comparison of the sequences of the shared WD40 domains, as well as the region of the Daphnia protein corresponding to the sixth of the seven Drosophila domains, revealed high degrees of amino acid conservation in these regions of the two proteins: WD40 1, 87.2% identity/100% similarity; WD40 2, 90.2% identity/100% similarity; WD40 3, 90.0% identity/95.0% similarity; WD40 4, 89.5% identity/94.7% similarity; WD40 5, 87.2% identity/100 similarity; WD40 6 (no domain formally identified by SMART in Daphnia), 75% identity/100% similarity; WD40 7, 94.7% identity/100% similarity (Fig. 9C).

Reciprocal blasting of Dappu-TWS against all proteins curated in FlyBase identified TWS (Flybase No. FBpp0081671) as the D. melanogaster protein most similar to the Daphnia query (Table 2). Comparison of Dappu-TWS to all non-redundant protein sequences curated by NCBI identified a beetle Tribolium castaneum protein (EFA10095) as showing the highest similarity to the Daphnia query (Table 3); three Drosophila TWS isoforms were among the top five blastp hits (Table 3).

3.1.10. SHAGGY (SGG)

A single D. pulex gene (dappu-sgg) was identified as encoding a putative SGG protein via a query using a D. melanogaster SGG (AAN09084; Adams et al., 2000). The Genes 2010 gene model shows dappu-sgg to be located on Scaffold 76 of the genome, with a predicted length of 5712 nucleotides (Table 1).

Figure 10 shows the alignment of the protein deduced from dappu-sgg (Dappu-SGG; 439 amino acids in overall length) with that of the Drosophila query (514 amino acids long). Comparison of the sequence of Dappu-SGG with Drome-SGG revealed 64.8% amino acid identity/77.6% amino acid similarity between the two proteins. SMART analyses of Dappu-SGG and Drome-SGG identified a single serine/threonine kinase domain in each protein (Fig. 10); the two serine/threonine kinase domains are nearly identical in amino acid sequence, i.e. 89.5% identity/98.2% similarity. Reciprocal blasting of Dappu-SGG against all proteins curated in FlyBase identified SGG (Flybase no. FBpp0070450) as D. melanogaster protein most similar to the Daphnia query (Table 2). Comparison of Dappu-SGG with all non-redundant protein sequences curated by NCBI revealed a glycogen synthase kinase (of which SGG is family member) from the tick Rhipicephalus microplus (ABO61882) to be the most similar protein match (Table 3); no Drosophila proteins were among the top five blastp hits (Table 3).

Figure 10.

Putative Daphnia pulex SHAGGY (SGG) protein. Alignment of Drosophila melanogaster SGG (Drome-SGG) with D. pulex SGG (Dappu-SGG). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, serine/threonine kinase domains identified by SMART analyses are highlighted in red.

3.1.11. SUPERNUMERARY LIMBS (SLIMB)

A single D. pulex gene (dappu-slimb) was identified as encoding a putative SLIMB protein via a query using a D. melanogaster SLIMB (AAF55853; Adams et al., 2000). The Genes 2010 gene model shows dappu-slimb to be located on Scaffold 169 of the genome, with a predicted length of 2880 nucleotides (Table 1).

Figure 11 shows the alignment of the protein deduced from dappu-slimb (Dappu-SLIMB; 510 amino acids in overall length) with that of the Drosophila query (510 amino acids long). Comparison of the sequence of Dappu-SLIMB with Drome-SLIMB revealed 76.3% amino acid identity/91.8% amino acid similarity between the two proteins. SMART analyses of Dappu-SLIMB and Drome-SLIMB identified an FBOX domain and seven WD40 domains in each protein (Fig. 11); the amino acid sequences of each of these domains is highly conserved between the two species: FBOX domain, 80.0% amino acid identity/95.0% similarity; WD40-1, 89.5% amino acid identity/94.7% amino acid similarity; WD40-2, 84.2% amino acid identity/94.7% amino acid similarity; WD40-3, 89.5% amino acid identity/97.4% amino acid similarity; WD40-4, 94.7% amino acid identity/100% amino acid similarity; WD40-5, 97.4% amino acid identity/100% amino acid similarity; WD40-6, 94.7% amino acid identity/100% amino acid similarity; WD40-7, 94.7% amino acid identity/100% amino acid similarity.

