Multiplex microsatellite PCR panels and their application in genetic analyses of bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix)

In this study, three multiplex PCR panels involving twelve microsatellite loci were developed for cross-species amplification in bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix). The panels were verified using population genetic analyses, and species and simulated hybrid discriminations based on the genotype data of all individuals from two populations for each species. The number of alleles (Na), the observed and expected heterozygosity (Ho and He), and the polymorphic information content (PIC) of twelve loci were ranged from 5 to 20, 0.189 to 0.956, 0.177 to 901, and 0.169 to 0.887, respectively. Overall, almost all the microsatellite loci were detected as highly polymorphic across the two species. The populations of same species showed close genetic distances and similar genetic structures. Significant genetic differentiations were only detected between populations from different species, with a higher variation between species (21.17%) compared with that between populations within species (0.18%). In the species and simulated hybrid discrimination analyses, the individuals from different species were separated distinctly, and the simulated hybrids could almost be separated with the true individuals. In brief, the results indicated the high efficiency of the microsatellite multiplex panels developed in this study, which provide a practicable tool for further genetic analyses of H. nobilis and H. molitrix.     

Sixteen polymorphic microsatellites in bighead carp (Aristichthys nobilis) and cross-amplification in silver carp (Hypophthalmichthys molitrix)

A (GT)(n) enriched partial genomic library of bighead carp (Aristichthys nobilis) was constructed by employing the (fast isolation by AFLP of sequences containing repeats) FIASCO protocol. Sixteen loci exhibited polymorphism with two to seven alleles/locus (mean 3.263) in a test population and the observed heterozygosity ranging from 0.100 to 0.690 (mean 0.392). Eleven of the 16 bighead carp microsatellites were found to be also polymorphic in silver carp. These polymorphic loci should provide sufficient level of genetic diversity to evaluate population structure of bighead carp.     

Comparative studies on the feeding selectivity of silver carp Hypophthalmichthys molitrix and bighead carp Aristichthys nobilis

Silver carp and bighead carp exhibited size-selection for food particles in aquarium experiments, but did not select their preferred species of plankton actively when they were distributed evenly in the water. They also possessed the capacity of selection for feeding area. The removal rates (% g⁻¹ fish weight) of silver carp for smaller plankton (phytoplankton) were higher than those of bighead carp. The removal rates by the latter for bigger plankton (zooplankton) were higher than those of silver carp, but for plankton about 70 μm dia. the rates by the two species were almost equal.     

The effect of light stimuli on dark-adapted visual sensitivity in invasive silver carp Hypophthalmichthys molitrix and bighead carp H. nobilis

Non-physical barriers, including the use of underwater strobe lights alone or paired with sound or bubbles, are being considered as a means to prevent the upstream migration of invasive silver carp Hypophthalmichthys molitrix and bighead carp H. nobilis. To optimize potential optical deterrents, it is necessary to understand the visual sensitivity of the fishes. Dark-adapted H. molitrix and H. nobilis were found to possess broad visual sensitivity between 470 to 620 nm with peak spectral sensitivity at 540 nm for H. molitrix and 560 nm in H. nobilis. To assess the effect of a strobe light on vision, dark-adapted H. molitrix, H. nobilis and common carp Cyprinus carpio, were exposed to three different 5 s trains (100, 200, or 500 ms on–off flashes) of white light and the recovery of visual sensitivity was determined by measuring the b-wave amplitude of the electroretinogram (ERG). For all species, the longest recoveries were observed in response to the 500 ms flash trains (H. molitrix mean ± SE = 702.0 ± 89.8 s; H. nobilis 648.0 ± 116.0 s; C. carpio 480 ± 180.0 s). The results suggest that strobe lights can temporarily depress visual sensitivity, which may render optical barriers less effective.     

Suction volume and filtering efficiency of silver carp (Hypophthalmichthys molitrix Val.) and bighead carp (Aristichthys nobilis Rich.)

