REPRODUCTIVE TRAITS IN COMMON TOAD BUFO BUFO FROM THE VICINITY OF BELGRADE

Common toad Bujo bufo is a species with wide geografical and altitudinal distribution in Europe (A I' n old 2002). Although it prefers forests, mainly deciduous, it also occurs in fields, meadows, gardens and human settlements (R ado van 0 vic 1951). The aims of this study were to analyse the annual reproductive cycle of Bufo bufo, as a part of the study on reproductive traits in amphibian populations from human-altered habitats, with special emphasis on sites in the vicinity of Belgrade; to compare seasonal changes in relative organ weights in both sexes; to examine variation in fecundity and relationship between fecundity and body size.

Analyses were performed on the sample of 26 females and 43 males collected in March, April, June, September and November from localities Trenja and Zuce.The study sites are near Belgrade, in agricultural area, and thus under the impact of human activities.Trenja is a small artificial lake, known as an important breeding site for several amphibian species, while the other site is a moderate-size pond near the Zuce village.
Measure points and sample size were chosen to represent seasons (and the most distinct phases of the annual reproductive cycle), avoiding, at the same time, to endanger natural populations.Body length (L) was measured with a dial caliper to 0.1 mm precision.Total body weight (W) was taken, as well as the weights of the following organs: gonads (ovaries and testes), fat bodies and liver.Organs were weighted with an electronic balance to 0.001 g precision.In addition, ovaries were immersed in 70% ethanol, and total number of oocytes was counted.
Organ weights were significantly correlated with body size, so L-adjusted weights were used (e.g.Cas till a et al. 1992Cas till a et al. , C vet k 0 vic et al. 1996)), and analyses were performed on residuals from least-squares regression of log transformed organ weights on log transformed body length.

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Differences between females and males in parameters of body size were statistically highly significant (t-test, p < 0.001), indicating a high level of sexual size dimorphism (SSD).In amphibians in general, intersexual size difference is a widespread phenomenon (e.g. Lee et al. 2002), and it was estimated that females were larger than males in approximately 90% of the analysed anuran species (S h i n e 1979).In addition, fecundity was significantly correlated with female body length (R = 0.5, p < 0.02), indicating the role of fecundity selection in the maintenance of SSD.
Seasonal changes in female reproductive cycle are shown in Fig. 1a.Relative ovaries weight exhibits significant seasonal variation (ANOYA, F = 3.27, P < 0.05), with lowest values in April and June.Seasonal variation in liver weight was highly significant (ANOYA, F = 6.84,P = 0.001), with a maximum in June.Surprisingly, nonsignificant changes in fat bodies weight (ANOYA, F = 2.36, p>0.05) were found, probably due to small sample size for this particular trait, i.e. small number of females having measurable fat bodies.
Seasonal changes in male reproductive cycle are shown in Fig. lb.Yariation in relative testes weight was not significant (ANOYA, F = 2.49, P = 0.059), though certain changes due to sperm release and postnuptial gametogenesis can be observed.Contrary to the pattern found in females, liver weight in males shows nonsignificant changes (ANOYA, F = 2.22, P > 0.05), and fat bodies show highly significant seasonal variation (ANOYA, F = 6.53,P < 0.001).
Significant annual variation in ovaries weight is related to seasonal activity of gonads, with minimum values in period following mating and eggs release.In female amphibians, liver is involved in synthesis of phospholipid vitellogcnin, a precursor of yolk (L 0 f t s 1984), and thus, also susceptible to seasonal changes.Fat bodies are important as an energy store, especially during hibernation, but data is also available on their role in reproduction (C vet k 0 vic et al. 1996).In males, fat bodies are important for spermatid formation and mating activities (e.g.Y e I' I' e I et al. 1986).In accord with this, our results show that fat bodies weight reached a minimum value in spring.

Figure 1a
Fig. l b Seasonal changes in L-adjusted organ weights in males.GON gonads, LV liver, FB fat bodies.