Figure 11.

Putative Daphnia pulex SUPERNUMERARY LIMBS (SLIMB) protein. Alignment of Drosophila melanogaster SLIMB (Drome-SLIMB) with D. pulex SLIMB (Dappu-SLIMB). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, FBOX and WD40 domains identified by SMART analyses are highlighted in dark gray and dark red, respectively.

Reciprocal blasting of Dappu-SLIMB against all proteins curated in FlyBase identified SLIMB (Flybase No. FBpp0083434) as the D. melanogaster protein most similar to the Daphnia query (Table 2). Interestingly, comparison of Dappu-SLIMB to all non-redundant protein sequences curated by NCBI identified vertebrate proteins, all apparent members of the E3 ubiquitin ligase family, as the top five blastp hits, with a mouse protein (BAE26547) being the best match with the Daphnia sequence (Table 3).

3.1.12. TIMELESS

Eight D. pulex genes (dappu-tim a, dappu-tim b, dappu-tim c, dappu-tim d, dappu-tim e, dappu-tim f, dappu-tim g, and dappu-tim h) were identified as encoding putative TIM proteins via a query using a D. melanogaster TIM (AAN10371; Adams et al., 2000). The Genes 2010 gene model shows dappu-tim a, dappu-tim b, dappu-tim c, dappu-tim d, dappu-tim e, dappu-tim f, dappu-tim g, and dappu-tim h to be located on Scaffolds 24, 6, 10, 6, 6, 24, 75, and 91 of the genome, respectively, with lengths of 14166, 5052, 3650, 5438, 5542, 11674, 2798, and 4537 nucleotides (Table 1).

Figure 12 shows the alignments of the protein deduced from dappu-tim a (Dappu-TIM A; 1197 amino acids in overall length) with that of the Drosophila query (1421 amino acids in length). Comparison of the amino acid sequences of these two proteins revealed 29.8% identity/60.1% similarity between the two TIMs. Comparisons of the other Daphnia TIMs with Drome-TIM showed varying levels of amino acid conservation, with some nearly as high as that seen between Dappu-TIM A and Drome-TIM, and others considerably lower: Dappu-TIM B, 23.6% amino acid identity/52.0% amino acid similarity; Dappu-TIM C, 23.7% amino acid identity/47.8% amino acid similarity; Dappu-TIM D, 26.4% amino acid identity/55.9% amino acid similarity; Dappu-TIM E, 23.4% amino acid identity/47.9% amino acid similarity; Dappu-TIM F, 22.3% amino acid identity/51.1% amino acid similarity; Dappu-TIM G, 14.2% amino acid identity/30.8% amino acid similarity; and Dappu-TIM H, 21.3% amino acid identity/45.6% amino acid similarity (alignments not shown). Alignment of the eight Daphnia TIM proteins to one another shows considerable variation in sequence composition between the proteins (in the interest of space, this alignment is provided only as an online supplemental figure [Supplemental Figure 1]). Table 3 provides pairwise comparisons of the amino acid identity/similarity of Dappu-TIM A-H. No functional domains were identified by SMART analyses in Drome-TIM or any of the Dappu-TIMs. Reciprocal blasting of eight Dappu-TIMs against all proteins curated in FlyBase identified an isoform of TIM as the D. melanogaster protein most similar to each Daphnia query (Flybase nos. FBpp0291971, FBpp0291971, FBpp0291971, FBpp0077254, FBpp0291970, FBpp0077254, FBpp0291970 and FBpp0291970, respectively; Table 2). Comparison of the Dappu-TIMs with all non-redundant protein sequences curated by NCBI revealed each to be most similar to an insect TIM isoform (i.e. the butterfly Danaus plexippus [AAR15505], the moth Antheraea pernyi [AAF66996], the beetle T. castaneum [EFA04644], T. castaneum [EFA04644], D. melanogaster [ADV36936], D. melanogaster [P49021], the fruit fly Drosophila virilis [O17482], and the cricket Gryllus bimaculatus [BAJ16356] for Dappu-TIM A-H, respectively; Table 3). In fact, the top five hits for each of the Daphnia proteins were insect isoforms of TIM (Table 3).

Figure 12.

