Comparative study of biochemical parameters in response to stress in Oreochromis aureus, O. mossambicus and two strains of O. niloticus

This study was aimed at detecting differences among tilapia species in immunological and biochemical parameters that are indicators for fish health. In addition, six blood parameters that are indicators of stress were measured, glucose concentration, ceruloplasmin activity, lysozyme activity, respiratory burst activity, haematocrit and leucocrit, and their levels were compared among groups. A calibration experiment was conducted with commercial stock of tilapia to optimize a protocol for measuring stress response in tilapia. An air exposure stress was induced to six groups of fish and blood samples were taken at six different times: 15 min, 1, 2.5, 4.5, 6 and 24 h, after inducing stress. The highest responses to stress were observed after 2.5–4.5 h. A second experiment was conducted with four tilapia species: Oreochromis aureus (Steindachner), O. mossambicus (Peters), a red strain and the wild type of O. niloticus L. Levels of serum biochemical components and parameters of the innate immunity response to stress were compared in ten fish from each species. Significant differences were observed. Oreochromis aureus differed from the other three species (notably from O. mossambicus) in most of the measured immune response traits (glucose concentration, lysozyme activity, haematocrit and levels of total protein and IgM after stress) and serum biochemical components (protein, albumin, triglyceride, cholesterol, calcium, magnesium, phosphorus, bilirubin and β‐hydroxybotiric acid). The identified differences suggest that hybrid families from O. aureus and O. mossambicus may be used to construct a segregating population for genetic analysis of the innate immune response to stress. Thus, these two species were bred for segregating F₂ population, suitable for quantitative trait loci studies for the innate immune response to stress.     

Dynamics of piscine francisellosis differs amongst tilapia species (Oreochromis spp.) in a controlled challenge with Francisella noatunensis subsp. orientalis

A 25‐week immersion challenge was conducted exposing Oreochromis mossambicus, Oreochromis aureus and Oreochromis urolepis hornorum to Francisella noatunensis subsp. orientalis (Fno). Two populations were compared for each fish species; ‘resident fish’ were defined as fish maintained in tanks since week 0 of challenge, whereas ‘naïve fish’ were defined as fish added to tanks once temperature in water reached <26 °C at 21 weeks post‐challenge. Fno genome equivalents (GEs) in water were similar in all treatments 1 h post‐challenge; however, significantly lower Fno GEs were detected 2 weeks post‐challenge in all tanks, and the only treatment with detectable Fno GE after 4 weeks of challenge were the O. mossambicus tanks. Twenty‐one weeks post‐challenge, naïve fish were stocked with ‘resident’ cohorts. Over a 4‐week period, mortalities occurred consistently only in O. mossambicus naïve cohorts. Overall presence of granulomas in spleen of survivors was similar (>55%) in all resident populations; however, in naïve populations, only O. mossambicus presented granulomas. Similarly, only O. mossambicus presented viable Fno in the spleen of survivors, and Fno GEs were only detected in O. mossambicus, and in resident O. aureus. In conclusion, the results of this study suggest different susceptibility of tilapia species to piscine francisellosis.     

Growth,salinity tolerance and microsatellite analysis of the F2 reciprocal hybrids of Oreochromis niloticus×Sarotherodon galilaeus at different salinities

Oreochromis niloticus (O), the sixth generation of Genetic Improvement of Farmed Tilapia, shows rapid growth but poor salt tolerance, while Sarotherodon galilaeus (S) exhibits opposite traits. To combine the traits, F₁ progeny was obtained through artificial fertilization. Fertile F₁ produced F₂ by natural spawning. The mean survival times, the median survival time (ST₅₀) or the survival rate of hybrids was greater than O. niloticus in a gradual or an acute salinity change. Plasma osmolarity, [Na⁺] and [Cl^?] of the hybrids fluctuated in 32 g L^?1 water during a 24‐h period, but eventually reached levels similar to fish in freshwater. O. niloticus♀×S. galilaeus♂ (OS F₂) or S. galilaeus♀×O. niloticus♂ (SO F₂) showed the fastest growth at 22.5 g L^?1, equal to about 78.2% or 69.7% of O. niloticus at 0 and 3.87 or 3.45 times that of S. galilaeus at their individual optimum growth. Growth in OS F₂ was 12% faster than SO F₂. Microsatellite analysis showed that F₂ had more alleles, a higher polymorphism information content and greater observed and expected heterozygosity than O. or S. Population differentiation was not detected between F₁ and F₂. All the results indicated that F₂ could be exploited for commercial production under saline conditions.     

