Molecular Evolution of Two Circadian Genes in Fish

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Circadian rhythms are roughly 24-hour cycles of biochemical, physiological, and behavioral processes which occur in living organisms including plants, animals, fungi and cyanobacteria. However, in the absence of evidence, it cannot be verified that the eukaryotes and cyanobacterial circadian systems originated from a common ancestor . Circadian rhythms enhance the fitness of organisms in both constant and changing environments . The cryptochrome (Cry) and period (Per) are two key components that control the circadian oscillators of animals .

1.1 Study of Cryptochrome

Cryptochromes are flavoproteins, which are homologous to photolyases. Photolyases are light-dependent DNA repair enzymes. They are activated by blue light and repair UV induced DNA damage by removing pyrimidine dimers. There are two types of photolyases, CPD photolyases which repair cyclobutane pyrimidine dimmers and 6-4 photolyases which repair 6-4 pyrimidine pyrimidone. These two photolyases together with cryptochromes constitute the photolyase/cryptochrome superfamily .

The first identified cryptochrome gene is Arabidopsis Cry1 (AtCry1) . Cryptochrome was then found ubiquitously in the plant and animal kingdoms, including algae, bryophyte, ferns, seed plants and various animal lineages . It was initially thought that eukaryote possesses both cryptochrome and photolyase, whereas prokaryote has only the latter one, until a new subclass of cryptochrome (CRY-DASH) was discovered in cyanobacteria and plants. This finding suggests that cryptochrome evolved before the divergence of eukaryotes and prokaryotes . Later, members of the CRY-DASH subfamily were found in fungi and vertebrates .

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In recent years, molecular evolution studies of circadian genes have attracted great attention. Compare with prokaryotes circadian genes, molecular evolution of eukaryotes circadian genes has yet to be comprehensively studied, and such study has a very promising prospect.

1.1.1 Structure of cryptochromes

Most cryptochromes, except Cry-DASH, are composed of 2 domains, an N-terminal photolyases-related (PHR) region and a C-terminal domain of variable length; Cry-DASH proteins lack the C-terminal domain. . The variation in the length of the C-terminal domains results in functional diversity within the cryptochrome family. The C-terminus of mammal cryptochrome possesses a nuclear localization domain that drives Cry/Per fusion molecule into the nucleus, and deletion of the C-terminus prevents mammal Cry from negatively regulating the transcription of other circadian components . Cryptochromes possess two chromophores: pterin (in the form of 5, 10-methenyltetrahydrofolate (MHF)) and flavin (in the form of flavin adenine dinucleotide (FAD)), both of them are bound to the PHR region, as cofactors that absorb light . Photolyases also have two chromophores, one of them is FAD, and the other can be either pterin or deazaflavin .

The 3D-architectures of PHR regions of the photolyase/cryptochrome superfamily are highly similar. All of them fold into 2 domains, an α/β domain and a helical domain. These 2 domains are connected by a variable loop and the 2 lobes of the helical domain form a groove, which is called FAD-access cavity. FAD embeds in this "molecular pocket", and may be resolved at the bottom .

The PHR region of the Arabidopsis Cry1 (AtCry) has several structural features distinct from same regions of photolyase and Cry-DASH . First, photolyase and Cry-DASH have exterior positive electrostatic potentials around the FAD-access cavity to interact with DNA, while the PHR region of the Arabidopsis Cry1 does not have this structural feature, suggesting that it is not binding double-strand DNA . Additionally, the PHR of the Cry1 has a predominantly negatively charged surface around the FAD-access cavity . Third, an ATP analog is able to merges inside the FAD-access cavity, yet photolyase and Cry-DASH have not reported to bind ATP .

1.1.2 Other functions of cryptochrome

Photolyases and cryptochrome are defined to be evolutionarily related flavoproteins that perform distinct physiological functions . Although cryptochromes share a structural similarity to DNA photolyases, they lack DNA repair activity except Cry-DASH . Besides being a key circadian gene, cryptochrome have developed a functional diversity in the long evolutionary history.

1.1.2.1 Plant photomorphogenesis

Arabidopsis cryptochrome plays a key role in plant photomorphogenesis, for instance, de-etiolation in seed germination, inhibiting hypocotyl and stem elongation and stimulating leaf expansion by blue light, regulating floral initiation by day length . Cryptochrome controls morphological changes in plants by changing gene expression, and this modulation reacts to the light .