Putative Daphnia pulex TIMELESS (TIM) protein. Alignment of Drosophila melanogaster TIM (Drome-TIM) with D. pulex TIM A (Dappu-TIM A). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure.

3.1.13. VRILLE (VRI)

A single D. pulex gene (dappu-vri) was identified as encoding a putative VRI protein via a query using a D. melanogaster VRI (AAF52237; Adams et al., 2000). The Genes 2010 gene model shows dappu-vri to be located on Scaffold 92 of the genome, with a predicted length of 2225 nucleotides (Table 1).

Figure 13 shows the alignment of the protein deduced from dappu-vri (Dappu-VRI; 676 amino acids in overall length) with that of the Drosophila query (729 amino acids long). Comparison of the sequences of Dappu-VRI and Drome-VRI revealed 27.7% amino acid identity/49.5% amino acid similarity between the two proteins. SMART analyses of Dappu-VRI and Drome-VRI identified a single basic region leucine zipper domain in each protein (Fig. 13); the amino acid sequence of this domain is highly conserved between the two VRIs, i.e. 76.9% amino acid identity and 96.9% amino acid similarity.

Figure 13.

Putative Daphnia pulex VRILLE (VRI) protein. Alignment of Drosophila melanogaster VRI (Drome-VRI) with D. pulex VRI (Dappu-VRI). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, basic region leucine zipper domains identified by SMART analyses are highlighted in dark blue.

Reciprocal blasting of Dappu-VRI against all proteins curated in FlyBase identified VRI (Flybase no. FBpp0289297) as the most similar D. melanogaster protein to the Daphnia query (Table 2). Comparison of Dappu-VRI with all non-redundant protein sequences curated by NCBI revealed a VRI from the butterfly D. plexippus (ATT86041) to be most similar to Daphnia VRI (Table 3); two Drosophila VRIs are among the top five blastp hits for this query (Table 3).

3.2. Input pathway proteins

3.2.1. CRYPTOCHROME (CRY)

Four D. pulex genes (dappu-cry a, dappu-cry b, dappu-cry c, and dappu-cry d) were identified as encoding putative CRY proteins via a query using a D. melanogaster CRY (AAC83828; Emery et al., 1998). The Genes 2010 gene model shows that these genes are located on Scaffolds 40, 18, 10, and 7 of the genome, respectively, with lengths of 2706, 4661, 3072 and 2052 nucleotides (Table 1).

Figure 14A shows the alignment of the protein deduced from dappu-cry a (Dappu-CRY A; 525 amino acids in overall length) with that of the Drosophila query (542 amino acids long). Comparison of the sequence of Dappu-CRY A with Drome-CRY revealed 44.8% amino acid identity/76.4% amino acid similarity between the two proteins (Figure 14A). Alignments of Dappu-CRY B, Dappu-CRY C and Dappu-CRY D with Drome-CRY also revealed high levels of structural homology between the proteins: Dappu-CRY B vs. Drome-CRY, 37.6% amino acid identity/69.1%; Dappu-CRY C vs. Drome-CRY, 38.2% amino acid identity/69.9% amino acid similarity; Dappu-CRY D vs. Drome-CRY, 24.4% amino acid identity/59.6% amino acid similarity (alignments not shown). Figure 14B shows the alignment of the four Daphnia CRYs with one another. As this panel shows, the four proteins show considerable variation in amino acid composition. Table 4 provides pairwise comparisons of the amino acid identity/similarity of Dappu-CRY A-D. No functional domains were identified by SMART analyses in Drome-CRY or any of the Dappu-CRYs.

Figure 14.

Putative Daphnia pulex CRYPTOCHROME (CRY) proteins. (A) Alignment of Drosophila melanogaster CRY (Drome-CRY) with D. pulex CRY A (Dappu-CRY A). (B). Alignment of Dappu-CRY A–D. In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure.

Table 4.

Matrix of percent amino acid identity/similarity between putative Daphnia pulex TIMELESS (TIM) proteins

	TIM-A	TIM-B	TIM-C	TIM-D	TIM-E	TIM-F	TIM-G	TIM-H
TIM-A
TIM-B	47.1/70.9
TIM-C	47.7/68.3	44.1/71.2
TIM-D	48.6/73.9	46.0/70.3	41.7/67.8
TIM-E	45.0/65.8	45.0/68.8	44.0/72.4	58.7/75.8
TIM-F	31.5/55.2	26.5/49.3	25.8/44.6	27.0/50.3	22.8/42.4
TIM-G	28.8/45.9	31.9/53.1	33.9/54.2	26.6/46.4	28.3/50.5	16.7/29.9
TIM-H	37.8/62.2	36.9/63.4	37.8/68.5	36.5/64.2	38.1/68.3	19.8/40.1	26.8/48.9

Values shown are percent amino acid identity/similarity.

