Aspects of the reproductive biology of the toadfish , Halobatrachus didactylus ( Schneider , 1801 ) ( Pisces : Batrachoididae ) *

The toadfishes (Family Batrachoididae) include 12 genera and 40 species of shallow water bottomdwelling fishes that live in soft sand, rocky bottoms and reefs (Cervigón, 1980). Some batrachoid species have traditionally been used as laboratory animals in the field of physiology (Hopkins et al., 1997; Gilmour et al., 1998; Perry et al., 1998; Paert et al., 1999), toxicology (Gutiérrez et al., 1978; Sinovcic et al., 1980; Sarasquete et al., 1982), ethology (Ament et al., 1997; Bass, 1998), neurobiology (Rabbitt et al., 1995; Fine et al., 1996; Hirsch et al., 1998), cardiology (Benítez et al., 1994a,b; Coucelo et al., 1996), and endocrinology (Fine et al., 1996; Knapp et al., 1999). Owing to this, their biology has been extensively studied in most of species. Along the Iberian Peninsula, the Batrachoidids are represented by only one species, Halobatrachus didactylus. Its distribution extends from the Bay of Biscay to Ghana and the west Mediterranean (Roux, 1986). In the Bay of Cádiz, it is abundant and represents an important component of the fish communities (Arias, 1976). SCI. MAR., 65 (2): 131-138 SCIENTIA MARINA 2001


INTRODUCTION
The toadfishes (Family Batrachoididae) include 12 genera and 40 species of shallow water bottomdwelling fishes that live in soft sand, rocky bottoms and reefs (Cervigón, 1980).Some batrachoid species have traditionally been used as laboratory animals in the field of physiology (Hopkins et al., 1997;Gilmour et al., 1998;Perry et al., 1998;Paert et al., 1999), toxicology (Gutiérrez et al., 1978;Sinovcic et al., 1980;Sarasquete et al., 1982), ethol-ogy (Ament et al., 1997;Bass, 1998), neurobiology (Rabbitt et al., 1995;Fine et al., 1996;Hirsch et al., 1998), cardiology (Benítez et al., 1994a,b;Coucelo et al., 1996), and endocrinology (Fine et al., 1996;Knapp et al., 1999).Owing to this, their biology has been extensively studied in most of species.Along the Iberian Peninsula, the Batrachoidids are represented by only one species, Halobatrachus didactylus.Its distribution extends from the Bay of Biscay to Ghana and the west Mediterranean (Roux, 1986).In the Bay of Cádiz, it is abundant and represents an important component of the fish communities (Arias, 1976).H. didactylus is generally found in shallow water -tidal channels and creeks, inlets, beaches-down to about 50 m.It is a sedentary and solitary species that lives partly buried in soft sand and mud, hidden between weeds and sea grasses or concealed in rock crevices.It is an euryhaline species, capable of colonizing diverse biotopes, from brackish waters of river estuaries such as those of the Guadalquivir (Spain), Mira, Sado, and Tagus (Portugal) to hypersaline waters of the salt ponds (Arias and Drake, 1990;López de Cunha, 1994;Costa et al., 1995;Fernández-Delgado et al., 2000).
The biology of H. didactylus has received special attention in recent years because of its use as a laboratory animal in toxicological and cardiological experiments (Benítez et al., 1994 a,b;Coucelo et al., 1996).Cárdenas (1977), andCosta et al. (1995) examined its feeding habits; and Arias and Drake (1990) studied juvenile distribution in Cádiz Bay.Abundance and distribution in the Mira, Sado and Tagus Rivers estuaries was studied by Costa (1993), Costa et al. (1995) and López de Cunha (1994), who considered this species as one of the most important and abundant fish in those habitats; biochemical and histochemical features of different organs were assessed by Gutiérrez et al. (1987), Establier et al. (1986), and González de Canales et al. (1989Canales et al. ( , 1990)); and haematology has been studied by Gutiérrez and Sarasquete (1985), and Sarasquete (1983Sarasquete ( , 1984)).Published information on its reproductive biology is scarce.There only exist reports on the histological, histochemical, and biochemical structure of the gonads (Blanco, 1991;González de Canales et al., 1992;Rosety et al., 1992).
The objective of this study was to investigate some reproductive traits (sex ratio, size at sexual maturity, spawning period, fecundity, length-weight relationship and condition factor) of H. didactylus.

