Water hardness inuenced reproductive dynamics in two freshwater sh species; Poecilia reticulata and Betta splendens

Hardness of in the form of CaCO3 affect reproductive potential in various sh species, differently. The study evaluates the effect of water hardness on growth and reproduction of two aquarium shes, Poecilia reticulata (Ovo-viviparous sp.) and Betta splendens (Oviparous sp.) by growing them under 150 (control), 320, 540 and 900 ppm CaCO3 levels in semi natural aquaria.


Introduction
Water hardness is a crucial factor for the growth, reproduction and embryo development of sh [1]. It affects the growth and reproductive biology of oviparous and ovoviviparous sh species differently due to variations in their requirement of CaCO3 in the reproductive process [1][2][3][4][5]. Considering the importance of hardness in the hatchability, larval growth and survival of sh eggs, it is recommended to maintain the hardness levels of the sh culture ponds above 20 mg/L CaCO3 [6,7].
Water hardness in some areas of the Dry Zone of Sri Lanka exceeds even 1000 mg/L. Vavuniya District reports remarkably high hardness values varying in between 100-1000 mg/L [8]. In Vavuniya, aquarium trade for ornamental sh keeping mainly depended on ground water sources. Thus, evaluating the effect of higher hardness on sh health is of prime economic and ecological importance.
Freshwater species, Poecillia reticulata (Guppy) and Betta splendens (Siamese ghting sh) were ovoviviparous and oviparous shes, respectively and were popular verities in the aquarium sh trade. They can be easily reared and bred under laboratory conditions.
The study anticipates to evaluate the effect of water hardness on ovi-paraous & ovo-viviparaous sh biology in semi-natural aquaria by measuring the growth performance of adults and larvae, and reproductive performances in terms of gonadosomatic Index (GSI), fecundity, fertility, hatchability and larval survival by using two aquarium sh species; Poecillia reticulata (Guppy) and Betta splendens (Siamese ghting sh) as model organisms.

Preparation of Aquaria
Glass aquaria of the size of 25x13x12 cm3 (24 tanks) were used for the exposure. Being an aggressive sh, male Betta splendens were planned to keep in 20 separate cubic aquaria (12x12x12 cm3) at a rate of each male per cube. The interior of aquaria was prepared as a simulation of natural pond using stones, aquatic plants and aeration.
The experiment composed of control (tap borne water 150 ppm CaCO3) and three treatment setups; 320, 540 and 900 ppm prepared by adding analytical grade CaCO3 to aged tap water. The hardness of the medium was determined by EDTA titrimetric method [9] and the treatment setups were monitored weekly basis to maintain the water hardness. Experiment setups were maintained at the Environmental Biology Laboratory (EBL) at the Vavuniya Campus in Sri Lanka. All the experiments were conducted as per the guidelines given by the research review panel of the Department of Bioscience, University of Jaffna.

Introduction of shes to Aquaria & Maintenance
Ovo-viviparous sh as Poecilia reticulata and oviparous shes as Betta splendens were chosen for the experiment. The virgin shes of both species (males and females) were purchased from a nearby aquarium in Vavuniya and were transferred to the EBL. Sexing was done by examining external morphology as both species showed sexual dimorphism. Initial weight and standard length were measured. Then the shes were acclimatized to aquarium condition and introduced to the experimental setups (150 ppm (control), 320, 540 and 900 ppm CaCO3 and reared for 1½ month. In each experimental setup 15 females were introduced to 5 males of B. splendens, separately. To avoid aggression each males of B. splendens were kept separately in the smaller aquaria. For, P. reticulata, 25 females were introduced to 5 males. The exposure was conducted with three replicates.

Determination of growth performance
Weight and length of adult sh were measured, and the length weight relationship analysis was conducted, using W= a TLb: (Log W = log a + b log TL) [10] to obtain the linear regression.
Determination of reproductive potential Two gravid females from each aquarium were randomly tested for fecundity (the number of ripening eggs found in the female just prior to spawning) and Gonadosomatic Index (GSI-the ratio of sh gonad weight to body weight). Euthanizing was conducted with 0.02% MS222 solution.
To estimate the bubble nest size of B. splendens, clean breeding aquarium (size-60x30x30 cm3) were prepared without arti cial bottom stones and aeration. A oating plant leaf was put on the air water interface to facilitate the nest formation. After adding the male in to the breeding aquarium, a clean glass cube containing female sh was placed near it to stimulate the nest building. Then the bubble nests built by male B. splendens were measured (diameter) by a ruler [11].
Hatchability of B. splendens was estimated after allowing successful courtship with a gravid female inside the breeding aquarium. Repeated introductions of gravid females were required to obtain a successful breeding. Without disturbing the bubble nest, the number of eggs released was counted. At the end of the matting, female was removed, and male was allowed for 24 -48 hour for pre hatched parental care. Next, the hatched eggs were counted, and the hatchability was determined, as the number of larvae hatched over the total number eggs [12].
Fertility of P.reticulata was determined by counting the intra-follicular embryos inside the female sh by sacri cing few shes at the 21 st day after mating [13]. Breeding tank of P. reticulata was formed with the arti cial aquarium stones and Vallisneria to provide hiding place for young ones. After female broods release young ones, they were separated from their parents and counted.
Larval survival rate was considered after one week of exposure to the water hardness treatments by counting the number of surviving one weeks-old larvae divided by the total number of hatched eggs / released young ones [12]. The larval growth performance was estimated in every 10 days interval by measuring the length gain.