Percent identity = number of amino acids identically conserved between the two proteins divided by the total number of amino acids in the longer protein.

Percent similarity = number of identical and similar amino acids in the two proteins divided by the total number of amino acids in the longer protein

As discussed in Section 1, along with CLK, CRY is the only other circadian protein for which a crustacean family member is known, i.e. an isoform from the Antarctic krill E. superba (Mazzotta et al., 2010). Alignments of the Daphnia CRYs with Eupsu-CRY, show similar levels of amino acid conservation to that seen for alignments with the Drosophila protein: Dappu-CRY A vs. Eupsu-CRY, 36.0% identity/69.5% similarity; Dappu-CRY B vs. Eupsu-CRY, 67.1% identity/88.6% similarity; Dappu-CRY C vs. Eupsu-CRY, 47.2% identity/76.5% similarity; Dappu-CRY D vs. Eupsu-CRY, 26.8% identity/58.9% similarity (alignments not shown).

Reciprocal blasting of four Dappu-CRYs against all proteins curated in FlyBase identified members of the CRY/6-4 photolyase family as the most similar D. melanogaster proteins to the Daphnia queries. For Dappu-CRY A, CRY (Flybase no. FBpp0083150) was found to be the most similar Drosophila protein to the query sequence, while the remaining three sequences were found to be most similar to 6-4 photolyase (Flybase no. FBpp0080935). Comparison of the Dappu-CRYs with all non-redundant protein sequences curated by NCBI revealed each to be most similar to a CRY protein, though all are more similar to isoforms from other species than they are to Drosophila proteins (i.e. the cricket Dianemobius nigrofasciatus [BAF45421], the mosquito Anopheles darlingi [EFR20390], the clawed frog Xenopus tropicalis [AAI66277], and the European seabass Dicentrarchus labrax [CBN81995]) for Dappu-CRY A-D, respectively). Based on the results of our blastp analyses (Table 3), it would appear that Dappu-CRY A is a homolog of the Drosophila-type or CRY1 subfamily, with Dappu-CRY B being a homolog of the vertebrate-type or CRY2 subfamily; alignments of Dappu-CRY A and B with CRY1 and CRY2 of the butterfly D. plexippus, respectively, are shown in Figure 15. Dappu-CRY D appears most similar to members of the CRY DASH subfamily (Table 3). It is unclear as to which subfamily of the CRY/6-4 photolyase superfamily Dappu-CRY C is a member (Table 3).

Figure 15.

Alignment of Daphnia pulex CRYPTOCHROME (CRY) A and B with their Danaus plexippus homologs. (A) Alignment of D. plexipus CRY1 (Danpl-CRY1) with D. pulex CRY A (Dappu-CRY A). (B). Alignment of D. plexipus CRY2 (Danpl-CRY2) with D. pulex CRY B (Dappu-CRY B). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure.

3.3. Output pathway proteins

3.3.1. PIGMENT DISPERSING HORMONE RECEPTOR (PDHR)

A single D. pulex gene (dappu-pdhr) was identified as encoding a putative PDHR via a query using a D. melanogaster pigment dispersing factor receptor (PDFR; AAF45788; Adams et al., 2000). The Genes 2010 gene model shows dappu-pdhr to be located on Scaffold 1 of the genome, with a predicted length of 4621 nucleotides (Table 1).