Multiple haemoglobins in three tilapiine species of the genus Oreochromis and in eight strains of O. niloticus (L.)

Among three tilapiine species, Oreochromis aureus (Steindachner), O mossambicus (Peters) and O. niloticus (L.), fifteen banding positions of haemoglobin were resolved using citric acid-aminopropylmorpholine pH 7.0 buffer. O. mossambicus and O. aureus displayed 7 and 8 fixed band locations, respectively, whereas eight strains of O. niloticus from Asian cultured and African wild populations showed a total of 14. Variability in the O. niloticus strains, manifested as presence or absence of haemoglobin bands, was observed in banding positions 3, 4, 6, 7, 8, 9, 10, 13 and 14. Six banding positions in O. niloticus were shared with O. mossambicus and 8 with O. aureus. A dendrogram constructed from distance coefficients between popu-lation pairs using the presence or absence data showed the close affinity among O. niloticus strains belonging to the O. niloticus niloticus subspecies. A slight separation of Kenya O. niloticus from the O. niloticus niloticus strains is consistent with its subspecific distinction as O. niloticus vulcani. Among three species, O. niloticus had a closer affinity to O. aureus than to O. mossambicus. The phylogenetic relation-ships using differences in haemoglobin patterns and the higher genetic variability of O. niloticus reflect similar results obtained from existing studies using allozymes. In general, the presence of multiple fish haemoglobins is attributed to their adaptability to haeterogeneous environments. This attribute is typical of tilapias.     

DNA riboprinting analysis of Tilapia species and their hybrids using restriction fragment length polymorphisms of the small subunit ribosomal DNA

Morphometric, meristic and DNA riboprinting analyses of Tilapia species and their hybrids inhabiting the River Nile were examined. Morphometric data showed striking similarities and overlapping among Tilapia species, making it impossible to differentiate these species. Meristic characteristics revealed that Tilapia species could be identified into four major groups (Oreochromis niloticus, O. aureus, Sarotherodon galilaeus and Tilapia zillii). The lateral line scales differed significantly between the four Tilapia species, while the number of fin rays in the dorsal and anal fins differed significantly, differentiating three species (but not between O. niloticus and O. aureus). Restriction fragment length polymorphisms (RFLPs) of nuclear small sub‐unit ribosomal RNA (18S srRNA) gene were used to differentiate the species. Polymerase chain reaction‐restriction fragment length polymorphisms data provided a unique pattern for each species with a specific restriction enzyme. Two hybrids of Tilapia designated H₁ and H₂ were detected. The endonucleases SacII and ApaI differentiated H₁ and H₂. This research revealed a monophylogenetic relationship among all the studied Tilapia species.     

Artificial Spawning of Tilapia Eggs1

A stripspawning methodology was evaluated for tilapia (oreochromid) species. This technique achieved an average hatching success of 68.6 ± 3.6% (N= 31). Female fecundity and spawning frequency were dependant on both genetical and husbandry factors. Egg yields for Oreochromis niloticus, O. mossambicus, and O. niloticus±O. mossambicus hybrids averaged 4.54, 10.86 and 10.36 eggs/g female/spawn, respectively. Female broodstock that were adapted to an intensive spawning regime exhibited a significant increase in fecundity. Additionally, egg survival was not affected by hydration for up to 15 minutes prior to fertilization. Results suggest that the strip spawning of tilapia species may be an efficient method of providing viable gametes for hatchery purposes.     

