Proceedings of 6th International Fruit Symposium 6–10 May 2002, Stellenbosch, South Africa pp. 259–263

Variation in symmetrical patterns of development in grandis and Ceratitis capitata (Diptera: )

A.O. Pires, D. Selivon* & A.L.P. Perondini Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, R. do Matão 277, 05508-900 São Paulo, SP, Brazil

This paper reports preliminary results on the asymmetrical variation in the number of supra- fronto-orbital and frontal setae in the fruit Anastrephagrandis (Macquart) and Ceratitiscapitata (Wiedemann). Brazilian samples of 100 females and 100 males of A. grandis and 50 females and 50 males of C. capitata were analysed. In A. grandis the number of frontal setae varied from two to six on each side,and about 57% of the flies were asymmetrical.The number of orbital setae varied from one to three on each side, and 27% of the individuals were asymmetrical. The scores of asymmetry (R–L) were symmetrically distributed around 0 (zero). For the frontal setae the distribution did not deviate from a normal curve, while for the orbital setae the distribution was leptokurtic. The variation of frontal setae is thus in accordance to the fluctuating asymmetry model, and for the orbital setae it is not clear if the variation does not include a covariation between sides.In C.capitata the number of frontal setae was two and only one male with three setae was found in the sample.The supra-fronto-orbital setae varied between two and three on each side,84% of the males and 92% of the females being symmetrical.The anterior supra-fronto-orbital setae of C.capitata showed sexual dimorphism,being plumose in males.These setae did not present variation.The degree of variation for both types of setae was so low that it does not allow inferences about the mode of asymmetrical deviations.

INTRODUCTION at random between the right and left sides of the Development of is brought about by the body. FA has been studied in a wide number of action of a multitude of genes that function , and it was found to affect different coordinately to construct the phenotype in a structures of the body (Parsons 1990; Markow precise way. In bilateral animals, perfect symmetry 1995). Although it is clear that FA reflects an insta- for bilateral characters are expected since the bility of developmental processes, and thus is a phenotype of an individual is the product of the measure of the capacity of an individual to respond same genotype (Thoday 1956). However, variation to genetic and environmental perturbations, it is in the morphology of animal body parts is common, not precisely known how and when these stressors ranging from subtle alterations to substantial cause detectable effects (reviews in Parsons 1990; modifications in traits. As the expression of the Møller & Pomiankowski 1993; Markow 1995). As genome is also under the influence of the environ- pointed out by Markow (1995), it is necessary that ment,phenotypic variation may be due to genetics, studies be done across taxa with different environmental disturbances or both (Parsons 1990; biological attributes in order to get a better insight Markow 1995). One form of variability in animals into the phenomenon. with bilateral symmetry is the differences between In the course of an analysis to characterize popu- homologous structures on the left and right sides lations of fruit flies of the genus Anastrepha of the body.Asymmetry of this sort can be of three (Selivon 1996; Selivon & Perondini 1998; Selivon types, directional asymmetry, antisymmetry and et al. 1999; Selivon et al. 2004), the presence of fluctuating asymmetry (Van Valen 1962; Parsons asymmetrical individuals for the frontal head setae 1990; Palmer & Strobeck 1986, 1992). In directional was observed in some species. Some species of asymmetry (DS), the structures on one side of the fruit flies are commonly found over extensive geo- body are almost always more variable than the cor- graphical areas, where they utilize a variety of host responding structures on the other side; fruits and are usually exposed to very different en- antisymmetry (AS) is characterized by the fact that vironmental conditions, even those disturbed by variability is so high that it can not be predicted human activity. Since FA may result from exposure which side will be more variable than the other;and to harmful environmental factors, it seems worth- in fluctuating asymmetry (FA) the differences occur while to analyse species of fruit flies for asymmetri-

*To whom correspondence should be addressed. cal variations of morphological traits, and E-mail: [email protected] eventually to correlate these variations to pertur- 260 Proceedings of the 6th International Fruit Fly Symposium