The Arabidopsis photomorphogenesis is regulated by the blue light receptors cryptochromes (Cry1 and Cry2) and the red/far red photoreceptors phytochromes (PHYA to PHYE), they transduce the environmental signal (light) to the central oscillator . But the catalytic mechanism of plant cryptochrome has yet to be expounded.

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Cryptochromes may affect nuclear gene expression changes by 2 mechanisms . First, plant cryptochrome may affect transcription directly by interacting with proteins associated with this machinery. A study shows that Arabidopsis Cry2 binds chromatin in a DNA sequence-independent conformation, but its mechanism is still unclear . The other possible mechanism is indirect regulation of transcription by Cry through interaction with proteins, which are associated with other cellular functions. For example, the C-terminal domain of Arabidopsis cryptochrome was shown to bind COP1, a component of an E3 ubiquitin ligase .

1.1.2.2 Magnetoreception

Studies have shown that a wide variety of organisms, such as insects, fishes, and reptiles, are magnetically sensitive and take advantage of it in migration . The most comprehensively studied examples were migratory birds . Retina cryptochromes are shown to be possible transducers in avian compass . The mechanism of this compass may be interpreted by the radical-pair model . Recently, the occurrence of photo-induced radical pair was verified in Xenopus laevis cryptochromes . Magnetic fields also affect plant growth by affecting cryptochromes when exposed to red light or blue light .

1.1.2.3 Cryptochrome-DASH

The physiological function of cryptochrome-DASH is still unclear. It is once reported that CRY-DASH gene expression is mediated by circadian clock machinery in tomato Solanum lycopersicum . However, recently research in this area has a greater progress. Selby and Sancer conducted the electrophoretic mobility shift assays and found that the Cry-DASH can bind or repair the T-T dimer in single-stranded DNA, indicating that CRY-DASH photolyases specified for cyclobutane pyrimidine dimers in ssDNA .

1.1.3 Evolution of Cryptochrome

Circadian rhythm and DNA repair may have a common evolutionary origin . Escape from sunlight represented a major selective force for development of circadian rhythms . Geological studies indicates that in Precambrian times (3800~544 mya), the atmosphere contained little oxygen, and primitive organisms were exposed to high ultraviolet radiation during the daytime . There are 2 main strategies for organisms to avoid the harmful effects of UV radiation . The first one is repairing the UV-induced DNA damage which is the physiological function of photolyase. The other one is to avoid being irradiated, such as migrate to deeper water. These movements were observed by the diel vertical migrations of zooplankton, which initiated and controlled by light . Such migration also occurs in other marine and freshwater organisms such as water flea Daphnia magna , and sensitivity is related closely to the UV photoreceptors in its compound eye . These diel vertical migration may interpret the coevolution of photoreception and circadian rhythms, and the coevolution of their respective controlled genes .

Phylogenetic analysis of the photolyase/cryptochrome superfamily suggests that multiple gene duplications of an ancestral CPD photolyase gene caused their functional divergence . Figure 1 demonstrates the evolutionary relationships of different cryptochromes. According to the tree topology, previous classification of this gene family has been corroborated . Animal cryptochromes, plant cryptochromes and cryptochrome DASHs are clustered into different clades. The phylogenetic tree also indicates that the plant cryptochrome, animal cryptochrome and cryptochrome-DASH families have distinct evolutionary histories, the plant cryptochromes are evolutionary older than animal cryptochromes.

1.2 Study of Period

The fruit fly (D. melanogaster) period (Per) was the first animal circadian clock gene that has been identified . Period (Per) is a canonical circadian clock gene that not involving functional diversity.

The Per genes code a highly conserved domain which named PAS (Period-Arylhydrocabon receptor nuclear translocator-Single mind) . This domain is also contained in many sensory, signaling, and protein-protein interaction modules including factors Drosophila/mammal Clock (d/mClk), Drosophila Cycle (dCyc), and mammal BMAL1 .

The best-studied animal period genes are Drosophila per and mouse period. Mammals/mouse has three period homologues (mPer1, mPer2 and mPer3), while Drosophila only has one. Both the dPer and mPer encode two PAS domains (PAS-A and PAS-B).