冬夏两季五种经济鱼类组织脂肪酸含量及组成分析

为了解不同季节不同鱼类不同组织中的不同脂肪酸含量,科学地指导鱼类膳食消费,本实验研究了冬夏两季,采集自上海市场常见的5种经济鱼类:大黄鱼(海洋肉食性),银鲳(海洋杂食性),日本鳗鲡(淡水肉食性),莫桑比克罗非鱼(淡水杂食性),草鱼(淡水草食性),分别检测鱼背部肌肉、腹部肌肉、尾部肌肉、肝脏和腹腔脂肪组织的脂肪含量和脂肪酸绝对含量。结果显示,5种鱼肌肉脂肪酸谱存在显著差异,并与各自的生活环境及食性均有关系;在鱼的腹腔脂肪或肝脏中,饱和脂肪酸(SFAs)和单不饱和脂肪酸(MUFAs)含量较高,且与组织脂肪含量密切相关;而多不饱和脂肪酸(PUFAs)、n-3系多不饱和脂肪酸(n-3 PUFAs)和n-6系多不饱和脂肪酸(n-6 PUFAs)含量较低,且与组织脂肪含量关系不大;大黄鱼和银鲳各肌肉组织中的n-3 PUFAs、二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)含量以及n-3/n-6值高于莫桑比克罗非鱼和草鱼,并与组织脂肪含量呈正相关;冬季草鱼腹部肌肉、莫桑比克罗非鱼尾部肌肉以及日本鳗鲡和银鲳的肝脏中的n-3 PUFAs含量较夏季高。研究表明,脂肪酸组成与物种、食性、水域环境以及季节温度和组织部位均有关系。从补充n-3 PUFAs摄入的角度分析,日本鳗鲡、大黄鱼和银鲳营养价值高于莫桑比克罗非鱼和草鱼,冬季鱼类的营养价值高于夏季。     

A genome scan of a four-way tilapia cross supports the existence of a quantitative trait locus for cold tolerance on linkage group 23

A genome scan, searching for quantitative trait loci (QTL) for the traits cold tolerance and body weight in tilapia, was performed on a cross between a (Oreochromis niloticus×Sarotherodon galilaeus) male and a (O. mossambicus×O. aureus) female. Fifty‐four microsatellites and 23 amplified fragment length polymorphism (AFLP) primer combinations were genotyped and tested for marker–trait associations. Sex‐specific linkage maps were constructed from this data. Twenty‐three point‐wise significant marker–trait associations were found in the genome scan, and putative QTL were subsequently tested in another (On×Sg) × (Om×Oa) family. None of the putative QTL from the first experiment were significant in the second experiment. However, one microsatellite, UNH130, found to be associated to weight in the first experiment, was found to be strongly associated to cold tolerance in the second experiment. Since QTL for cold tolerance and body weight were recently found on the linkage group containing UNH130 (linkage group 23) in another study, this linkage group was investigated more closely using interval mapping. The results provide indications, but not conclusive evidence, of a QTL for cold tolerance on linkage group 23.     

Electrophoretic evidence for extensive hybrid gene introgression into commercial Oreochromis niloticus (L.) stocks in the Philippines*

Abstract. The usefulness of isozyme and protein markers in identifying tilapia species and their hybrids is demonstrated Genetic characterization of Oreochromis niloticus populations from commercial farms, experimental stations, and government hatcheries in Luzon, Visayas and Mindanao in the Philippines indicates well-established introgression with O mossambicus Genetic differentiation of the O niloticus stocks, measured by Nei's genetic distance, was highly correlated with O mossambicus gene content The implications of these results for tilapia genetic improvement are discussed.     

The obvious heterosis and genetic characters of intergeneric cross and backcross juveniles between blunt snout bream (Megalobrama amblycephala) and topmouth culter (Culter alburnus)