Figure 16 shows the alignment of the protein deduced from dappu-pdhr (Dappu-PDHR; 516 amino acids in overall length) with that of the Drosophila query (669 amino acids long). Comparison of the sequence of Dappu-PDHR with that of Drome-PDFR revealed 32.7% amino acid identity/55.0% amino acid similarity between the two proteins. SMART analyses of Dappu-PDHR and Drome-PDFR identified seven transmembrane domains (TMDs) in each protein (Fig. 16). The amino acid sequences of these TMDs are highly conserved between the two proteins: TMD1, 52.2% amino acid identity and 87.0% amino acid similarity; TMD2, 72.2% amino acid identity and 94.4% amino acid similarity; TMD3, 72.7% amino acid identity and 95.5% amino acid similarity; TMD4, 47.4% amino acid identity and 78.9% amino acid similarity; TMD5, 55.5% amino acid identity and 95.0% amino acid similarity; TMD6, 72.2% amino acid identity and 83.3% amino acid similarity; TMD7, 81.8% amino acid identity and 100% amino acid similarity. In addition, a single hormone receptor domain was identified in Dappu-PDHR; this domain is absent in Drome-PDFR, though the corresponding region of this protein is 35.8% identical/58.9% similar to its Daphnia counterpart (Fig. 16).

Figure 16.

Putative Daphnia pulex PIGMENT DISPERSING HORMONE RECEPTOR (PDHR) protein. Alignment of Drosophila melanogaster pigment dispersing factor receptor (Drome-PDFR) with D. pulex PDHR (Dappu-PDHR). In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure. In this figure, hormone receptor and transmembrane domains identified by SMART analyses are highlighted in black and light gray, respectively.

Reciprocal blasting of Dappu-PDHR against all proteins curated in FlyBase identified PDFR (Flybase No. FBpp0099841) as the D. melanogaster protein most similar to the Daphnia query (Table 2). Comparison of Dappu-PDHR to all non-redundant protein sequences curated by NCBI identified a PDHR from the penaeid shrimp Marsupenaeus japonicus (BAH85843) as the top protein match for the query; three Drosophila proteins were also among the top five blastp hits identified via the Daphnia protein (Table 3).

4. Discussion

4.1. Genome mining identifies a putative set of putative circadian proteins in Daphnia pulex

Over the last decade, genome mining has become a major method for protein discovery in both vertebrates and invertebrates. At present, the only crustacean genome that has been fully sequenced and is available for public use is that of the cladoceran D. pulex (Colbourne et al., 2005; Bauer, 2007; Stollewerk, 2010; Colbourne et al., 2011; Tautz, 2011). Recently, this resource has been used for protein discovery in this species, providing detailed information on the structures of molecules involved in many physiological/behavioral processes, for example, steroid biosynthesis and innate immunity (Rewitz and Gilbert, 2008; McTaggart et al., 2009).

In the study presented here, we have used the D. pulex genome to mine for proteins that may be involved in the control of circadian rhythmicity in this species. Specifically, we used the sequences of known Drosophila circadian proteins to query the Daphnia genome for putative ortholog genes and their encoded proteins. Using this strategy, a number of putative D. pulex circadian genes and their proteins were identified and characterized, including those likely involved in the establishment of the core clock, i.e. PER, TIM, CLK, CYC and CRY2, as well as proteins in their post-translational modifications and degradation, i.e. DBT, CK2, SGG, PP2A, PP1 and SLIMB. Moreover, genes and proteins putatively involved in setting the phase and amplitude of the core clock were identified, i.e. PDP1, VRI and CWO, as were orthologs of the blue-light receptor protein CRY1, which likely function as input pathways to the core clock, synchronizing it to the solar day, and PDHR, which may serve to transduce one of the clock’s output signals. Taken collectively, this collection of genes/proteins represents the first putative set of circadian proteins thus far described from any crustacean.

4.2. Several Daphnia circadian genes appear to exhibit extensive gene duplications

Overall, Daphnia have an unusually high level of gene duplication in comparison with the other arthropods for which genomic databases exist (Colbourne et al., 2011). In fact, only in aphids has a similar level of gene duplication been noted (Huerta-Cepas et al., 2010; Ollivier et al., 2010; Shigenobu et al., 2010); both Daphnia and aphids are cyclical parthenogens (Cortés et al., 2008). The purpose of gene redundancy in these and other species is generally not well understood. For some genes, the encoded protein isoforms may be nonfunctional, may have different kinetic properties for the same substrate, or may have novel functions (Force et al., 1999). It is also possible that the protein isoforms may be expressed in a life stage-specific manner (development, diapause, reproduction), or that their expression is tissue-specific. Multiple genes, and hence protein isoforms, may help an organism adjust to shifting environmental conditions, e.g. changes in salinity, oxygen levels, or temperature.