Mechanism of circadian oscillator in animals

In animal, central oscillators occur in the brains which control the circadian behavior of the organism and peripheral oscillators in some tissues . Fruit fly and mammal circadian oscillators have been extensively studied .

Animal Cry proteins are functionally various. Two distinct groups of animal Cry have been identified base on their roles in circadian clocks . Drosophila-like type 1 Cry is UV-A/blue light receptors in circadian oscillator, while vertebrate-like type 2 Cry is thought to be negative regulators of the clock's transcriptional negative-feedback loop . There are several transcription factors involved in the feedback loop, including Per , Tim, Bmal1, and Clk . Cryptochrome alters the transcriptional regulation of these components by physically interacting with them . The regulation mechanisms of fly and mammal circadian clocks are different to some degree. Contrary to fly cryptochromes, mammal cryptochromes are components of the negative-feedback loop, and the interaction between mammalian cryptochromes and other clock components is not be affected by light . The fly Cry binds to Tim, and results in degradation of it, thus inhibits the activity of Per/Tim dimmer. Without Cry, the Per/Tim dimmer is able to enter nucleus and repress the transcription of other clock genes. Mammal Cry interacts with Per, then inhibits the activity of Clk and Bmal1 and repress expression of clock genes.

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Fish

Fish is a large and primitive group of vertebrate, fishes are adapted in nearly all aquatic environments, from the muddy coastal swamps, to the deepest oceans. Around 31,500 fish species are identified, which exhibit greater diversity than any other group of vertebrates. There are two main groups of fish (Pisces), Chondrichthyes and Osteichthyes.

1.4.1 Fish-specific genome duplication

In the evolution history of vertebrates, the genomes were duplicated twice (2R duplication), and a third genome duplication occurred in the basal group of ray-finned fishes (Actinopterygii) later (~350 mya), which is named the fish-specific genome duplication (FSGD or 3R) . Therefore, the ray-finned fish genomes possessed twice as many genes as the lobe-finned fishes (Sarcopterygii), Cartilaginous fishes (Chondrichthyes) and tetrapods initially, then most duplicated genes were secondarily lost or evolved new functions .

The FSGD was first identified by the studies on HOX cluster genes . Recent molecular evolution studies reveal that extra circadian genes of teleost fishes such as period (Per), clock (Clk) and Bmal1 were derived from this chromosome doubling event . However, the cryptochrome seems to departure this theory. Except Cry-DASH, zebrafish (Danio rerio) has 7 cryptochrome genes, while the amphibian Xenopus (Silurana) tropicalis has 3 copies, one Cry1 and two Cry2; the chicken Gallus gallus, also has 3 cryptochrome copies, one Cry1, one Cry2 and one Cry4; and the mammals, including Homo sapiens and Mus musculus, only have one Cry1 and one Cry2.

1.4.2 Study of zebrafish as model organism

The zebrafish (D. rerio), a tropical freshwater fish belonging to Cyprinidae, became an important vertebrate model organism in evolutionary research and circadian study . Studies suggest that zebrafish exhibit robust circadian rhythmicity both on physiological and behavioral activities in response to light .

In addition, cryptochrome gene sequences offered by zebrafish genome sequencing project enable researchers to have background knowledge of this gene family. Recently, the Sanger Institute has released the latest vision Zv9.

2. Material and Method

2.1 Taxa selection and sample collection

20 fish species are employed in our research (Tab. 1). Fish tissues (brain, gut, heart, liver) are collected and transferred to RNAlater (or Sample Protector) immediately, then stored at -80℃.

abbreviation

habitat &habit

sampling site

Scoliodon laticaudus

sco

shallow water, marine

Chiloscyllium plagiosum

chi

shallow water, marine

Dasyatis sp

das

shallow water, marine

Latimeria chalumnae

lac

marine, intermediate depth

Lepisosteus oculatus

lep

freshwater

Ceratodontiformes

cer

freshwater shallow

Acipenseriformes

aci

Gymnothorax saxicola

mur

reef, night active, marine

Puntius tetrazona

pun

freshwater

Doryrhamphus pessuliferus

dor

day active, shallow, marine

Monacanthus chinensis

mon

day-active, marine

Anomalops katoptron

ano

deepwater marine

Myripristis

myr

reef, marine, night active

Epinephelus fuscoguttatus

epi

day active, marine

Gomphosus varius

gov

Amphiprion clarkii

amp

commensal with sea anemone, day active, reef

Gobiidae (fresh water)

gof

freshwater

Gobiidae (marine)

gom

marine

Synchiropus splendidus

sys

highly light sensitive in activity levels, reef

Sebastiscus marmoratus

seb

marine, shallow

2.1.1 Description of fish species

1. Class: Chondrichthyes

Subclass: Elasmobranchii

(1) Scoliodon laticaudus (spadenose shark)