In this study, we used artificial insemination to generate hybrid groups of fish [MC‐F₁(MA♀×CA♂) and MC‐F₂(MC‐F₁♀×♂)] by intergeneric crosses of Megalobrama amblycephala (MA) and Culter alburnus (CA); sequential backcrosses [CAM‐B₁ (CA♀×MC‐F₁♂) and MCC‐B₁ (MC‐F₁♀×CA♂)] were also performed. All these hybrids showed high rates of fertilization, hatching and survival (p > 0.05). For genetic traits, compared with those of the M. amblycephala and C. alburnus parental lines (Table 1), the fertilization rate, hatching rate and 7‐day survival rate of MC‐F1(MA♀×CA♂), MC‐F2(MC‐F1♀×♂), CAM‐B1 (CA♀×MC‐F1♂) and MCC‐B1 (MC‐F1♀×CA♂) by artificial insemination exhibited similar high rates (p > 0.05). The morphology of the four hybrids MC‐F₁/F_2, CAM‐B₁ and MCC‐B₁ were intermediate between those of their parents. Compared with their parents of MA and CA, weight gain rate (WG), specific growth rate (SGR) and protein efficiency ratio (PER) of hybrids MC‐F₁/F₂, CAM‐B₁ and MCC‐B₁ were significantly (p < 0.05) increased and feed conversion ratio (FCR) was significantly (p < 0.05) decreased after 3 months feeding. Moreover, protein content of muscle for MC‐F₁/F₂, CAM‐B₁ and MCC‐B₁ was significantly (p < 0.05) higher and carbohydrate content of muscle was significantly (p < 0.05) lower than their parents. The females and males of the four hybrids had normal gonadal development. In this study, we successfully generated intergeneric and backcross hybridization lines with fertile potential among fish of the Cultrinae subfamily and these hybrids had obvious heterosis in terms of growth performance, feed utilization and muscle quality.     

Mass production of hybrid magur and its culture potential in Bangladesh

Fertility, survival rate and hatching rate of hybrids of local magur Clarias batrachus and African catfish C. gariepinus were compared with the purebred species. Hybridization was conducted by hormone injection of purebreds and F₁ hybrids (F₁‐A), and artificial fertilization. Pure parental crosses as well as possible hybrid combinations were obtained. The fertility of F₁ hybrids (F₁‐A), purebred C. batrachus and C. gariepinus was 71.0%, 46.5% and 84.0% respectively. The fertility of an F₂ hybrid was 32.0%. The timing of embryonic developmental stages of F₁ hybrids (F₁‐A) was similar to that of purebred species. The hatching rate and survival rate of F₁ hybrids (F₁‐A) up to first feeding stage were 33.0% and 48.3%, respectively, which were significantly higher (P > 0.05) than the purebred C. batrachus. Only three embryos of an F₂ hybrid hatched successfully, and only one of these survived. The embryonic development of F₁ hybrids (F₁‐A) and their hatchability and survivability ensured the viable capacity of their progeny for mass culture. Hybrid morphology, both external and internal, was intermediate to that of the parents.     

Impact of Oreochromis mossambicus × O. niloticus (Pisces: Cichlidae) hybridization on population reproductive potential and long-term influence on a reservoir fishery

Abstract Exotic cichlids, introduced to Asia during the second half of the twentieth century, contribute significantly to the reservoir fisheries in the region. The two major cichlid species, Oreochromis mossambicus (Peters) and O. niloticus (L.), tend to interbreed easily. In Sri Lankan reservoirs, introgressive hybridization of these two cichlids takes place. The reproductive biology of O. mossambicus, O. niloticus and their hybrids in three reservoirs in Sri Lanka was evaluated. An imbalance in sex ratio with male dominance was evident. The estimated fecundity for 20-cm fish, using fecundity-total length relationships for various populations, indicated that there was a decline in fecundity in hybrid forms. It is hypothesized that the long-term effect of crosshybridization between the two cichlids might lead to a decline in fish yields in perennial reservoirs of Sri Lanka. The importance of the findings of the present study for the management of the reservoir fisheries in Asia is highlighted.     

Genetic diversity and differentiation of Nile tilapia (Oreochromis niloticus) revealed by DNA microsatellites

To assess the genetic diversity of Nile tilapia Oreochromis niloticus, a total of 250 fish from five Egyptian populations were genotyped using six microsatellite markers. Heterozygosity and Wright's F‐statistics (F_IS, F_ST, and F_IT) were calculated to determine the genetic variation within and between these populations. Observed heterozygosities were in the range of 0.4 (Burullus) to 0.96 (Qena), with F_IS values ranging from 0.082 to 0.282. The mean F_ST showed that approximately 96.5% of the genetic variation was within‐population and 3.5% was among populations. Standard genetic distances were used to classify the five populations into two major groups. The deeper lotic river Nile populations of Assuit and Cairo formed one group and the shallow less lotic Delta lakes populations of Manzalla and Burullus formed the second group, with the upstream Nile Qena population being an outgroup. The findings from the current study help understanding of the broad‐scale population structuring of the Nile tilapia (O. niloticus) allowing the population groupings identified to act as potential sources of genetic variation. These populations could be included in future Marker‐Assisted‐Selection programs for economically desired production traits.     

