A First Chromosomal Characterization of Black - Spotted Pufferfish, Arothron Nigropunctatus (Tetraodontifrom; Tetraodonidae)

Vol.6, No.2 pages 27-35 Science Technology and Engineering Journal (STEJ) Research Article

A First Chromosomal Characterization of Black - Spotted Pufferfish, Arothron nigropunctatus (Tetraodontifrom; Tetraodonidae)

Montri Reungsing1, Alongklod Tanomtong2, Nattasuda Donbundit2 and Khunapat Sonsrin3*

1 Biotechnology Program, Faculty of Science and Technology, Rajamangala University of Technology Tawan - ok, Siracha, Chonburi 20110, Thailand 2 Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 3 Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Muang, Surin 32000, Thailand * Corresponding author: [email protected]

(Received: 16th June 2020, Revised: 23rd July 2020, Accepted: 5th October 2020)

Abstract-The present study aims to analyze concerned karyotype and idiogram of black - spotted pufferfish (Arothron nigropunctatus Bloch & Schneider, 1801). Chromosome was prepared from kidney tissues of fish reared at Phuket Marine Biological Center, Phuket province. The mitotic chromosomes were harvested by the colchicine - hypotonic - fixation - air drying method. Conventional staining and Ag - NOR banding techniques were applied to stain the chromosomes. The results showed that A. nigropunctatus has diploid (2n)=38, the fundamental number (NF) was 72. The types of chromosomes are two large metacentric, ten medium metacentric, eight medium submetacentric, two small metacentric, 12 small submetacentric, and four small telocentric chromosomes. This is the first report of marker chromosome; found that NORs located at the short arm region of the submetacentric chromosome. No cytologically distinguishable sex chromosome was observed. The karyotype formula could be deduced as follows:

