Inheritance of molecular markers and sex in the Australian freshwater crayfish, Cherax destructor Clark

Inheritance of three kinds of molecular genetic markers (mtDNA, random‐amplified polymorphic DNAs (RAPDs) and allozymes) and sex were investigated in crossbreeding experiments between three populations of the Australian freshwater crayfish Cherax destructor. Crossbreeding did not disrupt the ively maternally inherited, and allozyme and RAPD markers were transmitted following expected Mendelian principles for co‐dominant and dominant traits respectively. Unlike these three markers, sex ratios were found to be distorted by crossbreeding in some families. Two crossbred families produced only females. The implications of these findings for freshwater crayfish population genetics, taxonomy and aquaculture are discussed.     

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.     

Use of microsatellite loci and AFLP markers to verify gynogenesis and clonal lines in Nile tilapia Oreochromis niloticus L.

To develop an effective system for parentage analysis in gynogenetic and clonal progeny of Nile tilapia, Oreochromis niloticus L., polymorphic microsatellite loci and amplified fragment length polymorphisms (AFLPs) were investigated in several gynogenetic families and clonal lines. Six microsatellite loci were screened in two meiotic gynogenetic families to look for loci with high gene–centromere recombination rates, which can be used to discriminate meiotic from mitotic gynogenetics. Microsatellite loci UNH189 and UNH211 showed 96.7% and 92.0% heterozygosity, respectively, in these families, while other loci showed lower recombination frequencies. Scoring both UNH189 and UNH211 would give a very low probability of an individual meiotic gynogenetic being homozygous for both loci. Multiplex polymerase chain reaction of microsatellite loci was used to verify parentage in four families of mitotic gynogenetics and five fully inbred clonal lines. The genotype of each clonal line should serve as a unique identifier. Twelve AFLP primers were also investigated and 26 diagnostic AFLP bands were identified to follow inheritance in mitotic gynogenetic individuals. Amplified fragment length polymorphisms were found to be effective for this purpose but microsatellites were more appropriate since they are co‐dominant, while AFLPs are dominant markers. A multiplex of the microsatellite loci used in this study would be useful for general parental assignment as well as for the analysis of the products of chromosome set manipulations.     

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.     

Inheritance and reliability of random amplified polymorphic DNA-markers in two consecutive generations of common carp (Cyprinus carpio L.)

Random amplified polymorphic DNA (RAPD) markers have been used in a variety of genetic studies in fisheries and aquaculture. Most population studies are performed without preliminary data demonstrating the Mendelian inheritance and reproducibility of RAPD markers. In this study, the inheritance and reproducibility of RAPD markers was examined in two consecutive generations of common carp, Cyprinus carpio L. Variability and segregation of RAPD markers were investigated in one F₁ progeny and three F₂ progenies. Seventy-four RAPD markers were generated by five primers using DNA extracted from the initial ornamental (koi) common carp female and wild-type colour common carp male. Fifty-five of these RAPD markers were transmitted to the F₁ progeny and the inheritance patterns were analysed. Twenty RAPD markers were fully reproducible and demonstrated dominant simple Mendelian inheritance patterns in two consecutive generations. Twenty-four RAPD markers were not reproducible in all progenies. Thirteen markers displayed inheritance ratios in the progenies that did not fit simple Mendelian inheritance patterns. Non-reproducibility of RAPD markers and distorted ratios may be caused by the absence of amplification, poor amplification or by the appearance of artefact bands. Random amplified polymorphic DNA markers with poor reproducibility and non-Mendelian inheritance can lead to misinterpretations of data in population studies, resulting in errors in the estimation of genetic diversity within and between individual populations. Therefore, it is recommended to first identify the set of reproducible RAPD markers that demonstrate Mendelian inheritance before application of the RAPD technique in population studies.     