Evaluation of some red tilapia strains for aquaculture: growth and colour segregation in hybrid progeny

Four red tilapia hybrids were evaluated for growth in earthen ponds and for colour distribution: ‘red’O. niloticus (L.) x O. niloticus (L.) or O. aureus (Steindachner). and O. niloticus or O. aureus x ‘white’ segregate of a Philippine red tilapia (Oreochromis sp.). The best growth was obtained in the ‘red’O. niloticus x O. niloticus and O. aureusx‘white’ hybrids, although they constituted only a little over 50% males. Of these two hybrids, the former is all-red, while the latter segregates into 35% red and 65% less attractive bronze-coloured individuals. Ways for establishing all-male populations of these two hybrids, for possible improvement of their performance, are discussed. Incompatibility between ‘red’O. niloticus females and O. aureus males, limiting fry production of this hybrid, was observed. The few fish of this hybrid obtained and tested proved to be all-male and all-red.     

Production and Processing Trait Comparisons of Channel Catfish,Blue Catfish,and Their Hybrids Grown in Earthen Ponds

Fingerling HS‐5 channel catfish, Ictalurus punctatus, NWAC 103 channel catfish, D&B blue catfish, Ictalurus furcatus, HS‐5 female channel × D&B male blue catfish F₁ hybrids, and NWAC 103 female channel × D&B male blue catfish F₁ hybrids were stocked into twenty‐five 0.04‐ha earthen ponds at 12,500 fish/ha and grown for 277 d. Fish were fed daily at rates from 1.0 to 3.0% biomass based on feeding activity and temperature and adjusted weekly assuming a feed conversion ratio (FCR) of 1.8 and 100% survival. At harvest, 40 fish from each pond were sampled, and all other counted and weighed. Mean survival, growth rate indexes (a), FCR, and skin‐on fillet percentages were not significantly different. Mean harvest weights and net production were higher for HS‐5 channel and its hybrid than for the NWAC 103 channel, NWAC 103 hybrid, and D&B blue catfish, partially because of their larger mean stocking weights. D&B blue catfish was more uniform in size than NWAC 103 channel and NWAC 103 hybrid. D&B blue catfish was the easiest to seine. HS‐5 hybrids and NWAC 103 hybrids had lower mean head percentage and a better processing yield than their parent channel catfish.     

Sensitivity of Tilapia (Oreochromis mossambicus) to Clostridium botulinum toxins

The sensitivity of Oreochromis mossambicus to Clostridium botulinum toxin types A–E was investigated. All five toxin types were toxic to O. mossambicus. In the case of toxin types A–D, O. mossambicus was considerably more resistant than mice and, in the case of type E toxin, fish were more sensitive. The minimum intraperitoneal (i.p.) dose of type E toxin for fish was half the minimum lethal dose for mice. The results of the study suggest that good hygiene should be maintained in fish/shrimp farms to keep contamination at a low level.     

Detection of hybrids between masu salmon <Emphasis Type="Italic">Oncorhynchus masou masou</Emphasis> and amago salmon <Emphasis Type="Italic">O. m. ishikawae</Emphasis> occurred in the Jinzu River using a random amplified polymorphic DNA technique

Identification of masu salmon Oncorhynchus masou masou and amago salmon O. m. ishikawae was accomplished using a random amplified polymorphic DNA (RAPD) technique. Of 80 primers screened, three primers amplified subspecies-specific fragments (OPA11-1095 ^*, OPB5-1618^* and OPD5-2038 ^*). Based on fragment patterns, 33 hybrids were detected among 150 upstream-migrating individuals in the Jinzu River, central Japan, a long-time habitat of masu salmon, to which amago salmon were recently introduced. All of the individuals examined in the 2000–2002 cohorts were identified as F ₁ and F _x as well as pure masu and amago forms. These results indicated continuing hybridization, the genetic pollution of the native masu salmon population by amago salmon possibly being a serious problem in the Jinzu River. Both standard length and body weight in F ₁ hybrids tended to be less than in pure masu salmon. However, F _x hybrids showed similar body sizes to masu salmon.     