Statistical analyses
One-way ANOVA and Tukey pairwise comparison were used to analysis the signi cant differences of weight, standard length, fecundity and Gonadosomatic Index, Laval growth (length) of the shes. The linear regression analysis was used to nd the length weight relationship (LWR) of the sh to know the growth pattern. In LWR linear regression analysis, the slope of regression lines explicit the exponent coe cient value 'b'. Signi cant variation in the estimates of 'b' for the sh species was examined from the expected value (ideal value 'b' = 3) was tested by t-test [14,15]. Students t-test was employed by dividing the difference between 'b' and '3' by standard error of 'b' [16].

Results
Growth performance of P. reticulata and B. splendens in various water hardness levels Growth improved with the increasing water hardness by showing signi cantly high weight values, particularly (p<0.05) above 540 ppm treatments, reaching 80% and 40% weight increments, respectively for P. reticulata (N=30) and B. splendens (N=20) in the highest hardness level (900 ppm). Similarly, length of the shes was also increased with higher hardness levels, though only 14-17 % increments were recorded.
When the growth pattern was estimated P. reticulata showed isometric growth at 540 and 900 ppm levels while B. splendens showed isometric growth in all hardness levels. (Table 1).
Fecundity of P. reticulata increased with the water hardness, reporting a signi cantly higher value at 900ppm. (16.20 ± 3.90, p=0.016). Unlike the fecundity of P. reticulata, that of B. splendens declined considerably by hardness treatment, showing only a slight increment in 540ppm (824 ± 175 eggs compared to 743 at the control, Figure 1).
Bubble nest surface area of B. splendens showed signi cant decline along the hardness series, reporting 108.58 cm2 at the control and 26.5 cm2 at 900 ppm. In the hard water environment 540 & 900ppm, bubbles were blasted and male sh was unable to rebuild the nest.
Fertility of P. reticulata, given as the number of intrafollicular embryos, was increased along the hardness series, reporting 88% in 900ppm compared to 74% at the control, though the increment was not signi cant (p>0.05). Hatchability of B. splendens declined signi cantly in higher hardness levels above 540ppm reporting only 25% success at 900 ppm compared to 78% in the control (p = 0.006). The percentage of larval survival of both species declined gradually with the increasing hardness level, reporting signi cant decline above 320 ppm (p<0.0009) hardness. However, the larval growth of P. reticulata was improved by higher hardness levels reaching a signi cant growth at 900 ppm (p=0.006), though the effect on B. splendens larvae was insigni cant.

Discussion
Water hardness in the form of CaCO3 had differential effects on P. reticulata and B. splendens growth and reproduction. Growth in the form of body weight and length, and reproduction by means of gonadosomatic index, fecundity, bubble nest diameter, fertility were improved in P. reticulata while those were degraded in B. splendens. These observations are compatible with previously reported studies for ovoviviparous and oviparous shes, conducted elsewhere.
According to Shim and Ho [17], dissolved Calcium is essential for growth of live bearer (ovoviviparous) sh especially P.reticulata. They also found that rearing P.reticulata in extreme water hardness (2500ppm) showed 10 times higher body weight gain than in soft water (167 ppm). In the same way, James et al., [18] found that Xiphophorus helleri (live bearer) reared in 1018ppm water hardness level exhibited better growth performance. Thus, higher water hardness positively favour the growth of P. reticulata in semi-natural aquarium. This observation is further reiterated by weight and length relationship, which revealed isometric growth occurred only above 540ppm hardness level. On the other hand, increasing water hardness showed no apparent effect on B. splendens resulting isometric growth in all hardness levels.
Reproduction of P. reticulata was enhanced showing faster sexual maturation, with higher hardness conditions in compliance with James [18] and Stratton [19], who reported higher and faster sexual maturation of X. helleri, in exceptionally high hardness medium. In line with this observation, Shim and Ho, [17] suggested that dissolved calcium is essential for sexual maturation of P. reticulata. As an oviparous sh, B. splendens showed retarded reproductive potential under higher hardness. This is reiterated by previous work, Ratinam [5] who found suppressed gonad development and maturity of Pterophyllum scalare and aborted maturation in Barbus conchonius and Barbus letrazona beyond 120 ppm hardness. Further it was reported that high calcium in hard water deposit on the surface of the sh eggs, blocking the water absorbed into the perivitelline [4,20] leading to dehydration and shrinking of the eggs [3]. Thus, being an oviparous sh B.spledens is suitable to the soft water environment than the hard water environment.
Increasing water hardness caused high mortality in larvae of both species. Newborn are unable to tolerate the adverse environmental factors like extreme hard water [21] due to the stress condition in the physiology created by excess amount of calcium. Numerous studies carried on various sh species; Clarias gariepinus, Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and brown trout (Salvelinus fontinails), Rutilus frisii kutum (kutum), reiterated this nding [3,4,[22][23][24]. Thus, soft water is preferable for larval rearing for both P. reticulata and B. splendens.
Hence, it may conclude that P. reticulata requires more calcium for the growth and reproduction than B. splendens which grow and reproduce well in soft water environment.

Limitations
This study was not intended to describe mechanism/s of action of CaCO3 in mediating growth and reproductive alteration of P. reticulata and B. splendens.