The expression of selected genes linked to metabolic homeostasis in obese pansteatitis‐suffering Mozambique tilapia,Oreochromis mossambicus (Peters)

The Oreochromis mossambicus (Peters) population inhabiting Lake Loskop, South Africa, is characterized by a high incidence of obesity and pansteatitis. We investigated potential links between the impaired health of Lake Loskop O. mossambicus and the endocrine system by assessing the expression of selected genes associated with the thyroid and adrenal endocrine axes as well as peroxisome proliferator‐activated receptor gamma (pparg). Moreover, contaminant‐induced thyroid and/or metabolic modulation in Lake Loskop water was evaluated using juvenile O. mossambicus in laboratory exposures. The expression of thyroid hormone receptor alpha (thra) and type 2 deiodinase (dio2) was higher in Lake Loskop O. mossambicus than fish from another population, suggesting a degree of thyroid disruption. The altered gene expression may be a consequence, rather than cause of obesity. Expression of dio2 and pparg was higher in juvenile O. mossambicus exposed to unfiltered compared to filtered lake water, and our data suggest fasting as causative factor. Micro‐organism abundance can therefore be a confounding factor in studies applying molecular markers to test for thyroid modulation by environmental waters. Pansteatitis was not a significant source of variance in the expression of any of the genes investigated, suggesting that the disease is not associated with disrupted endocrine signalling.     

Aflatoxin B1 induced alterations in the stability of the lysosomal membrane in Oreochromis mossambicus (Peters, 1852)

This article elucidates the effect of crude dietary aflatoxin B₁ (AFB₁) on the stability of the lysosomal membrane in tilapia (Oreochromis mossambicus), and the analysis was made at the biochemical and cellular level. The liver lysosomal acid phosphatase activity was used to evaluate the functional integrity of the lysosome. Three experimental groups were fed on diets containing 0.375, 2.5 and 6 mg kg^?1 of AFB₁ for a time period of 6 weeks, whereas a fourth group fed on the semi‐purified diet formed the control. Significant changes were observed in the stability of the lysosomal membrane in aflatoxin exposed groups. This is the first report, so far, of changes induced by AFB₁ on the stability of lysosomal membrane in tilapia (O. mossambicus).     

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.     

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.     

Genetic characterization of wild Dutch common carp (Cyprinus carpio L.)

Six male carp, caught in the water system surrounding the Anna Paulowna (AP) Polder in The Netherlands, were characterized using allozyme and microsatellite markers. At the sMDH‐A1,2* loci an allele was found, which has previously only been found in wild River Rhine and wild Vietnamese common carp. Microsatellite allele frequencies showed that these AP carp were significantly different from a group of carp originating from several different domesticated strains. Based on both allozyme and microsatellite data, the AP carp probably originated from a wild or feral self‐sustaining population.     

An investigation of the possible causes for the loss of productivity in genetically improved farmed tilapia strain in Fiji: inbreeding versus wild stock introgression

Four microsatellite markers and a mitochondrial DNA (mtDNA) fragment were used to investigate two possible explanations for a reported decline in productivity of genetically improved farmed tilapia (GIFT) in Fiji: (i) a decline in genetic diversity (GD) and (ii) genetic introgression from feral tilapia populations. Genetic diversity was estimated using θ and allelic richness, while Bayesian clustering was used to assign individuals to genetic groups (K=2 or 3) to test for introgression. Differentiation among groups was estimated using F_ST analysis. Results indicate that genetic diversity had declined compared with a GIFT reference stock from WorldFish Centre, while there was little evidence for introgression from feral tilapia populations. Loss of genetic diversity most probably resulted from practices that have not actively managed genetic resources in the hatchery. While GIFT is considered to be an improved line of Nile tilapia (Oreochromis niloticus), mtDNA analysis here revealed haplotypes assigned previously to three discrete Oreochromis species (O. niloticus, Oreochromis mossambicus and Oreochromis aureus) in both the Fijian strain and the WorldFish Centre strain. Possible sources for the three divergent lineages are discussed. Results have implications for the management and future expansion of the tilapia culture industry in Fiji as well as in other Pacific island nations.     