As discussed in Section 1, a defining parameter of circadian rhythms is temperature compensation, the molecular basis of which is not well understood (Salomé and McClung, 2005; Salomé et al, 2010). Tomaiuolo et al. (2008) constructed a mathematical model based on two splice variant isoforms of the β-subunit of Drosophila CKII that differ in kinetic rates of PER phosphorylation. This model suggests that through dynamic regulation of the proportions of the two β-subunit isoforms expressed, an increase in robustness of the circadian clock can be predicted. Two per alleles exist in wild populations of D. melanogaster, with population differences in frequency that vary by latitude (Sawyer et al., 1997). The PER proteins have different thermokinetic properties that may be involved in the circadian clock temperature compensation. Likewise, in a northern-latitude Drosophila population, the tim mutation, ls-tim, has been shown to adjust photoresponsiveness in this more seasonally-variable environment (Sandrelli et al., 2007). The multiple Daphnia PP1, PP2A-WBT and TIM variants predicted here may likewise possess different kinetic parameters to offset temperature, salinity, oxygen, and/or other environmental variables.

4.3. Conservation of structural domains suggests Daphnia possesses an insect-like molecular clock, but organized more like that of butterflies and mosquitoes than of Drosophila

In our study, putative homologs to most of the known Drosophila circadian proteins were identified in D. pulex. Structural domains in both the Daphnia and Drosophila proteins were analyzed via the online program SMART (Schultz et al., 1998; Letunic et al., 2009). While we realize that not all of the domains/functional regions that have been reported for the Drosophila circadian proteins are detected via this program (e.g. Saez and Young, 1996; Ousley et al., 1998; Chang and Reppert, 2003; Lin and Todo, 2005), those that were, for the most part, appear to be highly conserved between the two species’ putative homologs; significant amino acid variation was noted outside of functional regions for several proteins. Even where discrepancies were noted, e.g. a PAC domain identified in Drome-PER but not in Dappu-PER, the corresponding regions of the two proteins were often very similar in amino acid composition (in the case of the PER PAC domain 72.7% identical/93.2% similar). Interestingly, blast analyses of the Daphnia sequences show them to be, for the most part, more similar to proteins identified from other species than they are to Drosophila. Moreover, the presence of both CRY1 and CRY2 in Daphnia suggests that its molecular clock is likely organized more similar to that recently described for butterfly and mosquito (e.g. Zhu et al., 2005; Yuan et al., 2007; Zhu et al., 2008) where CRY1 is proposed as a photosenstive input to the clock and CRY2 is core clock protein (functioning to repress of CLK/CYC-mediated transcription), than it is to Drosophila (which possesses only CRY1).

4.4. Potential cellular locus and output signals of a Daphnia neuronal clock

All circadian systems have three functional components: a core clock, which is responsible for time keeping, input pathways that act to synchronize the clock to the environment, and output pathways that transmit the timing information from the clock for the control of physiology and behavior. Here we have identified a protein that may function as the input to a Daphnia clock, i.e. an isoform of CRY1, as well as proteins that may act to establish the core molecular clock itself, i.e. PER, TIM, CLK, CYC, CRY2, DBT, SGG, VRI, etc. To be determined, however, are possible output pathways from the Daphnia clock that would signal the timing information necessary to establish circadian rhythms in physiology and behavior in this species.

As the cellular location of the core circadian clock (or clocks) in D. pulex is unknown, it is difficult to postulate how output signals would be generated in, and transmitted from, this timekeeper. This said, work conducted on other species (both invertebrate and vertebrate) would suggest that circulating hormones are an important part of the Daphnia clock’s output pathway, mediating the expression of the overt circadian rhythms present in this species. One possibility is that hormones are released directly from the clock cells themselves; alternatively, the clock cells may project to and innervate relay sites, likely endocrine organs, which are the sources of the hormonal signals. Regardless of locus, in insects, several peptide hormones have been shown to be key components of circadian signaling systems, particularly pigment dispersing factor (PDF), a member of the pigment dispersing hormone (PDH) family; PDF is present in a number of known circadian clock neurons in Drosophila (for review see: Allada and Chung, 2010; Tomioka and Matsumoto, 2010).