m m sm m sm t 2n (38) = L 2 + M 10 + M 8 + S 2 + S 12 + S 4 Keywords: Arothron nigropunctatus, Chromosome, Karyotype 28 Montri Reungsing, Alongklod Tanomtong, Science Technology and Engineering Journal (STEJ) Nattasuda Donbundit shallowand Khunapat-water Sonsrin environments (Santini et al., Figure 1. General characteristic of the 2013; Oliveira et al., 2006). Marine black-spotted pufferfish (Arothron pufferfish of the family Tetraodontidae is nigropunctatus). 1. Introduction well known to possess athe potent four frontalneurotoxin, teeth fused (Santos, 1992; tetrodotoxin (TTX) in theirSabrah viscera, et al., spines2006). 2. Materials and methods The order Tetraodontiformeson the representbody, and one the four frontal Most teeth marine fused fi shes2.1 studied Chromosome have preparation of the most peculiar radiations(Santos, 1992 of teleost; Sabrah eta aldiploid., 2006 complement). of 48Four acrocentric male and female of A. fishes. Tetraodontiform fishesMost comprises marine fisheschromosomes studied have (Sola aet al., 1981;nigropunctatus Klinkhardt were obtained from farming approximately 412 species,diploid divided complement into ten etof al.48, 1995;acrocentric Brum, 1996).the However,Institute of Phuket Costal Fisheries families as follows: Triacanthodidaechromosomes includ- (Sola et alin., 1981;some Klinkhardttaxa, close speciesResearch have been and Development and Phuket ing 23 species in 11 genera, Triacanthidae et al., 1995; Brum, 1996).reported However, showing changesin inMarine chromosome Biological Center, in Phuket comprising seven species in four genera, some taxa, close speciesnumber have been and reported formula (CaputoProvince, et al., 1997; Thailand. The 0.01% colchicine Balistidae composing showingof 37 species changes in 12 in chromosome number Corrêa and Galetti Jr., 1997)(1 and mL different/100 g body weight) was injected into genera, Monacanthidaeand composing formula of(Caputo 102 et al., 1997; Corrêa sex chromosome systems the(Morescalchi abdominal cavity and left for one hour. species in 27 genera, Aracanidaeand Galetti compos- Jr., 1997)et and al., 1992;different Gomes sex et al., 1994; Cano et ing of 13 species in sixchromosome genera, Ostraciidae systems (Morescalchi et al., Chromosomes were prepared from the al., 1996). Moreover, some kidneyspecies show cells by the squash technique. composing of 22 species1992; in Gomesfi ve genera, et al ., 1994;supernumerary Cano et chromosomes al., (Pauls et al., Kidney tissues were cut into small pieces Triodontidae monotypic,1996). Tetraodontidae Moreover, some1996). species In family show Tetraodontidae, the diploid then mixed with hypotonic solution (0.075 composing of 184 speciessupernumerary in 27 genera, chromosomes numbers (Pauls ranged et from al., 28 to 46 chromosomes M KCl). After discarding all large pieces of Diodontidae comprising1996). 18 In species family Tetraodontidae, in and most t heof diploidthem (30 species) have the seven genera, and Molidaenumbers comprising ranged fi ve fromfundamental 28 to numbers 46 rangedtissues, from 367 tomL of cell sediments were species in three generachromosomes (Matsuura, 2015). and most78 (Arai, of them2011). (30 transferred to a 15-mL centrifuge tube and species) have the fundamental numbers Although half of the tetraodontiform incubated for 45 min. Hypotonic solution ranged from 36 to 78 (Arai, 2011 In )the. present study is the first fi shes distributed along the reef - associated was discarded from the supernatant after In the present cytogenetic study is the study ﬁrst on A. nigropunctatuscentrifugation at 1,200 rpm for 8 min. Cells (Alfaro et al., 2007), the ten recognized (Fig. 1) accomplished with the Ag - NOR cytogenetic study on A. nigropunctatus were fixed in a fresh cool fixative (3 families inhabit a wide(Fig. variety 1) ofaccomplished habitats. stainingwith the technique. Ag-NOR Our results provide new absolute methanol: 1 glacial acetic acid) to In addition, family Tetraodontidaestaining technique. is found Our cytogeneticresults provide information new for further study which up to 7 mL were gradually added predominantly on coralcytogenetic reefs, sea grasses,information on for taxonomy further study and evolutionary on relationship. and other tropical,taxonomy shallow -and water evolutionary Moreover, relationship. we provide basicbefore and beinguseful centrifuged again at 1,200 rpm environments (Santini etMor al.eover,, 2013; Oliveirawe provide information basic and for useful the conservation, for 8 min,breeding at which time the supernatant was et al., 2006). Marine pufferfiinformation sh of the for family the conservation,and chromosome breeding evolution discarded. study of thisThe fixation was repeated until Tetraodontidae is welland known chromosome to possess evolution fish. study of this the supernatant was cleared and the pellet a potent neurotoxin, tetrodotoxinﬁsh. (TTX) was mixed with 1 mL fixative. The mixture in their viscera, spines on the body, and was dropped onto a clean by a plastic pipette followed by air-dry technique (Ditcharoen et al., 2019; Supiwong et al., 2019).

2.2 Chromosome staining

The slide was conventionally stained with

20% stock Giemsa’s solution for 15 min. Ag-NOR banding was conducted by adding 5 cm four drops of 50% silver nitrate and two Figure 1. General characteristic of the black - spotted pufferfi sh (Arothron nigropunctatusdrops of). 2% gelatin on slides, in order. The slides were covered with cover glasses and incubated at 60ºC for 7 min. After that, the slides were soaked in distilled water until Vol 6. No 2, July-December 2020 A First Chromosomal Characterization of Black-Spotted Pufferfi sh, 29 Arothron nigropunctatus (Tetraodontifrom; Tetraodonidae)