MATERIALS AND METHODS
Samples of fish were obtained monthly from artisanal fishermen's catches in Cádiz Bay (South-Western Spain) from February 1999 to March 2000.
All specimens were measured (total length, L T ), and weighed (body weight, W T ; eviscerated weight, W E ; gonad weight, W G ; and liver weight, W L ).Sex and maturity (I, undeveloped; II, ripening or recovering; III ripe, gamete running; and IV, spent) were also recorded.
Sex was established by macroscopic examination of the gonads.A Chi-square test (Steel and Torrie, 1985) with Yates correction for continuity was used to compare sex ratios in each month, and at each 20mm length interval with a hypothetical 1:1 ratio.
To estimate the mean total length at maturity (L 50 ) for males and females, the fraction of mature fish in each 10 mm interval was fitted to a logistic function (Gaertner and Laloé, 1986) by using the Marquardt method (Draper and Smith, 1966).
The spawning season was inferred from the study of monthly variation in gonadosomatic index and hepatosomatic index and the relative frequencies of gonad maturity stages through the study period.Gonadosomatic index (GSI) and hepatosomatic index (HSI) were calculated as: GSI = 100(W G .W E -1 ); HSI = 100(W L .W E -1 ). Fecundity was estimated using 73 ripe ovaries preserved in Gilson's fluid.Relative fecundity (RFL T : fecundity related to total length; RFW T : fecundity related to total weight; RFW E : fecundity related to eviscerated weight; RFW G : fecundity related to gonad weight) for each fish was calculated as: RFL -1 and RFW G = F. W G -1 .Fecundity was regressed on body length, on body weight and ovary weight by linear regression on logarithms using model II regression as suggested by Ricker (1973).The oocyte diameter (mm) was estimated from 140 randomly chosen ripe oocytes, measured by the ocular micrometer of a binocular microscope.
Length-weight relationship was modeled by linear model II regression analysis on the log-transformed variables (Ricker, 1973).Relative condition factor (Kr) for adult specimens was calculated as the percentage ratio of the observed weight of a fish to the weight expected from the calculated lengthweight relationship (Le Cren, 1951).
Monthly changes in relative condition factor, gonadosomatic index and hepatosomatic index were assessed by using a one-way analysis of variance (ANOVA).Significant ANOVAs (p≤ 0.05) were followed by Duncan's multiple range test (Steel and Torrie, 1985).GSI data were log 10 transformed to satisfy the assumptions of the analysis.

Sex ratio
Sex ratios did not differ (p >0.05) from 1:1 except in May 1999 when females were favored significantly, and February 2000 when males predominated (Table 1).
The number of males and females was equal at lengths <220 mm L T , and between 260 and 300 mm L T , but females outnumbered males at 220-260 mm L T .Males dominated in specimens >300 mm and all individuals with lengths over 380 mm were males (Table 2).

Size at sexual maturity
The smallest mature fish were a 191 mm L T female and a 160 mm L T male.Fifty percent maturity occurred at 262 mm L T for females and at 302 mm L T for males (Fig. 1).
According to these results, adults were defined as those individuals at/or exceeding the minimum size at sexual maturity and at least stage II.

Gonadosomatic Index (GSI)
GSI increased with stage of development.GSI for stage I and II females remained below 19.13.For mature females, GSI was between 3.98 and 40.12.Immature males had a GSI of 0.10-4.07,while for mature fish it ranged from 0.59 to 11.37.GSI was significantly higher in females and showed a high variability throughout the year in both sexes.
GSI of adult females ranged between 0.33 and 40.12, and varied between months (F = 71.82;p <0.01).Higher GSI values occurred in March-May 1999 and declined sharply thereafter, indicating that oocytes were released.Low values occurred in July-November.In December, the gonad cycle started again (Fig. 2a).
Spawning periods for males were not as marked as for females.However, male GSI values decreased in June-July coincident with those of females.GSI values varied from 0.01 to 11.37, and showed significant monthly differences (F = 15.00;p <0.01).Mean GSI REPRODUCTION OF H. DIDACTYLUS 133 for males remained high and variable in February-May, and low in July-November (Fig. 2b).

