Atlas Journal of Biology 2 (1): 84–87, 2012 doi: 10.5147/ajb.2012.0062 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Inheritance of Humeral Spotting in the Croaking Gourami (Os- phronemidae: Trichopsis vittatus) Jack S. Frankel*, Lisa Adler-Golden, and Andre N. Porter Department of Biology, Howard University, Washington, DC 20059, USA Received: January 28, 2012 / Accepted: March 11, 2012 __________________________________________________ * Corresponding author: jfrankel@howard.edu 84 Abstract The croaking gourami (Trichopsis vittatus) exhibits two phe- notypes associated with humeral spotting. Fish possess a prominent, dark humeral patch or spot located behind the operculum or lack this spotting pattern. Segregation patterns observed from the progenies of eleven different crosses sup- port the hypothesis that the inheritance of humeral spotting in T. vittatus is controlled by the action of a single autosomal locus, with complete dominance of the allele controlling the spotted phenotype. Key Words: Trichopsis vittatus, croaking gourami, humeral spot- ting, Osphronemidae. Introduction Labyrinth fishes of the teleost families Osphronemidae, He- lostomatidae, Belontiidae, and Anabantidae comprise the tra- ditional anabantoids, a group of approximately 80 relatively small African and southeast Asian species (Linke, 1991). The anabantoids are quite popular with aquarium hobbyists for their interesting reproductive behaviors, with males of most species brooding eggs in their mouths or in a floating bubblenest (Linke, 1991; Axelrod & Vorderwinkler, 1995). The croaking goura- mi, Trichopsis vittatus (Cuvier, 1831), is named for its ability to produce an audible chirping noise, accomplished via the utili- zation of specialized adaptations of the pectoral fins (Hengl- muller & Ladich, 1999). These chirping sounds are produced by both sexes during breeding and well-mated individuals can continue producing sounds for a few hours. Males alone may also produce such sounds during the establishment of dominance hierarchies (Ladich, 1998; Ladich et. al., 1992). The croaking gourami is an Asian anabantoid with a widespread distribu- tion throughout Sumatra, the Sunda Islands, Malaysia, Thailand, and Vietnam (Linke, 1991; Axelrod & Vorderwinkler, 1995). This species occupies bodies of water ranging in size from pond-like accumulations of water to small rivers. In addition to its unique sound-producing ability, both female and male fishes in natu- SHORT COMMUNICATION A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 85 ral populations of T. vittatus typically exhibit a black patch or spot located caudally to the operculum. While this humeral spot is a feature of most individuals, there is an alternate pheno- type lacking this humeral spot. The mode of inheritance of this spot is of particular interest, as it likely serves as an eyespot to confuse predators. As a continuation of our interest in the inheritance of both banding and spotting patterns in freshwater teleosts (Frankel, 1985, 1991, 1998, 2001, 2002, 2004, 2009, 2011), the present study was undertaken to ascertain the mode of inheritance of humeral spotting in the croaking gourami. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Table 1. Probable genotypes (PG), observed phenotypic numbers, expected ratios, degrees of freedom (df), chi-square values (X2) and probability of fit (P) for crosses amongst humeral spotted and unspotted Trichopsis vittatus. * (H) = humeral spotted parental fishes; (N) = unspotted parental fishes; (F) = F1 offspring. + The probability for all X2 tests is > .05; thus, results fit expected ratios according to Mendelian inheritance. Cross No. Parents* Phenotypic Numbers Exp. ratio df X 2 P + ♀ (PG) x ♂ (PG) Humeral Spotting No Spotting 1 HI (AA) x H1 (AA) 24(FI&F1) 0 1:0 2 HII (AA) x H2 (AA) 17(FII&F2) 0 1:0 