Experiments were conducted to measure the suction volume of silver carp and bighead carp of age 1 + with respiratory chamber, and to calculate the suction volume and the filtering efficiency with respect to changes in concentrations of food particles. Suction volume (B. ml/mouth) and filtering efficiency (E. %) were calculated using the following formula: C ₁=C₀(1-BE/v)ⁿ where C₀ and C₁ were the concentrations of specific food particles at the beginning and at the end of experiment, respectively, V was the volume (ml) of experimental water, and n was the total number of observation of suction made during the experimental period. The relationships between suction volume (ml/mouth) of age I+ silver carp (B_h) and bighead carp (B_a) and their standard lengths (L, cm) were: B _h=0.561L-8.94, B_a= 0.627L-7.48 while those of the fingerlings were: B _h= O.l70L-0.837, B_a= 0.157L-0.418. The suction volume of the fingerlings was mainly affected by fish size, the function of temperature between 15 and 25° C being negligible. However, temperature affected filtering rate (filtered volume per unit time) through its effect on filtering frequency. The filtering efficiency of the fishes for rotifers (Brachionus caliciflorus) was 100 per cent. The relationships between filtering efficiency and sizes of food particles smaller than or equal to that of a rotifer were: E _h=25.1 ln e.s.d. -13.6, E_a=22.2 In e.s.d. -33.1 where E_h and E_a were filtering efficiency of silver carp and bighead carp, respectively, and e.s.d. was the equivalent spherical diameter (μm) of food particles.     

Experimental evaluation of calcein and alizarin red S for immersion marking of silver carp Hypophthalmichthys molitrix (Valenciennes, 1844)

Calcein (CAL) and alizarin red S (ARS) at concentrations of 50–200 and 150–300 mg/L, respectively, were used for immersion marking of juvenile silver carp Hypophthalmichthys molitrix (79.65 ± 2.11 mm total length, mean ± SD). The marked fish were kept in separate tanks (0.012 individuals per litre, rearing temperature 18.4–25.7°C) for 360 days. Each experimental treatment group consisted of three replicates (16 individuals per replicate). Immersion for 24 h produced detectable marks in the sagittae, lateral line and non‐lateral line scales, and fin rays (dorsal, pectoral, ventral, anal, and caudal) at 360 days post‐marking. Acceptable marks in the sagittae were observed for CAL at concentrations of 150–200 mg/L and for ARS at concentrations of 200–300 mg/L. Fluorescent marks were poorly visible in all non‐lateral line scales from both the CAL‐ and ARS‐treated groups. Acceptable fluorescent marks in the lateral line scales and fin rays were detected for CAL at concentrations of 150–200 and 100–200 mg/L, respectively, and for ARS at concentrations of 200–300 and 150–300 mg/L, respectively. In particular, optimal marks were observed at the highest concentrations investigated in lateral line scales (200 mg/L CAL, 300 mg/L ARS) and fin rays (200 mg/L CAL, 200–300 mg/L ARS). There was no significant difference in the survival or growth of marked fish compared to controls throughout the experiment (P > 0.05).     

Development of microsatellite DNA markers of silver carp (Hypophthalmichthys molitrix) and their cross‐species application in bighead carp (Aristichthys nobilis)

Forty‐four microsatellite DNA markers were developed for silver carp, and used to investigate polymorphisms of 41 wild silver carps and seven wild bighead carps collected from Jingzhou fragment of Yangtze River. In silver carp, 40 markers were polymorphic. A total of 297 alleles were detected at 40 polymorphic loci. The number of alleles per locus varied from two to 16 with an average of 7.4 and the expected heterozygosities of each locus ranged from 0.07 to 0.91 with an average of 0.69. All markers amplified both silver carp and bighead carp DNAs.     

Observations on the blood of the Chinese silver carp Hypophthalmichthys molitrix (Valenciennes)

The ranges and mean values for several blood parameters of adult specimens of Hypophthalmichthys molitrix were determined in order to establish a standard haematological profile for future investigations. Several significant correlations were established, the most important being between haematocrit, haemoglobin and erythrocyte counts. The data are discussed in relation to the general biology of this species.     

The hearing abilities of the silver carp (Hypopthalmichthys molitrix) and bighead carp (Aristichthys nobilis)

Concern regarding the spread of silver carp (Hypopthalmichthys molitrix) and bighead carp (Aristichthys nobilis) through the Illinois River has prompted the development of a Bio-acoustic Fish Fence (BAFF) to act as an acoustic fish deterrent. The application of this technology has resulted in a need to understand the auditory physiology of the target species, in order to maximise the effect of the barrier in preventing the migration of the non-indigenous carp species into Lake Michigan, whilst minimising the effect on indigenous fish populations. Therefore, the hearing thresholds of 12 H. molitrix and 12 A. nobilis were defined using the Auditory Brainstem Response (ABR) technique, in a pressure-dominated sound field generated by submerged transducers of the type used in the construction of the BAFF system. The results clearly show that these fish are most sensitive to sounds in a frequency bandwidth of between 750 Hz and 1500 Hz, with higher thresholds below 300 Hz and above 2000 Hz.     

Digestive enzyme pattern of two stomachless filter feeders, silver carp, Hypophthalmichthys molitrix Val., and bighead carp, Aristichthys nobilis Rich.