Hepatosomatic index (HSI)
HSI showed strong variations during the sampling period for both sexes (Fig. 2a,b).Differences between months were statistically significant (F = 8.04; p < 0.01) in females.Mean HSI values were significantly higher in December-March, and were minimum in April-November.Males also showed monthly differences in HSI (F = 6.01; p < 0.01).Highest values were found in February-April 1999, and lowest ones in June-July 1999.
The seasonal cycle of the HSI for females is not related to that of GSI.(r = -0.03;p > 0.05).That of males showed a slight correlation to GSI (r = -0.12;p < 0.05).

Monthly variation in the proportion of different maturity stages
Both ripe males and females were collected between March and September 1999 and December 1999-March 2000 (Fig. 2).
The percentage of mature females was higher in April (61%), and May (75%).The rest of the year, almost all fishes were ripening or recovering.Spent females were observed in low percentages between April and August.The highest proportion (75%) occurred in June 1999 (Fig. 2a).
The greatest proportions of ripe males were found in April (45 %), and May (64 %).Maturing males was found in high proportions throughout the year, except in April and May when ripe individuals dominated.Spent males were collected in May (Fig. 2b).
Peaks in GSI, along with the changes in percent frequency of ripe and spent individuals, indicates that H. didactylus spawns in March-August and has a reproductive peak in May-June.

Length-weight relationship
Length-weight relationships were derived from 901 specimens ranging from 122 to 470 mm and 30 to 2105 g.
Equations for the sexes differed (t = -2.70;p <0.01), with relationships represented in Figure 5.The exponents of the two regressions showed a positive allometry (t = 9.39, p <0.01 for males and t = 9.53; p <0.01 for females)

Condition Factor (Kr)
Mean Kr for males and females showed a similar seasonal pattern (Fig. 6).In adult males, Kr showed monthly differences (F = 5.40; p < 0.01).Kr was higher in February-April 1999, and decreased sharply thereafter.Kr for adult females also varied significantly between samples (F = 4.32, p <0.01).Highest condition was attained in March-April.The lowest values occurred in June.