Fig. 1. Heads of a male Anastrepha grandis (A) and of a male Ceratitis capitata (B) in frontal view. Note that this specimen of A. grandis is asymmetrical for the frontal setae (arrow). In C. capitata, the plumose form of the anterior orbital setae is seen (arrow). bations, either genetic or environmental. We RESULTS present here results on the type of variability of the frontal and orbital setae of Anastrepha grandis Anastrepha grandis (Macquart) and Ceratitis capitata (Wiedemann). The heads of adult Anastrepha species display a series of conspicuous seta. The orbital setae are MATERIALS AND METHODS located in front of the ocelli, and in the frons there A. grandis was collected from pumpkins (Cucur- are two rows of frontal setae,one on each side par- bita pepo) from an inland plateau in the state of São allel to the eyes in a dorsal/ventral orientation Paulo (22°35’S,46°50’W), while C. capitata was re- (White & Elson-Harris 1992),as shown in Fig. 1A for covered from infested fruits of Terminalia catappa A. grandis. (tropical almond) collected in the coastal area of Substantial variation in the number of both types the state of São Paulo (23°40’S,45°20’W). Adults of of setae per side was observed in A.grandis.For the both species were recovered in the laboratory and frontal setae, sides with 2–6 setae were found transferred to 70% ethanol for storage. (Fig. 2A).When sides were analysed independently, Samples of 100 adults of each sex of A. grandis it was found by a Kolmogorov test that there were and 50 of C. capitata were used. The heads were no significant differences in the distributions of the examined under a stereo-microscope and the number of setae between the left and right sides of number of orbital and frontal setae on each side male or female individuals (males, d = 0.03 < d5% = was recorded. Males and females were scored 0.192, n.s.; females, d = 0.08 < d5% = 0.192, n.s.), nor separately.The number of setae could be unambig- differences between males and females (pooled uously determined, even in cases where they were sides),as shown in Fig. 2 (d = 0.09 < d5% = 0.136,n.s.). lost or broken during collection or processing The mean number (±S.D.) of setae per side was of the specimens, owing to the presence of the 3.82 ± 0.67 in males and 3.85 ± 0.73 in females. For socket of setae implantation. From a sample of the orbital setae, sides bearing 1–3 setae were specimens, images of the head were taken by found,and those with two setae were the most fre- a Leica DC100 CCD camera coupled to the stereo- quent,as shown in Fig. 2B.There were no significant microscope. differences among males and females, the mean The frequency distributions of the number of number of orbital setae per side being 1.60 ± 0.49 setae per side, and between males and females, and 1.65 ± 0.47, respectively. were compared by a Kolmogorov-Smirnov test Symmetrical and asymmetrical specimens were (Sokal & Rohlf 1981). Asymmetry was evaluated by found, as illustrated in Fig. 1A. Since for both types the right minus left (R–L) scores (Van Valen 1962, of setae no significant differences between sexes Palmer & Strobeck 1986),and the parameters of the were observed, the data were pooled for the next distributions, means, standard deviations, skew- analysis. The scores of asymmetry (R–L) for both ness, kurtosis for signed (R–L) values of each trait, types of setae were distributed around 0 (zero), as and confidence intervals. Significant differences shown in Fig. 3. Parameters of these curves are were calculated according to Zar (1974). shown in Table 1. For the frontal setae (Fig. 3A) the Pires et al.: Variation in symmetrical patterns of development in Anastrepha grandis and Ceratitis capitata 261

Fig. 2. Frequency distributions of specimens of Anastrepha grandis according to the number of frontal (A)or orbital (B) setae per side.