2. Materials and methods cover glasses and incubated at 60ºC for 7 min. After that, the slides were soaked in 2.1 Chromosome preparation distilled water until the cover glasses were separated (Howell and Black, 1980). Four male and female of A. nigropunctatus were obtained from farming the Institute 2.3 Chromosome checking, karyotyping of Phuket Costal Fisheries Research and idiograming and Development and Phuket Marine Biological Center, in Phuket Province, Standardized karyotype and idiogram of Thailand. The 0.01% colchicine (1 mL/100 g this fi sh were constructed. Chromosome body weight) was injected into the abdominal checking was performed on mitotic cavity and left for one hour. Chromosomes metaphase cells under a light microscope. were prepared from the kidney cells by the The frequencies of chromosome number squash technique. Kidney tissues were cut per cell were counted. The maximum into small pieces then mixed with hypotonic frequency of chromosome number per solution (0.075 M KCl). After discarding cell is the diploid chromosome number all large pieces of tissues, 7 mL of cell of this fi sh. Ten cells of four male and sediments were transferred to a 15 - mL female with clearly observable and well centrifuge tube and incubated for 45 min. - spread chromosome were selected for Hypotonic solution was discarded from karyotyping. The length of short arm the supernatant after centrifugation at chromosome (Ls) and long arm chromosome 1,200 rpm for 8 min. Cells were fi xed in a (Ll) were measured and calculated to fresh cool fi xative (3 absolute methanol: 1 the length of total arm chromosome (LT, glacial acetic acid) to which up to 7 mL were LT=Ls+Ll). The relative length (RL), gradually added before being centrifuged the centromeric index (CI) and standard again at 1,200 rpm for 8 min, at which deviations (S.D.) of RL and CI were time the supernatant was discarded. The calculated. The CI (q/p+q) between 0.50 fi xation was repeated until the supernatant - 0.59, 0.60 - 0.69, 0.70 - 0.89, and 0.90 was cleared and the pellet was mixed with - 1.00 were classifi ed as the metacentric, 1 mL fi xative. The mixture was dropped submetacentric, acrocentric and telocentric onto a clean by a plastic pipette followed chromosomes, respectively. The by air - dry technique (Ditcharoen et al., fundamental number (number of 2019; Supiwong et al., 2019). chromosome arm, NF) was obtained by assigning a value of two to metacentric, 2.2 Chromosome staining submetacentric, acrocentric chromosomes, and one to telocentric chromosome. All The slide was conventionally stained with parameters were used in karyotyping. The 20% stock Giemsa’s solution for 15 min. idiogram was constructed using a model Ag - NOR banding was conducted by drawing of karyotype and accomplished by adding four drops of 50% silver nitrate a computer program (Sarasan et al., 2018; and two drops of 2% gelatin on slides, Chaiyasan et al., 2018). in order. The slides were covered with 30 Montri Reungsing, Alongklod Tanomtong, Science Technology and Engineering Journal (STEJ) Nattasuda Donbundit and Khunapat Sonsrin