Recent transcriptome and genome mining in D. pulex has identified a homolog of PDF/PDH in this species, NSELINSLLGLPRFMKVVamide (Gard et al., 2009; Christie et al., 2011). Moreover, immunohistochemistry using an antibody generated against β-PDH (NSELINSILGLPKVMNDAamide) labels a small set of neurons (~8 somata) that are distributed throughout the brain/optic ganglia of D. pulex (Gard et al., 2009). While currently speculation, the role of PDF as a signaling agent, and hence marker, for some clock cells in the brain of Drosophila suggests that PDH-immunopositive neurons in the brain of D. pulex may represent at least a subset of the cellular loci for a circadian neuronal pacemaker in this species, a hypothesis recently strengthened by the finding of circadian patterns of activity in at least some of these cells (Strauβ et al., 2011).

In addition to PDF, a number of other hormones, primarily peptides, have been implicated in circadian signaling in insects. For example, corazonin, crustacean cardioactive peptide (CCAP) and diapause hormone have all been suggested as possible output signals from insect clock systems (e.g. Sehadová et al., 2007); isoforms of both corazonin and CCAP have been predicted from the D. pulex transcriptome and/or genome (Gard et al., 2009; Christie et al., 2011). Likewise, several peptide hormones have been shown to, or are postulated to show, circadian rhythms in their cycling in decapod crustaceans (Strauss and Dircksen, 2010), i.e. red pigment concentrating hormone and crustacean hyperglycemic hormone. Transcriptome and genome mining in D. pulex suggests that these hormonal systems too are present in this species (Gard et al., 2009; Christie et al., 2011). In fact, via transcriptome and genome mining over 100 peptide hormones have recently been identified in D. pulex (Gard et al., 2009; Christie et al., 2011). Here we have identified a putative PDH receptor protein, which, if we are correct in the peptide being a circadian signal in Daphnia, may function to transduce at least one of the core clocks output signals for the control of physiology and behavior in this species.

5. Conclusions and future directions

Circadian rhythms in physiology and behavior have been documented in numerous crustacean species; however, little is known about the molecular and/or cellular machinery underlying them in any member of this arthropod subphylum (Strauss and Dircksen, 2010). This said, their well-mapped nervous systems and amenability to in-depth electrophysiological and molecular investigations make them an optimal group of animals for studying circadian biology. Moreover, the fact that many intertidal crustaceans exhibit both circadian and circatidal rhythms make these animals an ideal model to explore possible interactions between circadian and circatidal signaling systems, including whether these two timekeeping systems use common or distinct molecular and/or cellular components.

Clearly the first step toward understanding circadian signaling in any species is obtaining knowledge of the molecules required for the establishment of the core clock. Here, we have achieved this milepost for the cladoceran crustacean D. pulex using a strategy combining genome mining and phylogenetic comparisons to known previously identified circadian proteins. D. pulex is now the only crustacean for which a putative set of circadian genes and proteins are known. With these data, we are now positioned to begin functional studies directed at determining if the mRNAs of the identified genes cycle in a circadian fashion and, if so, whether these rhythms are similar to those seen in insects. Similarly, the proteins deduced from these identified genes now allows for the generation of Daphnia-specific antibodies to these molecules, which will be useful both for mapping the distribution of these proteins and for determining if they cycle in manners similar to their insect counterparts. Finally, the identification of the D. pulex circadian genes described here now provide targets for knockdown experiments designed to elucidate the functional roles their encoded proteins play in the establishment of circadian signaling in this and other crustacean species.

Supplementary Material

01

Supplemental Figure 1. Alignment of Dappu-TIM A-H. In the line immediately below each sequence grouping, stars indicated amino acids that are identically conserved, while single and double dots denote amino acids that are similar in structure.

Table 5.

Matrix of percent amino acid identity/similarity between putative Daphnia pulex CRYPTOCHROME (CRY) proteins

	CRY-A	CRY-B	CRY-C	CRY-D
CRY-A
CRY-B	37.4/70.7
CRY-C	38.3/71.2	48.1/78.7
CRY-D	26.3/61.0	26.4/58.8	27.7/64.3

Values shown are percent amino acid identity/similarity.

Percent identity = number of amino acids identically conserved between the two proteins divided by the total number of amino acids in the longer protein.

Percent similarity = number of identical and similar amino acids in the two proteins divided by the total number of amino acids in the longer protein