Fig. 3. The distribution of asymmetry scores (R–L) of frontal (A) and orbital (B) setae of Anastrepha grandis, and of orbital setae (C)ofCeratitis capitata. The expected normal distribution curve for each seta is superimposed on the graphs. distribution did not deviate from a normal curve, may show symmetrical distribution of R–L scores since skewness was non-significant (t = 0.1547; (Palmer et al. 1993). In the present case, however, 0.50 < P < 0.60), and the curve was mesokurtic, scatterplots were not very informative since the although at a marginal level (t = –1.705,0.025 < P < data are discrete variables (1, 2,..., 6 setae). Instead, 0.05). For the orbital setae (Fig. 3B), the curve was we scored the frequencies of the individual pheno- symmetrical (non-significant skewness, t = 0.326, types to get information on the association of sides 0.60 < P < 0.70) but it departed from normality bearing different number of setae. For the frontal being leptokurtic (t = 2.172, 0.01 < P < 0.025). setae, the most frequent phenotype was the Scatterplots of left and right values add relevant presence of four setae on each side (4 × 4), which informationaboutthetypeofasymmetrypresented was considered as the characteristic phenotype of by a character,since different kinds of FA variations the trait.Thus,the other classes were considered as

Table 1. Means, 95% confidence intervals (CI), skewness and kurtosis in fluctuating asymmetry (FA) of the number of setae of pooled male and female Anastrepha grandis.

Setae n Mean FA (S.D.) 95% CI (min/max) Skewness (P) Kurtosis (P)

Frontal 200 0.015 (0.8235) 0.9983 / 0.1298 0.0265 (>50.0) –0.5836 (P > 0.025) Orbital 200 0.040 (0.5194) 0.0324 / 0.1124 0.0559 (>60.0) 0.7433 (P < 0.025)* 262 Proceedings of the 6th International Fruit Fly Symposium

Table 2. Number of individuals of Anastrepha grandis Table 3. Number of individuals of Anastrepha grandis exhibiting different phenotypic classes according to exhibiting different phenotypic classes according to the number of frontal setae on each side of the head. the number of orbital setae on each side of the head.