3. Results and discussion The present study was accomplished by using the Ag - NOR staining technique. The present study revealed that the diploid The objective of this technique is to chromosome number of A. nigropunctatus determine NORs regions. Which represented was 2n=38 and the NF was 72 in male and the location of genes (loci) that function in female. The types of chromosomes are two ribosome synthesis (18S and 28S ribosomal large metacentric, ten medium metacentric, RNA), and a positive NOR will be detected eight medium submetacentric, two small when these genes are functioning (Sharma metacentric, 12 small submetacentric, and et al., 2002). From the result, the short arm eight small telocentric chromosomes (Figure region of the submetacentric chromosome 2). It was in agreement with the previous pair 9 showed clearly observable NORs reports from Japan (Arai and Nagaiwa, (Figure 3). Normally, most fishes have only 1976; Ojima, 1985). We compared with one pair of small NORs on chromosomes other fishes in the family Tetraodonidae (Mohannath G. et al., 2016). If some fishes (genus Arothron), A. hispidus, 2n=42 have more than two NORs, it may be (NF=82) (Natarajan and Subrahmanjan, caused by the translocation between some 1974); A. immaculatus, 2n=42 (NF=68) parts of chromosome which have NOR and (Choudhury et al., 1982); A. immaculatus, another chromosome. Furthermore, NOR 2n=42 (NF=72) (Arai and Nagaiwa, 1976); usually located close to the telomere of A. leopardus, 2n=40 (NF=68) (Choudhury et the chromosome. If NOR appears between al., 1982) and A. reticularis, 2n=42 (NF=68) the centromere and telomere (interstitial (Choudhury et al., 1982) (Table 1). NOR), it may be the result of the tandem fusion between this chromosome with Sex chromosome systems of Te- NOR and another one. However, it may be traodontidae have been described by male caused by the pericentric inversion between heterogamety, others by female heterogamety two telocentric chromosomes that one and others no cytologically distinguish- chromosome has NOR at the telomere able sex chromosome (Sá - Gabriel and (Galetti Jr. et al., 2000). Molina, 2004). Present study, no cytologically distinguishable sex chromosome was The asymmetrical karyotype of observed. It may be possible that the fish’s A. nigropunctatus and all three types of sex - chromosomes are at the initiation of chromosomes (metacentric, submetacentric differentiation. The results is inconsistent and telocentric chromosomes) that we with the report of Ojima (1985) that found the important chromosome markers. proposed functionally differentiated sex The idiogram shows the continuous length chromosomes, the multiple sex systems gradation of the chromosomes. The largest derive from the translocations. In the chromosome is approximately twice the size of the smallest chromosomes. The X1X1X2X2/X1X2Y sex system, the diploid value of females presented one chromosome chromosome markers of A. nigropunctatus more than males. The origin of this system are chromosome pair 1, the largest metacentric may be linked to the existence of centric and chromosome pair 19, the smallest fusions (Robertsonian rearrangements). telocentric chromosomes. The data of the chromosomal checks on mitotic metaphase cells of the A. nigropunctatus are shown in smallsmall NORs NORs on onchromosome chromosomes (sMohannath (Mohannath andand telocentrictelocentric chromosomes)chromosomes) thatthat wewe G. etG. al et., al2016., 2016). I)f. someIf some fishes fishes have have more more foundfound thethe importantimportant chromosomechromosome markers. markers. thanthan two two NORs, NORs, it