Contamination of palm kernel meal with Aspergillus flavus affects its nutritive value in pelleted feed for tilapia, Oreochromis mossambicus

An assessment of the nutritive value of palm kernel meal (PKM) and aflatoxin‐contaminated PKM (obtained by fermenting PKM with Aspergillus flavus) as a dietary ingredient in pelleted feed for tilapia, Oreochromis mossambicus Peters, was carried out in a 12‐week feeding trial. Seven isonitrogenous (40% crude protein) and isoenergetic (15.1 kJ g^?1) practical diets were formulated and fed close to apparent satiation to triplicate groups of 12 fish (mean initial weight 8.4 ± 0.1 g). The control diet contained 30% fish meal and 10% soybean meal (SBM) proteins. Four other experimental diets containing 20% and 50% of the SBM protein replaced by either PKM or fermented PKM, respectively, were formulated. Two additional diets containing either PKM or fermented PKM supplemented with a commercial aflatoxin adsorber (0.5% Sorbatox^TM) were also formulated. Measured aflatoxin B₁ levels in the fermented PKM‐based diets ranged from 75 to 100 µg kg^?1 diet. The growth performance and feed utilization efficiency of tilapia fed fermented PKM‐based diets were significantly lower than in fish fed the control diet at all inclusion levels (P < 0.05). Despite a small reduction, weight gains of tilapia fed PKM‐based diets were not significantly different compared with fish fed the control diet. The addition of 0.5% Sorbatox did not produce any beneficial or negative effects to the growth of tilapia. Under the dietary conditions of the present experiment, it was concluded that PKM can substitute up to 50% SBM in practical diets for O. mossambicus without much adverse effect to fish growth. However, when PKM was contaminated with A. flavus, its' incorporation into tilapia diets resulted in growth depression as a result of decreased diet digestibility and also possibly because of the presence of anti‐nutrients found in the contaminated PKM.     

Polymorphic microsatellite differences among four cultured populations of two selected tilapia strains

Genetic diversity plays a vital role for maintaining economically important traits in fish populations. The goal of this study was to assess the genetic variability among four tilapia populations in China. Two populations named XA (Oreochromis aureus, 30 samples) and AN (Egyptian Oreochromis niloticus, 24 samples) were gathered from Freshwater Fisheries Research Center (FFRC), and the other two named AL (O. aureus, 30 samples) and AJ (Egyptian O. niloticus, 30 samples) were from Guangxi Fisheries Research Institute (GFRI). GFRI introduced AL and AJ from XA and AN, and selected them for generations. We want to know if AL and AJ have genetic changes after selection and generations. Fifteen polymorphic microsatellite markers were used. Our results indicated that the four tilapia populations showed moderate genetic variability. The polymorphism information content (PIC) of the four populations was from 0.28 to 0.39; A_e was from 1.67 to 2.15; the H_e was from 0.35 to 0.46; the genetic similarity indexes were from 0.94 to 0.69 and also the clustering result showed that these four populations were highly related. There were no significant changes in genetic variation between late‐introduced population and early‐introduced population of O. aureus. Our results helped to understand the genetic variability of tilapia populations in China and for future marker‐assisted selection for important economic traits.     

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.     

Enhancement of non‐specific immune responses and disease resistance on oral administration of Nyctanthes arbortristis seed extract in Oreochromis mossambicus (Peters)

The immunomodulatory effect of the seed extract of an Indian medicinal plant Nyctanthes arbortristis L. (NAT) on non‐specific immune responses and disease resistance of tilapia, Oreochromis mossambicus was investigated. Chloroform extract was administered orally as a feed supplement at doses of 0.01%, 0.1% or 1% for 3 weeks. Non‐specific immune parameters such as serum lysozyme and alternate complement haemolytic (ACH₅₀) activities, and cellular reactive oxygen species (ROS), reactive nitrogen intermediate (RNI) and myeloperoxidase production and disease resistance against live virulent Aeromonas hydrophila were investigated after 1, 2 or 3 weeks of feeding with chloroform extract‐supplemented diet. The results of this study indicated that feeding tilapia for 2 weeks with selected doses of chloroform extract of NAT seeds significantly enhanced serum lysozyme, alternate complement activities and cellular ROS, RNI and MPO production. It was evident from the disease resistance test that feed supplemented with NAT seed extract at 0.1% or 1% level significantly reduced the mortality of O. mossambicus and a 3‐week feeding with 0.1% extract‐supplemented diet appears to be the optimal regimen for maximal disease resistance. Thus, the study indicates the scope of using the NAT extract as an immunoprophylactic in finfish aquaculture.     