Order: Carcharhiniformes

Family: Carcharhinidae

Characteristic:

A moderately stout, with head broad, greatly depressed, and trowel shaped shark. Snout bell-shaped in dorsoventral view. Preoral length greater than internarial space and mouth width. Eyes small and without posterior notches. Spiracles absent. Teeth similar in upper and lower jaws. Teeth of anteroposteriors with slender oblique cusps and distal blades but no cusplets or serrations. 25-33/24-34 rows of teeth. Interdorsal ridge absent. First dorsal origin over or behind pectoral rear tips, its midbase much closer to pelvic bases than to pectorals and its free rear tip about over pelvic midbases. Second dorsal fin height 1/3 of 1st height or less. Pectoral fin origin under interspace between 4th and 5th of gill slits. Anal fin much larger than 2nd dorsal fin, with short preanal ridges. Colour light grey, yellowish or brownish grey above, without any colour pattern.

Scoliodon laticaudus is a small, stocky species, the spadenose shark has a broad head with a distinctive highly flattened, trowel-shaped snout. The eyes and nares are small. The corners of the mouth are well behind the eyes and have poorly developed furrows at the corners. There are 25-33 tooth rows in the upper jaw and 24-34 tooth rows in the lower jaw; each tooth has a single slender, blade-like, oblique cusp without serrations. The first dorsal fin is positioned closer to the pelvic than the pectoral fins, which are very short and broad. The second dorsal fin is much smaller than the anal fin. There is no ridge between the dorsal fins. The back is bronze-gray in color, and the belly is white. The fins are plain but may be darker than the body. The maximum known length is 74 cm (29 in), though there are unsubstantiated reports of individuals reaching 1.2 m (3.9 ft).

The spadenose shark is a small, stocky species, which has a broad head with a distinctive highly flattened, trowel-shaped snout.

Habitats:

A common tropical and subtropical shark of continental and insular shelves

Distribution:

Indo-West Pacific including Tanzania, Pakistan, India, Sri Lanka, Malaysia, Singapore, Thailand, Java, Borneo, China, Taiwan, Japan

(2) Chiloscyllium plagiosum

Order: Orectolobiformes

Family: emiscylliidae

Characteristic:

Body fairly stout. Mouth well in front of eyes. A lateral ridge present on each side of trunk. Dorsal fins usually shorter and more elevated, length of 1st dorsal fin base 3/5~4/5 its distance from 2nd dorsal fin. Dorsals without projecting free rear tips. First dorsal origin over or behind pelvic fin bases. A prominent colour pattern of numerous white spots on a dark brown background, with darker brown or blackish transverse bands.

Dorsal fins with convex posterior margins. Color pattern of white and dark spots, with dark bands and a brown body. The coloration is very unique in this family making it very simple for identification. The teeth of bamboo sharks are not strongly differentiated. Each tooth has a medial cusp and weak labial root lobes with 26-35 teeth on the upper jaw ans 21-32 teeth on the lower jaw.

Habitats:

This is a common but little-known inshore bottom shark species

Distribution:

Indo-West Pacific including India, Sri Lanka, Singapore, Thailand, Indonesia, Viet Nam, China, Taiwan, Japan, the Philippines.

(3) Dasyatis

Order: Myliobatiformes

Family: Dasyatidae

2. Class: Osteichthyes

Subclass: Sarcopterygii Coelacanthimorpha Crossopterygii

(4) Latimeria chalumnae (coelacanth)

Order: Coelacanthiformes

Family: Latimeriidae

3.