mayit may be be caused caused by by the the TheThe idiogramidiogram shows thethe continuouscontinuous length length translocationtranslocation between between somesome partparts s ofof gradationgradation ofof the chromosomes. The The largest largest chromosomechromosome which which have have NOR NOR and and another another chromosomechromosome is approximatelyapproximately twicetwice the the chromosome.chromosome. Furthermore, Furthermore, NOR NOR usually usually sizesize ofof thethe smallest chromosomes.chromosomes. TheThe locatedlocated close close to tothe the telomere telomere ofof thethe chromosomechromosome markers ofof A.A. nigropunctatus nigropunctatus chromosome.chromosome. If NORIf NOR appears appears between between the the areare chrchromosomeomosome pairpair 1,1, thethe largestlargest centromerecentromere and and telomere telomere (interstitial (interstitial NOR), NOR), metametacentricentric and chromosome pair pair 19 19, ,the the it mayit may be thebe theresult result of ofthe the tandem tandem f usionfusion smallestsmallest telocentrictelocentric chromosomes.chromosomes. The The data data betweenbetween this this chromosome chromosome w itwhit hNOR NOR and and ofof thethe chromosomalchromosomal checkschecks onon mitoticmitotic anotheranother one. one. However, However, it mayit may be be caused caused by by metaphasemetaphase cells ofof thethe A.A. nigropunctatusnigropunctatus smallthesmall the pericentricNORs NORspericentric on on chromosome chromosomeinversion inversion sbetween s( Mohannath(betweenMohannath twotwo andandareare telocentricshownshowntelocentric in ( Table chromosomes)chromosomes) 22)).. ((FigFig. .4 4) )showsthat showsthat we thewe the G.telocentricG. et telocentricet al al.,., 2016 2016 )chromosomes.) .I fchromosomesI fsome some fishes fishes have thathavethat more moreoneone foundfoundidiogramidiogram the the important importantfromfrom conventional chromosome chromosome stainingstaining markers. markers. and and thanchromosomethanchromosome two two NORs, NORs, has it has it mayNOR may NOR be beat caused atcausedthe the telomere by telomereby the the TheTheAgAg idiogram- -NORidiogramNOR shows bandingshows the the continuous techniquescontinuous techniques .length .length TheThe translocation(Galettitranslocation(Galetti Jr. etJr. al etbetween.,between al 2000., 2000) . )some.some partparts s ofof gradationgradationkaryotypekaryotype of of formuthe the chromosomes. chromosomes.lala forfor A.A. nigropunctatus nigropunctatus The The largest largest is is TheThe asymmetrical asymmetrical karyotype karyotype of of A. A. asas follows: follows: chromosomechromosome which which have have NOR NOR and and another another chromosomechromosomemm isis approximatelymapproximatelysmsm mm twicetwicesmsm thethet t chromosome.nigropunctatuschromosome.nigropunctatus Furthermore, Furthermore,and and all all three NORthreeNOR types usuallytypesusually o fo f sizesize22nn (38) (38)ofof =the= the LL 2smallest +smallest M 1010 ++ Mchromosomes.Mchromosomes.88 + + S S2 2+ + S S 12 12 The+The +S 4S . 4. locatedchromosomeslocatedchromosomes closeclose (metacentric, toto (metacentric, thethe telomeretelomere submeta submeta ofofcentric centricthethe chromosomechromosome markers markers of of A. A. nigropunctatus nigropunctatus chromosome. chromosome. If If NOR NOR appears appears between between the the areare chrchromosomeomosome pairpair 1,1, thethe largestlargest A. A. B.B. centromerecentromere and and telomere telomere (interstitial (interstitial NOR), NOR), mm metametacentricentric cand and chromosome chromosome pair pair 19 19, ,the the it may be the result of the tandem fusion smallest telocentric chromosomes. The data it may be the result of the tandem fusion smallest11 22 telocentric 3 chromosomes. 