3 HIII (AA) x H3 (AA) 12 0 1:0 4 HIV (AA) x H4 (AA) 26 0 1:0 5 HV (AA) x H5 (AA) 20 0 1:0 Pooled 99 0 1:0 6 NI (aa) x N1 (aa) 0 22 0:1 7 NII (aa) x N2 (aa) 0 21(FIII&F3) 0:1 8 NIII (aa) x N3 (aa) 0 19(FIV&F4) 0:1 9 NIV (aa) x N3 (aa) 0 14(FV&F5) 0:1 Pooled 0 76 0:1 10 HI (AA) x F1 (AA) 25 0 1:0 11 FI (AA) x H1 (AA) 27 0 1:0 12 HIV (AA) x F2 (AA) 16 0 1:0 13 FII (AA) x H4 (AA) 18 0 1:0 Pooled 86 0 1:0 14 NII (aa) x F3 (aa) 0 12 0:1 15 NIV (aa) x F5 (aa) 0 10 0:1 16 NIII (aa) x F4 (aa) 0 15 0:1 17 FIII (aa) x N2 (aa) 0 11 0:1 18 FV (aa) x N3 (aa) 0 22 0:1 Pooled 0 70 0:1 19 HII (AA) x N1 (aa) 21(FVI&F6) 0 1:0 20 HIII (AA) x N2 (aa) 32(FVII&F7) 0 1:0 21 HV (AA) x N3 (aa) 10(FVIII&F8) 0 1:0 22 NI (aa) x H2 (AA) 12(FIX&F9) 0 1:0 23 NIII (aa) x H5 (AA) 15(FX&F10) 0 1:0 Pooled 90 0 24 FVI (Aa) x F9 (Aa) 15 6 3:1 1 0.1429 0.7054 25 FVII (Aa) x F8 (Aa) 19 7 3:1 1 0.0513 0.8208 26 FVIII (Aa) x F7 (Aa) 12 5 3:1 1 0.1765 0.6743 27 FIX (Aa) x F6 (Aa) 16 5 3:1 1 0.0159 0.8996 28 FX (Aa) x F10 (Aa) 15 4 3:1 1 0.1579 0.691 29 FVI (Aa) x F10 (Aa) 22 7 3:1 1 0.0115 0.9146 30 FVII (Aa) x F6 (Aa) 21 8 3:1 1 0.1034 0.7477 Total 7 0.6593 0.9986 Pooled 120 42 3:1 1 0.0741 0.7854 Heterogeneity 6 0.5852 0.9966 31 FVI (Aa) x F4 (aa) 18 15 1:1 1 0.2727 0.6015 32 FVII (Aa) x F5 (aa) 14 15 1:1 1 0.0345 0.8526 33 FIV (aa) x F7 (Aa) 12 10 1:1 1 0.1818 0.6698 34 FV (aa) x F8 (Aa) 9 11 1:1 1 0.2 0.6547 Total 4 0.689 0.9526 Pooled 53 51 1:1 1 0.0385 0.8444 Heterogeneity 3 0.6505 0.8847 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 86 Materials and Methods Healthy adult specimens of T. vittatus were obtained from a wholesale distributor in Maryland, USA, and maintained in separate 76 liter holding tanks equipped with aerators at 25o C. Male and female fishes exhibiting either the characteristic humeral spotting phenotype or an aberrant, unspotted pheno- type were selected at random from stock specimens, placed in separate 76 liter tanks, and allowed to develop at 25oC until sexually mature. Optimal water conditions were provided for all fish (i.e. low water hardness of 5o dGH, pH 7.5, and tempera- ture 25oC). Sexually mature fishes were transferred to 36 liter tanks with temperature raised to 30oC (Linke, 1991). All progeny for this study were obtained utilizing artificial fertilization techniques. Artificial fertilizations were performed at a constant temperature of 30oC. Eggs were collected and inseminated in fish Ringer’s solution prepared with 6.50 g NaCl, 0.250 g KCl, 0.20 g NaHCO3, and 0.30 g CaCl2 in one liter of distilled water. Gametes were obtained by cycling individual females through at least one natural breeding period in the presence of three males. Each gravid female was transferred to a 250 ml fingerbowl containing Ringer’s solution. A small piece of nylon mesh netting was used to secure the female, bellyside down, against the side of the fingerbowl. Gentle and even pres- sure was then applied to the abdominal flank, resulting in the release of eggs. Sperm was obtained by gently compressing the abdomen of a male fish against the side of a 500 ml fin- gerbowl containing tank water. The milt, clearly visible at the urogenital aperture as a thin white stream, was collected using a narrow-mouthed pipette and expelled over the eggs. The total volume of sperm suspension, collected as “dry” as possible, was approximately 0.5 ml. The time interval from egg collection to sperm addition was between 20 and 40 seconds. Parental fishes, exhibiting either the spotted (H) or unspot- ted (N) phenotype, along with F1 progeny (F), were used in a series of 34 crosses (Table 1). Embryos from all crosses were transferred to 250 ml fingerbowls containing tank water and incubated at 30oC. Dead or developmentally arrested individu- als were removed daily until fry were free-swimming. Progeny groups then were placed in