Right side Right side Number of setae 23456Number of setae 1 2 3

Left side Left side 2 00100 142310 3 0 26 38 3 0 2 22 104 1 4 0 33 53 15 0 3000 5022170 6 00100 asymmetrical. Leptokurtosis is thus explained by low variability of the character, thus concentrat- phenotypic variants, including symmetrical (3 × 3, ing the specimens in the perfect symmetry class 5 × 5) and asymmetrical (3 × 4;4 × 3;5 × 3,etc.) indi- (score 0). viduals. It was observed that 26.5% of the individuals presented the normal phenotype (4 × DISCUSSION 4), symmetrical variants represented 16.5% of the The results of the present analysis of variability of sample, and the other 57.0% were asymmetrical setaenumberexcludeddirectionalasymmetryand (Table 2). antisymmetry for the two types of setae in A. A different situation was observed for the orbital grandis and C. capitata, because no indications of setae. For these setae the most frequent individu- skewness in the distributions of asymmetry scores als (52.0 % of the sample) were 2 × 2,considered as were found. Hence, the data would indicate these the normal phenotype of the character,21.0% were as cases of fluctuating asymmetry. However, it is symmetrical with variations (1 × 1), and the other known that symmetrical distributions of asymme- 27.0% were asymmetrical. Leptokurtosis observed try scores may encompass other kinds of variation for the asymmetry of orbital setae may be ex- than pure fluctuating asymmetry, if the latter is plained by the large number of individuals with takenas‘smallfluctuationduetorandomdeviation perfect symmetry (73% of the specimens), which of development’ (reviews in Palmer & Strobeck include the normal phenotypes plus the symmetri- 1986,1992).Palmer et al.(1993) have shown several cal variant individuals (Table 3). of these cases, e.g. positive and negative covariant asymmetry, which may indicate that a genetic Ceratitis capitata component is related to the asymmetrical varia- This species has two frontal setae which were tion. This reasoning may be applied to the orbital almost invariable in the present sample: only setae of A. grandis since leptokurtosis of the asym- one side in a male individual had three setae. The metry distribution may be attributed to a co- number of orbital setae varied from two to three variation of sides generating a large number of per side, with most (92%) individuals exhibiting variants, albeit symmetrical specimens, signalling two setae. The anterior orbital setae, also called that factors other than random fluctuations may be supra-fronto-orbital setae or bristles (see Hunt etal. involved in the variation of this trait.Hence,it is not 2002), are sexually dimorphic, being plumose in clear if the asymmetrical variation of the orbital males (White & Elson-Harris 1992), as shown in setae results from pure fluctuating asymmetry. On Fig. 1B. Since there were no significant differences the contrary, the asymmetry of the frontal setae between sexes (data not shown), the next analysis may be considered as pure fluctuating asymmetry was made with pooled male and female data. The since the distribution of R–L scores does not devi- distribution of asymmetry scores (R–L) for the or- ate from a normal curve. bital setae, shown in Fig. 3C, was symmetrical Traits under strong selection express less observ- (mean ± S.D. = 0.00; skewness = 0), but departed able fluctuating asymmetry, as was shown in from a normal distribution, being leptokurtic (g2 = Drosophila nigrospiracula (Polak 1993) and in 5.6724; P < 0.001). In this sample, however, varia- other animals and plants (review in Møller & tion was very low, with 86% of the individuals ex- Pomiankowski 1993). This could explain the low hibiting the normal phenotype (2 × 2), 2% being variability of the orbital setae in C. capitata since symmetrical variants 3 × 3), and the other 12% this structure presents a sexual dimorphism, and Pires et al.: Variation in symmetrical patterns of development in Anastrepha grandis and Ceratitis capitata 263 seems to be involved in the sexual behaviour of the try: measurement, analysis, patterns. Annual Review flies (review in Hunt et al. 2002), and is therefore of Ecology and Systematics 17: 391–421. PALMER, A.R. & STROBECK, C. 1992. Fluctuating asymme- considered to be under selective pressure. The try as a measure of developmental stability: implica- cause of the great homeostasis presented by this tions of non-normal distributions and the power of trait, as analysed by Hasson & Rossler (2002), is not statistical tests. Acta Zoologica Fennica 191: 57–72. of concern in this paper. PALMER, A.R., STROBECK, C. & CHIPPINDALE, A.K. 1993. In conclusion, and based on the premises of Bilateral variation and the evolutionary origin of macroscopic asymmetries. Genetica 89: 201–218. Palmer et al. (1993), this study indicates that the PARSONS, P.A. 1990. Fluctuating asymmetry: an epi- frontal setae of A.grandis,exhibiting what seems to genetic measure of stress. Biological Review 65: 131– be pure fluctuating asymmetry, may be used as a 145. parameter to assess developmental instability POLAK, M. 1993. Parasites increase fluctuating asymme- (Parsons 1990; Markow 1995). However, neither try of male Drosophila nigrospiracula: implications the orbital setae nor the homologous setae of for sexual selection. Genetica 89: 255–265. SELIVON, D. 1996. Estudo sobre a diferenciação popu- C. capitata should be used for this purpose. It lacional em Anastrepha fraterculus (Wiedemann) remains to be ascertained whether the asymmetry (Diptera, Tephritidae). Doctoral dissertation, Departa- of frontal setae we have observed in other species mento de Biologia, Universidade de São Paulo, São of Anastrepha are also instances of pure fluctuating Paulo. asymmetry. SELIVON, D. & PERONDINI, A.L.P.1998. Eggshell morphol- ogy in two cryptic species of the Anastrepha frater- ACKNOWLEDGEMENTS culus complex (Diptera, Tephritidae). Annals of the Entomological Society of America 91: 473–478. The research was supported by the Brazilian SELIVON, D., MORGANTE, J.S. & PERONDINI, A.L.P. 1999. Agencies, FAPESP (grants 98/10107-4 and Haldane’s rule and other aspects of reproductive 01/07049-8) and CNPq. A.P.O. has a doctoral isolation observed in the Anastrepha fraterculus scholarship from CNPq, and D.S. and A.L.P.P. are complex (Diptera, Tephritidae). Genetics and Molecu- research fellows of CNPq. lar Biology 22: 507–510. SELIVON, D., VRETOS, C., FONTES, L. & PERONDINI, A.L.P. 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