44 5 5 6 The6 data 7 7 between between this this chromosome chromosome w witithh NOR NOR and and ofof thethe chromosomalchromosomal checkschecks onon mitoticmitotic another another one. one. However, However, it it may may be be caused caused by by metaphasemetaphase cellscells ofof thethe A.A. nigropunctatusnigropunctatus thethe pericentricpericentricVol 6. No 2, July-December inversioninversion 2020betweenbetweenA Firsttwotwo Chromosomal sm sm are8are8 Characterizationshown shown 99 in in 10( Table( Table of Black-Spotted 1111 2 2 ) . ) . ( Fig (12 Fig12 . Pufferfi . 4 4 ) ) 13shows 13shows sh, 14 the14 31 the Arothron nigropunctatus (Tetraodontifrom; Tetraodonidae) telocentric telocentric chromosomeschromosomes thatthat oneone idiogramidiogram fromfrom conventionalconventional stainingstaining andand chromosomechromosome hashas NORNOR atat thethe telomeretelomere AgAg-NOR-NOR banding banding techniques techniques. . TheThe small small NORsNORs(Table onon2). chromosome chromosome(Figure 4) showsss ((MohannathMohannath the idiogram 15and15and telocentric 16telocentric16 2n (38) 17 = Lchromosomes)chromosomes)m + Mm + M smthatthat + S mwewe (Galetti Jr. et al., 2000). karyotype formula for2 A. nigropunctatus10 8 2is (GalettiG. G. etet alal .,Jr.., 2016 2016et al).,).. 2000IIff somesome). fishesfishes havehave moremore karyotypefoundfoundsm thethe formu importantimportantt la for chromosome chromosomeA. nigropunctatus markers.markers. is from conventional staining and Ag - NORt t + S + S . thanthan twotwoTheThe NORs,NORs, asymmetrical asymmetrical itit maymay bebe karyotypekaryotype causedcaused by byofof theA. theA. asTheasThe follows: follows: idiogramidiogram12 4 showsshows thethe continuouscontinuous lengthlength banding techniques. The karyotype formula 1818 1919 mm mm smsm mm smsm t t nigropunctatus and all three types of 2n (38) = L 2 + M 10 + M 8 + S 2 + S 12 + S 4. nigropunctatustranslocation translocation betweenbetweenand all somethreesome typespartpartss oofoff 2gradationngradation (38) = L of2of + theMthe 10 chromosomes.chromosomes. + M 8 + S 2 + TheSThe12 largest largest+ S 4. chromosomeschromosomesfor A. nigropunctatus (metacentric, (metacentric, submeta issubmeta as follows:centriccentric chromosomeFigurechromosomeFigure 2. The 2. The which whichmetaphase metaphase havehave NORchromosomeNOR chromosome andand anotheranother plate plate (A.) (A.) and andchromosomechromosome karyotype karyotype (B.)isis approximately ofapproximately blackblack--spottedspotted twicepufferfishtwice pufferfish thethe chromosome.chromosome.(A. ( nigropunctatusA.Furthermore,Furthermore, nigropunctatus NOR),NOR 2),n 2=38 n =38usuallyusually by by conventional conventional sizesize staining stainingofof thethe technique,smallestsmallest chromosomes.chromosomes. sscalecale bar bar indicate indicate TheThes 5s 5 locatedlocated A. A.closeclose toto thethe telomeretelomere ofof thetheB.B. chromosomechromosome markersmarkers ofof A.A. nigropunctatusnigropunctatus micrometersmicrometers (m (m = =metacentric, metacentric,mm smsm == submetacentricsubmetacentric andand t t == telocentrictelocentric chromosome.chromosome. IfIf NORNOR appearsappears betweenbetween thethe areare chrchromosomeomosome pairpair 1,1, thethe largestlargest chromosomechromosomes). s ). 11 2 2 3 3 4 4 5 5 6 6 7 7 centromerecentromere andand telomeretelomere (interstitial(interstitial NOR),NOR), metametacentricentricc andand chromosomechromosome pairpair 1919,, thethe