(5) Lepisosteus oculatus (Spotted Gar)

Order: Lepisosteiformes

Family: Lepisosteidae

Characteristic:

The average length of Lepisosteus oculatus is 76 cm. This gar is covered with hard, diamond-shaped ganoid scales. Their bodies are spotted, including the top of the head and the fins.

Gars are long and cylindrical with elongated mouths. Spotted gar grow to a length of 3 feet (0.9 m), weighing 8 pounds (3.6 kg). Their upper body is brown to olive, and they have silver-white sides. Head, body, and fins have olive-brown to black spots that help camouflage the fish. A broad, dark stripe is on the sides of immature fish. Their long, snout-like mouth is lined with strong, sharp teeth, and their body is covered with thick, ganoid (diamond-shaped) scales. Spotted gar may be distinguished from other Texas gar species by the dark roundish spots on the top of the head, the pectoral fins and on the pelvic fins.

Habitats:

Spotted gar prefers shallow open waters, usually 3 - 5 m deep, as well as stagnant backwater. They are often found near the surface basking near fallen logs, trees, or brush. This species is also shoreline-oriented, meaning it can be found near banks that include some sort of brush covering. Spotted gars are rarely found in areas that do not include some form of brush covering.

Spotted gar prefer clear, quiet, vegetated waters of streams, swamps and lakes. They sometimes enter brackish waters along the Gulf Coast.

Distribution:

Spotted gars are very widespread, and can be found from central Texas east into western Florida. Their territory extends north through the Mississippi River drainage into Illinois, the lower Ohio River, and the Lake Erie drainage.

(6) lungfish

(7) sturgeon

(8) Gymnothorax saxicola (Ocellated Moray Eel, Black Edge Moray Eel)

Superorder: Elopomorpha

Order: Anguilliformes

Ocellated Moray Eels are nocturnal animals. Morays are members of the family Muraenidae. The 100 species identified by scientists range in size from 2-10 feet. The largest is the giant moray which reaches 10 feet in length and weighs 75 pounds whereas; Ocellated Moray Eels reach lengths of only about 1 foot. Morays have beautiful color patterns which help to camouflage them in the reef. Because morays keep their mouths open almost all of the time, the insides of their mouths are camouflaged also.

All Morays have muscular, snake-like bodies with thick skin. They have no scales, but a layer of mucus covers the body and protects the skin from germs and parasites. Pelvic and pectoral fins are not found on morays eels. Morays eels have one long dorsal fin that curves around and connects with the short caudal fin (tail fin). The only fin found on the belly is the long anal fin. In the moray eels, the dorsal, caudaul and anal fins are all connected.

Like all morays the Ocellated moray eels has poor eyesight but a very good sense of smell. Because it is a night hunter with poor eyesight, the moray relies on its keen sense of smell to locate prey hiding in the coral.

Ledges and caves within the coral reef are the favorite lairs for the eels.

Distribution:

Western Atlantic, including the Greater Antilles south to Brazil, including the Central American coast from Nicaragua to northern coast of South America

(9) Puntius tetrazona (Tiger barb; Sumatra Barb)

Order: Cypriniformes

Family: Cyprinidae

Characteristic:

(10) Doryrhamphus pessuliferus (Yellowbanded Pipefish)

Order: Syngnathiformes

Family: Syngnathidae

(11) Monacanthus chinensis (Centreboard Leatherjacket, Fan-bellied Filefish)

Order: Tetraodontiformes

Family: Monacanthidae

Characteristic:

To about 38cm, but those seen were 5-8cm long. Large triangular skin flap on the belly that can be greatly expanded. It has thin brown bands on its tail. The upper fin rays on the tail is produced into a filament. It has a concave snout profile and triangular back profile. Body with broad oblique bars on the sides, in some these bars may be indistinct. They come in all shades from brown to green.

Habitats:

Distribution:

Indo-Pacific: Malaysia and Indonesia to Samoa, north to southern Japan, south to northwestern Australia and New South Wales.

(12) Anomalops katoptron (flashlight fish)

Order: Beryciformes

Family: Anomalopidae

Characteristic

Two Dorsal fins with V and I spines, and 14-15 soft rays; anal fin with II spines and 10-11 soft rays. Body black brown, and all fins light, outside of all fins dark, excet for Pectoroal fin.