Changes in mitochondrial fatty acid composition following temperature acclimation of carp and their possible effects on FoF1-ATPase activity

Carp undergo temperature acclimation of respiratory function by altering mitochondrial ATP synthase (F_oF₁-ATPase) both quantitatively and qualitatively (Itoi et al. 2003). To address such acclimation temperature-dependent changes of F_oF₁-ATPase activity, we investigated in this study the correlation between the fatty acid composition and F_oF₁-ATPase activity in fast muscle of thermally acclimated carp. The quantities of saturated fatty acids of mitochondria from carp acclimated to 10 °C were significantly lower than those of carp acclimated to 30 °C. While mono- and poly-unsaturated fatty acids tended to increase with cold acclimation of carp, the molar concentration of 16:0 aldehyde in mitochondria from the 10 °C-acclimated carp were less than those from the 30 °C-acclimated fish. The specific activities of F_oF₁-ATPase in the 10 °C- and 30 °C-acclimated fish mitochondria were calculated to be 167±22 and 56±10 nmol/min ⋅ mg mitochondrial protein, respectively, the difference being significant at P<0.005. Taken together, the increase in F_oF₁-ATPase activity in fast muscle mitochondria of carp after cold temperature acclimation may be closely related to the increase of unsaturated fatty acids in mitochondria. Abbreviations: BSA - bovine serum albumin; DHA - docosahexaenoic acid; EGTA - ethyleneglycol bis(2-aminoethylether)tetraacetic acid; EPA - eicosapentaenoic acid; F_oF₁-ATPase - mitochondrial ATP synthase; α-F₁-ATPase - F_oF₁-ATPase α-subunit; β-F₁-ATPase - F_oF₁-ATPase β-subunit; HEPES - 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; SDS - sodium dodecyl sulfate; SDS-PAGE - SDS-polyacrylamide gel electrophoresis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reproductive ability of second generation ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids and characteristics of their offspring

The purpose of this study was to investigate the reproductive ability of second generation (F₂) koi (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids. Only diploid F₂ males and females were fertile and used in crosses. A significant increase was recorded in male fertility in F₂ versus F₁. In contrast with an earlier study in which only one fertile F₁ male was found, about 20% of F₂ males produced sperm. The observed reproductive ability of F₂ hybrids was similar to that demonstrated by the only fertile F₁ male and F₁ females. F₂ males produced diploid spermatozoa and generated triploids when crossed with koi females. All triploid fish in these progenies were males indicating that F₂ males had a sex chromosome constitution of XY. F₂ females produced diploid eggs and generated mostly triploids when crossed with koi males. In progenies obtained by crosses of F₂ males with F₁ and F₂ females, most of the surviving juveniles (63%–100%) were diploid; a minority of juveniles were aneuploid (ploidy ranged from 2.1n to 3.6n). Diploid fish in these progenies were presumably the result of spontaneous androgenesis and gynogenesis, by the same mechanisms observed earlier in progenies obtained by crossing the F₁ male with F₁ females.     

Fish-management relationships in Israeli commercial fish farming

To analyse the relationships among fish species performance and management procedures, a database was built up with data from 31 fish farms during the period 1976–1987 (1673 observations) and analysed through multivariate statistics (factor analysis). The data include nurseries, grow-out and operational ponds with mono- and polycultures of common carp, Cyprinus carpio, tilapia hybrid, Oreochromis niloticus × O. aureus, silver carp, Hypophthalmichthys molitrix, and mullet, Mugil cephalus. The main conclusions include the following. (1) The highest total yields and best tilapia performances were obtained in polyculture ponds where tilapia was the main species. (2) The best carp performances occurred in grow-out polyculture ponds where carp was the main species. Carp performance was improved in polycultures with mullet and silver carp, irrespective of whether tilapia were present or not. (3) Carp and tilapia yields increased as the nutritional inputs (feed pellets, sorghum pellets, manure), pond size and culture duration increased. The effect of the nutritional input was not linear, but logarithmic. (4) Growth rate of common carp was more affected by total density and stocking size than that of tilapia. Better carp and tilapia growth occurred in grow-out ponds when stocked at large sizes and cultured during short periods, mainly when both species were present. (5) Carp growth varied with the geographical region and size of fish pond, being better in smaller than in larger ponds due to reduced access to natural benthic food in deep ponds.