itit maymay bebe thethe resultresult ofof tthehe tandemtandem ffusionusion smallestsmallest telocentrictelocentric chromosomes.chromosomes. TheThe datadata

betweenbetween thisthis chromosomechromosome wwitithh NORNOR andandsmsm 88 of of the the 9 9 chromosomal chromosomal 10 10 11 11 12checks checks12 13 13on on mitotic 14mitotic 14

anotheranother one.one. However,However, itit maymay bebe causedcaused byby metaphasemetaphase cellscells ofof thethe A.A. nigropunctatusnigropunctatus the the pericentricpericentric inversioninversion betweenbetween twotwo areare shownshown inin ((TableTable 22)).. ((FigFig.. 44)) showsshows thethe 1515 16 16 17 17

telocentrictelocentric chromosomeschromosomes thatthat oneone idiogramidiogram fromfrom conventionalconventional stainingstaining andand chromosome chromosome hashas NORNOR atat thethe telomeretelomere t t AgAg--NORNOR banding banding techniques techniques.. TheThe

(Galetti (Galetti Jr.Jr. etet alal.,., 20002000)).. 1818karyotype karyotype 19 19 formuformulala forfor A.A. nigropunctatusnigropunctatus isis

TheTheFigure asymmetricalasymmetrical 2. The metaphase karyotypekaryotype chromosome ofof A.A. plate (A.) andasas follows: follows:karyotype (B.) of black - spotted pufferfi sh FigureFigure 2 2. The. The metaphase metaphase chromosome chromosome plate plate (A.) (A.) and and karyotype karyotypemm (B.) (B.) ofm mof black blacksm-smspotted-spottedmm pufferfish pufferfishsmsm tt nigropunctatus nigropunctatus (A. nigropunctatus), andand allall 2n=38threethree bytypestypes conventional ooff staining22nn technique, (38)(38) == LL 2scale2 ++ MM bar1010 indicates ++ MM 88 + +5 S Smicrometers22 ++ SS 1212 ++ SS44.. (A.(A. nigropunctatus nigropunctatus(m = metacentric,),), 2 2nn=38 =38sm =by by submetacentric conventional conventional and staining staining t = telocentric technique, technique, chromosomes). s calescale bar bar indicate indicates s5 5 chromosomes chromosomes (metacentric,(metacentric, submetasubmetacentriccentric micrometersmicrometers (m(m == metacentric,metacentric, smsm == submetacentricsubmetacentric andand t t == telocentrictelocentric

A.A.A.chromosome A.chromosome s).s). B.B.B. mm mm 11 2 2 3 44 5 5 6 6 7 7 11 22 33 44 55 66 77

smsmsm 888 9 99 10 1010 11 11 1111 1212 12 13 1313 13 14 14 14

151515 16 16 17 17 17

tt t 181818 1919 19

Figure 3. The metaphase chromosome plate (A.) and karyotype (B.) of black - spotted pufferfi sh FigureFigure 22.. (A.The The nigropunctatus), metaphasemetaphase chromosomechromosome 2n=38 by Ag -plate plateNOR (A.) banding(A.) andand technique, karyotype karyotype scale (B.)(B.) bars ofof indicates blackblack-- spottedspotted5 micrometers. pufferfish pufferfish A.A. ( (A.TheA. nigropunctatusnigropunctatus arrows indicate nuclear),), 22nn=38=38 organizer bybyB. conventionalB.conventional regions, NORs stainingstaining (m = metacentric, technique,technique, sm = ss calesubmetacentriccale barbar indicateindicate ss 55 and t = mtelocentricm chromosomes). micrometersmicrometers (m(m == metacentric,metacentric, smsm == submetacentricsubmetacentric andand tt == telocentrictelocentric chromosomechromosomess).). 11 2 2 3 3 4 4 5 5 6 6 7 7

smsm 88 9 9 10 10 11 11 12 12 13 13 14 14

1515 16 16 17 17

t t 1818 19 19

A.A. B.B. mm 11 22 33 44 55 66 77

smsm 88 99 1010 1111 1212 1313 1414

1515 1616 1717 tt 1818 1919

Figure 3. The metaphase chromosome plate (A.) and karyotype (B.) of black-spotted pufferfish (A. nigropunctatus), 2n=38 by Ag-NOR banding technique, scale bars indicates 5 32 Montrimicrometers. Reungsing, Alongklod The arrows Tanomtong, indicate nuclearScience Technology organizer and Engineeringregions, JournalNORs (STEJ) (m = Nattasudametacentric, Donbundit sm and = submetacentricKhunapat Sonsrin and t = telocentric chromosomes).

1 2 3 4 5 6 7

8 9 10 11 12 13 14

15 16 17

18 19

FigureFigure 4. Idiogram 4. Idiogram showingshowing lengths lengths and shapesand shapes of chromosomes of chromosomes of the black - spottedof the pufferfi black -shspotted (A. nigropunctatus), 2n=38 (A.). The arrow indicates NOR - bearing chromosome pair 9. pufferfish (A. nigropunctatus), 2n=38 (A.). The arrow indicates NOR-bearing chromosome pair 9.

Table 1. Review of cytogenetic publications in the genus Arothron (Tatraodonidae).