separate 36 liter rearing tanks, fed initially on Pure Aquatic Brand Premium Fry Food, and allowed to develop until their phenotype could be visually determined. Since humeral spotting is more defined in young adults, deter- mination of spotting was only scored for those individuals. Phe- notypic data of all progeny were recorded and subjected to chi-square analysis. Pooled and heterogeneity chi-square tests were also performed, treating the progenies from reciprocal F1 x F1 crosses as single large progenies in an analysis of overall goodness of fit. Results and Discussion Table 1 presents data for the proposed genotypes of paren- tal fishes, observed phenotypic numbers, expected ratios, and probability of fit for T. vittatus analyzed for the mode of inheri- tance of humeral spotting. Parental fishes and progeny from all crosses clearly displayed either the humeral spotted or unspot- ted phenotype. Females and males exhibiting a humeral spot HI, HII, HIII, HIV, HV and H1, H2, H3, H4, H5, respectively, were scored as homozygous dominants, as crosses involving these indi- viduals always resulted in spotted progeny (crosses 1-5, 10-13, 19-23). Parental fishes lacking a humeral spot (females NI, NII, NIII, NIV and males N1, N2, N3) were scored as homozygous recessives, as crosses amongst these individuals consistently bred true (crosses 6-9). Further, when FIII and FV females, and F3, F4, and F5 males were mated with their parent, the resulting offspring consistently lacked a humeral spot (crosses 14-18). In addition, reciprocal crosses between spotted and unspotted parental fishes always resulted in spotted progeny (FVI-FX and F6-F10) (crosses 19-23). Crosses amongst F1 fishes resulting from parental spotted (H) and unspotted (N) matings always resulted in a satisfactory fit to a 3:1 phenotypic ratio of F2 progeny (crosses 24-30), commen- surate with the action of a single autosomal gene locus exhibit- ing complete dominance for humeral spotting; the A_ genotype results in expression of humeral spotting. This mode of Men- delian inheritance was further supported by crosses between presumptive F1 homozygous recessives (FIV, FV, F4, F5) and F1 heterozygotes (FVI, FVII, F7, F8) (crosses 31-34). Both spotted and unspotted fry resulted from these crosses and, based on chi-square analyses, conformed to the expected 1:1 phenotypic ratio. Results of this study support the hypothesis that humeral spotting in the croaking gourami is controlled by a single lo- cus, with dominance at this locus required for the expression of the spotted phenotype. Segregation patterns for the spotted and unspotted phenotypes of T. vittatus clearly fit an autoso- mal pattern of inheritance, as chi-square tests do not deviate significantly from expectations. The data also suggest complete dominance for humeral spotting, since there is no discernable difference in the appearance of spotting between presumptive heterozygous (Aa) and homozygous (AA) fishes. Further, results of heterogeneity tests also support the acceptance of the null hypothesis for these data. This prominent, dark humeral spot in the croaking gourami most certainly serves as an eyespot and, therefore, would give a selective advantage to those individuals possessing this feature by providing them with an interspecific marking to minimize predation. A monogenic mode of inheri- tance has also been reported for the expression of a caudal pe- duncle marking in the labyrinth fish Pseudosphromenus cupanus (Frankel, 2001). Here, the presence of a caudally located band is also controlled by a dominant allele determining this eyespot phenotype. It is interesting to note, however, that markings serv- ing to provide fishes with disruptive patterns are not always the preferred or prominent phenotypic alternative. 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