Habitats

Hides during the day and venture out at night to feed, tending to occur along steep drop-offs near caves on dark, moonless nights. Feeds on zooplankton. The large, deep water form is occasionally collected by fishing at depths of 200 to 400 m. Caught with

Distribution

Pacific Ocean: Philippines and Indonesia to the Tuamoto Islands, north to southern Japan, south to the Great Barrier

(13) Holocentrus/Myripristis

Order: Beryciformes

Family: Holocentridae

(14) Epinephelus fuscoguttatus (Tiger grouper; Blotch grouper; Blotchy rockcod)

Order: Perciformes

Family: Serranidae

Characteristic:

Dorsal fin with XI spines and 14 or 15 rays, the third or fourth spine longest, its length contained 2.9 to 3.5 times in head length and distinctly shorter than longest dorsal-fin rays, the interspinous membranes distinctly incised; anal fin with III spines and 8 rays; pectoral-fin rays 18 to 20; pectoral-fin length contained 1.7 to 2.1 times in head length; pelvic fins not reaching anus, their length contained 2.0 to 2.5 times in head length; caudal fin rounded. Lateral-body scales of fish more than 10 cm standard length smooth, with auxiliary scales; lateral-line scales 52 to 58; lateral-scale series 102 to 115.

Habitats:

Occurs in lagoon pinnacles, channels, and outer reef slopes, in coral-rich areas and with clear waters. Juveniles in seagrass beds. Feeds on fishes, crabs, and cephalopods. May be ciguatoxic in some areas. Mainly active at dusk. Indo-Pacific: Red Sea and

Distribution:

Widely distributed in the Indo-Pacific region, including the Red Sea, but not known from the Persian Gulf, Hawaii, or French olynesia.

(15) Gomphosus varius (Purple club-nosed wrasse; Bird wrasse)

Order: Perciformes

Family: Labridae

(16) Amphiprion clarkii (Yellowtail clownfish)

Order: Perciformes

Family: Pomacentridae

Characteristic:

Dorsal rays X-XI, 15-17; Anal rays II, 12-15; pectoral rays 18-21; lateral-line scales 34-35; body depth 1.7-2.0 in standard length; posterior margins of opercle, interopercle and subopercle strongly serrated. Generally brown to blackish with three white bars on head and body.

Habitats:

Inhabits lagoons and outer reef slopes; found to 50-60 m, but also in shallow water. Omnivorous. A group consists of a large dominant female, few males, and several juveniles. Both female and male defend territory and guard eggs. Low- ranking males and ju

Distribution:

Distributed in the Indo-West Pacific from Persian Gulf to Western Australia, throughout the Indo-Australian Archipelago and in the western Pacific at the islands of Melanesia and Micronesia, north to Taiwan, southern Japan and the Ryukyu Islands.

(17) Rhinogobius duospilus ()

Order: Perciformes

Family: Gobiidae

(17) Gobiidae (marine)

(19) Synchiropus splendidus (Mandarinfish, Mandarin dragonet)

Order: Perciformes

Family: Callionymidae

Characteristic:

Body moderately depressed. Preopercular spine with processes on both inner and outer sides. First dorsal fin slightly high in males; almost all dorsal-fin soft rays trifurcate, 8 divided rays in second dorsal fin; anal-fin soft rays branched except first; pelvic fin without free ray; pectoral fin with 30 rays. Body orange or orangish brown with broad curved bands, elongate spots, and dashes of green; blue marking on head and blue margins on fins.

Habitats:

Inhabits shallow protected lagoons and inshore reefs. Found on silty bottoms with coral and rubble. Usually in small groups spread over small area. Has been reared in captivity.

Distribution:

Distributed in the Western Pacific from Ryukyu Islands to Australia. It is found in Lanyu, southeastern Taiwan.

(20) Sebastiscus marmoratus (Rockfish, Scorpionfish, Filefish)

Order: Scorpaeniformes

Family: Scorpaenidae

Characteristic:

Dorsal fin with XII spines and 10-12 soft rays; anal fin with III spines and 5 soft rays; pectoral fin with 17-19(18); lateral line scales 49-54. Body compressed, pectoral fin rays usually 18, pectoral fin shaped in pentagon. Caudal fin truncated. No spinule on upper margin of 2nd infraorbial bone. Body abdomen yellowish. Body color varies in relation with deoth of habitat, deeper ones suffused with red.