A. nigropunctatus 2n Chromosome formula NF Reference Arothron hispidus 42 36sm + 6t 78 Natarajan and Subrahmanjhan (1974) A. immaculatus 42 14m + 16sm + 12t 72 Arai and Nagaiwa (1976) 42 12m + 14sm + 16t 68 Choudhury et al. (1982) A. leopardus 40 14m + 14sm + 12t 68 Choudhury et al. (1982) TableA. nigropunctatus1. Review of cytogenetic38 14m +publications 20sm + 4t in the72 genusPresent Arothron study (Tatraodonidae). A. reticularis 42 12m + 14sm + 16t 68 Choudhury et al. (1982) Species 2n Chromosome formula NF Reference Remarks: 2n = diploid chromosome number, NF = fundamental number, m = metacentric, sm = Arothronsubmetacentrics hispidus and t = 42telocentric 36sm chromosomes. + 6t 78 Natarajan and Subrahmanjhan (1974) A. immaculatus 42 14m + 16sm + 12t 72 Arai and Nagaiwa (1976) Vol 6. No 2, July-December 2020 A First Chromosomal Characterization of Black-Spotted Pufferfish, 33 Arothron nigropunctatus (Tetraodontifrom; Tetraodonidae)

Table 2. Mean length of short arm chromosome (Ls), length long arm chromosome (Ll), length total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from 10 metaphase cells of the black - spotted pufferfish (A. nigropunctatus), 2n=38.

Chro.pair Ls Ll LT RL±SD CI±SD Chro.size Chro.type 1 0.98 1.03 2.01 0.097+0.007 0.511+0.005 Large Metacentric 2 0.68 0.71 1.39 0.067+0.004 0.513+0.008 Medium Metacentric 3 0.61 0.65 1.27 0.061+0.005 0.515+0.015 Medium Metacentric 4 0.62 0.65 1.26 0.061+0.004 0.511+0.012 Medium Metacentric 5 0.56 0.59 1.15 0.056+0.004 0.514+0.008 Medium Metacentric 6 0.52 0.53 1.05 0.051+0.005 0.509+0.005 Medium Metacentric 7 0.46 0.49 0.95 0.046+0.003 0.516+0.015 Small Metacentric 8 0.52 0.79 1.31 0.062+0.005 0.605+0.021 Medium Submetacentric 9* 0.43 0.70 1.13 0.055+0.003 0.622+0.022 Medium Submetacentric 10 0.37 0.70 1.07 0.052+0.003 0.651+0.018 Medium Submetacentric 11 0.34 0.68 1.02 0.049+0.002 0.671+0.025 Medium Submetacentric 12 0.34 0.66 1.00 0.048+0.004 0.662+0.025 Small Submetacentric 13 0.32 0.63 0.95 0.046+0.005 0.665+0.021 Small Submetacentric 14 0.32 0.63 0.94 0.045+0.003 0.666+0.033 Small Submetacentric 15 0.30 0.61 0.91 0.044+0.004 0.676+0.023 Small Submetacentric 16 0.27 0.59 0.86 0.042+0.004 0.687+0.024 Small Submetacentric 17 0.27 0.57 0.83 0.041+0.003 0.684+0.037 Small Submetacentric 18 0.00 0.80 0.80 0.039+0.004 1.000+0.000 Small Telocentric 19 0.00 0.80 0.80 0.039+0.003 1.000+0.000 Small Telocentric Remarks: * = NOR - bearing chromosome and Chro. = chromosome.

4. Acknowledgements 5. References

This work is supported by Science Alfaro, M. E., Santini, F. and Brock, C. D. achievement scholarship of Thailand (2007). Do reefs drive diversification (SAST) and Surindra Rajabhat University. in marine teleosts? Evidence We would like to thank the cytogenetic from the pufferfishes and their research group for the accuracy check of allies (order Tetraodontiformes). the report and valuable help. Evolution 61, 2104 - 2126. Arai, R. (2011). Fish karyotypes: A check list. Spring Tokyo, Japan. 34 Montri Reungsing, Alongklod Tanomtong, Science Technology and Engineering Journal (STEJ) Nattasuda Donbundit and Khunapat Sonsrin

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