RAPD fingerprinting of the eel-loaches Pangio filinaris and Pangio piperata: preliminary evaluation

Random amplified polymorphic DNA (RAPD) analysis was applied to two morphologically similar species of the increasingly popular ornamental eel‐loaches, Pangio filinaris and P. piperata, collected from Kemat River, Terengganu and Padang Sanai River, Kedah, West Malaysia. Forty primers were tested in a preliminary screening. Of these, five (OPA‐03, OPA‐09, OPA‐11, OPA‐13 and OPA‐18) were chosen for their ability to provide consistent amplification. RAPD analysis differentiated the two species, with each showing different RAPD patterns for each primer used. The five primers generated a total of 82 scorable loci with up to 83% and 60% polymorphism in P. piperata and P. filinaris respectively. The RAPD banding patterns of these two species ranged from 2 to 14 fragments, with a size range of 250–2000 bp. Genetic similarity within species ranged from 0.610 to 0.985 (mean 0.799±0.14) for P. piperata and from 0.824 to 0.934 (mean 0.885±0.04) for P. filinaris respectively. The interspecies genetic similarity ranged from 0.386 to 0.486 with an average value of 0.434±0.040. Our study revealed RAPDs as useful genetic markers for the taxonomic clarification and assessment of genetic variability of the eel‐loaches.     

Karyotype analysis of Oreochromis mossambicus,O. urolepis hornorum and their hybrid based on Cot‐1 DNA bands by fluorescence in situ hybridization

Chromosomal karyotypes of Oreochromis mossambicus and O. urolepis hornorum and their hybrid were analysed by means of Cot‐1 DNA bandings through fluorescence in situ hybridization (FISH). To identify all chromosomes, Cot‐1 DNA – which contains highly and moderately repetitive DNA – was extracted from genomic DNA, labelled as a probe with Dig‐11‐dUTP, and in situ hybridized to spreads of mitotic chromosomes of the three samples. The hybridized signals were detected by means of Cy3‐conjugated antidigoxigenin. The FISH results indicated that the three samples had the same diploid number (2n=44) of chromosomes. Specific fluorescence signal bands were detected on all individual chromosome pairs. On the basis of Cot‐1 DNA FISH banding patterns and chromosome morphology, the karyotypes of the three samples have been constructed; no remarkable differences were detected between the karyotypes of these species using this method. These results – which are similar to those reported previously, with respect to chromosome number, morphology and Cot‐1 DNA FISH patterns – suggest chromosomal stasis during speciation and hybridization of tilapia (Oreochromis, Cichlidae). Such a molecular cytogenetic procedure, if used in conjunction with other genomic research methods, could facilitate the study of genomic structure and be adapted for chromosome studies of other animal species.     

Inheritance mode of microsatellite DNA markers and their use for kinship estimation in kuruma prawn Penaeus japonicus

The allelic inheritance mode of microsatellite DNA markers was examined using seven copulated wild females and their offspring. Five microsatellite loci, CSPJ002 *, CSPJ010 *, CSPJ012 *, CSPJ014 *, and CSPJ015 *, were used in the study. At almost all family/locus combinations, one sire was determined and distributions of genotypes in offspring were consistent with the Mendelian segregation ratio. Distributions of genotypes were consistent with the ratio after assuming a null allele at some loci. Consequently, the alleles of CSPJ002 * and CSPJ012 * were inherited following the Mendelian inheritance mode in every family; however, the null allele was expected in CSPJ010 *, CSPJ014 *, and CSPJ015 * in some families. Thus, these loci should be used carefully in population genetic analysis, but siblings could be detected in the dendrograms based on unweighted pair-group method using arithmetic averages (UPMGA).     

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.