DISCUSSION
In fish, sex ratio varies considerably from species to species but in the majority of species it is close to 1:1.It differs from one population to another of the same species and may vary from year to year in the same population (Nikolsky, 1963).Monthly variations in sex ratio of H. didactylus samples apart from those of May 1999, and February 2000 showed no significant differences from that of the expected 1:1 ratio.The greater number of females compared to males in May, coincident with the reproductive peak, could be attributed to the fact that, as in other batrachoid species such as Opsanus beta, O. tau, and Amphichthys cryptocentrus, males actively guard the eggs, and maintain residence in the nests until young attain the free-swimming stage (Breder, 1941;Gray and Winn, 1961;Hoffman and Robertson, 1983), making them less exposed to capture.Costa et al. (1995) obtained an overall 1:1 sex ratio for H. didactylus in the Mira estuary, but these authors did not test the presence of a monthly or length related pattern.Granado and González (1988) also reported a 1:1 sex ratio for A. cryptocentrus, in tropical waters, during most of the year.
Size related differences in sex ratios were observed.Significant deviations from parity usually occurred among the medium and largest sizes, with females predominating the medium while the largest sizes were disproportionately male.The differences observed in the size-specific sex ratio of the toadfish have been reported for other fish species (Turner et al., 1983;Stergiou et al., 1996), and have been related to sexual differences in growth, mortality and energetic cost of reproduction.
Males and females of H. didactylus mature at different lengths.Males began maturing earlier than females, a fact that has been attributed to gains in fecundity with size (Stearns, 1992).L 50 for males was greater than for females, which may indicate that after first maturation females reduce growth and allocate energy mainly for the production of gametes while males, with a smaller reproductive effort, continue growing.
No previous estimates of size at maturity are available for the species investigated here.In A. cryptocentrus, a species that attains similar sizes to H. didactylus, males also mature at a larger size than females.Sizes at sexual maturity for both sexes of this species (males, 270 mm, and females, 310 mm) (Granado and González, 1988) are very similar to those of H. didactylus.
Our estimate of the spawning season in H. didactylus in Cádiz Bay is similar to that of Blanco (1991) who stated that this species spawns during spring.However, his finding that the maturation-spawning occurs in March-April in females and earlier (February-March) in males differs from the peak observed in the present study (May-June).This may indicate that in H. didactylus, as in other teleosts, the spawning season is affected by year to year climatic changes (i.e. in water temperature), that delay or advance maximum spawning.Gray and Winn (1961) indicated that local differences in shallow water and deepwater populations and yearly climatic differences might be expected, particularly if temperature is causative.
The spawning season of H. didactylus is similar to that reported by Gudger (1910) and Schwartz (1974) for O. tau in North Carolina and Chesapeake Bay.Gray and Winn (1961) observed that O. tau spawning in South Carolina begins in May and extends to early August.In the tropics, A. cryptocentrus, can reproduce throughout the year (Granado and González, 1988).
Although no significant correlation was observed between gonadosomatic and hepatic indices for females, the decrease in HSI in females during April-May, may indicate the use of liver reserves during gonad growth and maturation.It is known that in non-mammalian vertebrates, the principal events responsible for the enormous growth of oocytes involve the storing and packaging of a hepatically derived plasma precursor, vitellogenin, into yolk protein (Wallace and Selman, 1981).This suggests that liver reserves may accumulate prior to reproduction, being then transferred through the blood stream to the ovaries during oogenesis.Bustamante (1983) observed an increase in weight of the liver in fishes prior to reproduction, and pointed out that this was due to an increase in the synthesis of lipids and proteins necessary for gonad development.
As for other batrachoid species, fecundity in H. didactylus is very low.This must be compensated for by a high survival of eggs and juvenile fish that results from males guarding the nests until young are free-swimming.The high amount of yolk, and the great egg sizes may also be mechanisms to assure a high survival rate in embryos and larvae such as has been reported by Yáñez-Arancibia et al. (1976) and Palazón et al. (1994) for the Ariidae, Galeichthys caerulescens, and Bagre marinus, respectively.The fecundity estimates of H. didactylus and other Batrachoids show that these species have a reproductive strategy common to other fish families with parental care, i.e. they produce a low number of large eggs.
Even though the size of eggs in H. didactylus is similar to that reported for other Batrachoids, the fecundity of H. didactylus is higher than for S. barbatus (245-499;Hoffman and Robertson, 1983), and A. cryptocentrus (45-467 eggs;Granado and González, 1988).Relative fecundity in this later species was estimated as 0.39-1.01eggs/g, which is considerably smaller than the values observed in H. didactylus.
The increase in fecundity with body length and weight is consistent with the observations made in other fish species (Granado and González, 1988;Gartner, 1993;Barbin and McCleave, 1997).
Hepatosomatic index is a good indicator of recent feeding activity (Tyler and Dunns, 1976); the decrease in HSI during the spawning period suggests that H. didactylus may decrease its feeding activity during reproduction.The condition factor also showed a minimum value in the spawning season, which as stated by Maddock and Burton (1999) can be the result of mobilization of somatic energy reserves needed for reproductive development, and/or the use of reserves stored in the body as a source of energy in spawned fish, influenced by reduced feeding during this period.In a previous study, Cardenas (1977) observed that 4-5 % of stomachs were empty in summerautumn, with the value increasing to 10-17 % in winter-spring, the higher values being registered in spring.
FIG. 3. -Relationship between fecundity and body length in H.didactylus.

TABLE 1 .
-Number of male and female H. didactylus by month and results of the Chi-square test for a 1:1 ratio.

TABLE 2 .
-Number of male and female H. didactylus by length class and results of the Chi-square test for a 1:1 ratio ns= p> 0.05; *= p ≤ 0.05 FIG. 1. -Sexual maturity of male and female toadfish, H. didactylus and corresponding size-at-maturity, L 50 .

TABLE 3 .
-Absolute and relative fecundity statistics in the toadfish H. didactylus