Habitats:

Inhabits marine demersal marine environment, usually shallow rocky reef. Ovoviviparous fish, spwaning from winter to spring. Urogenital papilla present in matured male. Caught by sport angling on rocky shores.

Distribution:

Western Pacific: southern Hokkaido, Japan, Taiwan to the Philippines.

2.2 Isolation of total RNA

2.2.1 Preparing of equipments and reagents

Mortar (Covered by tinfoil and autoclaved by dry treating at 180℃)

Powder-free gloves

Liquid nitrogen

Refrigerated centrifuge

UV spectrophotometer

Ultra-low Temperature Freezer*

Centrifuge tube and tips (RNase-free)

DEPC-treated water (add 100 µL DEPC into 100ml ddH2O, sit at room temperature overnight, and autoclaved) or buy it from supplier

Trizol Reagent

70% ethanol (prepare with DEPC-treated water)

Isopropyl

Chloroform

2.2.2 Procedures

(1) Tissues are rinsed in DEPC-treated water twice and powdered on liquid nitrogen with a mortar and pestle (or homogenized with a rotor power homogenizer), and then homogenized in 1mL of Trizol reagent. Transfer the sample to a tube (RNase-free is not necessary at this moment), then incubate at room temperature for 5 minutes.

(2) Add 200 µL of chloroform (per 1mL Trizol), shake vigorously (not vortex) for 15 seconds to mix well, and incubate at room temperature for 5 minutes. Centrifuge samples at 12,000 rpm at 4℃ for 15 minutes to separate phases. Transfer upper aqueous layer to a new tube, and centrifuge again.

(3) Add 0.5mL of isopropanol to the aqueous layer, mix thoroughly by shaking for 15 seconds, and incubate at room temperature for 10 minutes. Centrifuge samples at 12,000 rpm at 4℃ for 10 minutes to pellet RNA.

(4) Carefully remove the supernatant & add 1mL 75% DEPC-ethanol and vortex on low for 5-10 seconds to wash the pellet thoroughly, then centrifuge at 6,000 rpm at 4°C for 5 minutes to re-pellet.

(5) Carefully remove the supernatant and air-dry the pellet at room temperature for 5-10 minutes. Dissolve the pellet in DEPC-treated water (30-100uL, depending on yield) by gentle pipetting and incubate at 55℃ for 5-10 minutes.

(6) RNA quantization on a spectrophotometer, A260/280 ratios should be between 1.8 and 2.1, A260/230 ratios should be at least 1.9, over 2.0 is preferable. A260/280 ratios outside this range indicate DNA or protein contamination. Low A260/280 ratios indicate carbohydrate, phenol, salt contamination

2.3 Reverse transcription (RT) and cDNA synthesis

The DNA templates were generated from total RNAs by reverse transcription (Invitrogen, Madison, WI).

2.4 Isolation of genomic DNA

In this study, a modified extraction protocol of fish genomic DNA with salt (NaCl) is employed , since the fish samples are not always suitable for RNA analyze.

2.3.1 Preparing of equipments and reagents

Lysis buffer (50 mM Tris-HC1, pH 8.0, 50 mM EDTA, 100 mM NaC1), with 1% SDS and 7 μL of 200 μg·mL-1 of proteinase K

TE buffer (10 mM of Tris pH 8.0 and 1 mM of EDTA)

2.3.2 Procedures

(1) Place each fish sample in a 1.5 mL microtube with 550 μL lysis buffer. then incubate the tube immediately in 50°C water bath for 12 h.

(2) Add 600 μL 5M NaCl in the before centrifuging for 10 min at 12,000 rpm. Transfer the supernatant to a new microtube, precipitate the DNA with 700 μL absolute cold ethanol and incubate at -20°C for 2 h.

(3) Centrifuge the DNA sample, rinse it with 700 μL 70% ethanol and resuspend in 50μL TE buffer, treate each sample with 30 mM RNAse in water bath for 40 min at 37°C.

(4) Preserve the obtained DNA sample at -20°C.

2.4 Primer design and PCR

3 pairs of degenerate primers for each cryptochrome gene were designed base on the D. rerio cryptochromes and aligned sequences using MUSCLE (Multiple sequence alignment).

2.5 Cloning and sequencing