Activities of digestive enzymes (trypsin and amylase) of microplanktophagous silver carp, Hypophthalmichthys molitrix , and macroplanktophagous bighead carp, Aristichthys nobilis , were investigated in relation to pH in 10 different segments of the gut. In addition, the possible existence of 'classical' lysozyme in both species and cellulase in silver carp were scrutinized, but no activities were detected. Tryptic and amylolytic activities decreased sharply from fore-gut to hind-gut indicating an efficient reabsorption mechanism. Trypsin and amylase of both species had a pH optimum at 8.3 and 7.0 respectively. The relatively low pH of 6.4 in the fore-gut of silver carp did not support an efficient operation of these enzymes, which was compensated by a higher enzyme concentration as compared to bighead carp. The latter, however, showed a higher pH of 7.3 in the fore-gut and thereby maintained similar activities as silver carp. The distinction of herbivorous and omnivorous fish in terms of digestion mechanisms is discussed.     

Development of silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) genetic maps using microsatellite and AFLP markers and a pseudo-testcross strategy

Silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) are two of the four most important pond-cultured fish species inhabiting the major river basins of China. In the present study, genetic maps of silver carp and bighead carp were constructed using microsatellite and AFLP markers and a two-way pseudo-testcross strategy. To create the maps, 60 individuals were obtained from a cross of a single bighead carp (female) and a single silver carp (male). The silver carp map consisted of 271 markers (48 microsatellites and 223 AFLPs) that were assembled into 27 linkage groups, of which 22 contained at least four markers. The total length of the silver carp map was 952.2 cM, covering 82.8% of the estimated genome size. The bighead carp map consisted of 153 markers (27 microsatellites and 126 AFLPs) which were organized into 30 linkage groups, of which 19 contained at least four markers. The total length of the bighead carp map was 852.0 cM, covering 70.5% of the estimated genome size. Eighteen microsatellite markers were common to both maps. These maps will contribute to discovery of genes and genetic regions controlling traits in the two species of carp.     

A comparison of complete mitochondrial genomes of silver carp Hypophthalmichthys molitrix and bighead carp Hypophthalmichthys nobilis: implications for their taxonomic relationship and phylogeny

Based upon morphological characters, Silver carp Hypophthalmichthys molitrix and bighead carp Hypophthalmichthys nobilis (or Aristichthys nobilis ) have been classified into either the same genus or two distinct genera. Consequently, the taxonomic relationship of the two species at the generic level remains equivocal. This issue is addressed by sequencing complete mitochondrial genomes of H. molitrix and H. nobilis , comparing their mitogenome organization, structure and sequence similarity, and conducting a comprehensive phylogenetic analysis of cyprinid species. As with other cyprinid fishes, the mitogenomes of the two species were structurally conserved, containing 37 genes including 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA (tRNAs) genes and a putative control region (D-loop). Sequence similarity between the two mitogenomes varied in different genes or regions, being highest in the tRNA genes (98·8%), lowest in the control region (89·4%) and intermediate in the protein-coding genes (94·2%). Analyses of the sequence comparison and phylogeny using concatenated protein sequences support the view that the two species belong to the genus Hypophthalmichthys . Further studies using nuclear markers and involving more closely related species, and the systematic combination of traditional biology and molecular biology are needed in order to confirm this conclusion.     

Biochemical genetic comparison of bighead (Hypophthalmichthys molitrix), silver carp (Aristichthys nobilis), and their hybrids reared in Czechoslovakia

Incidental and/or uncontrolled hybridization between silver carp ( Hypophthalmichthys molitrix ) and bighead ( Aristichthys nobilis ) represents a serious problem in Czechoslovak aquaculture. This fact affects fitness traits very negatively in successive generations of hybrids. To solve this problem, 1076 individuals in a total of both H. molitrix. A. nobilis , and their hybrids in 12 groups from six rearing facilities were analysed. Twelve protein systems representing 21 presumptive loci were studied to analyse the electrophoretic patterns of their products using horizontal starch gel electrophoresis of blood and tissue extracts. Both species displayed identical electrophoretic patterns in MYO-I* , LDH-A *, LDH-B *, sMDH-1 *, sIDHP-3*, GPI-1*, and CK-I * loci. For a reliable differentiation of both species products of the following nine loci are applicable ALB *, PA *, TF, sMDH-2 *, SOD *, NDH *, MYO-IF , MYO-III *, and CK-2 *. In addition, some polymorphic variants in slDHP-1 *, sIDHP-2 *, LDH-C *, EST-II *, and GPI-2 * loci are of use as auxillary markers while the other variants are common to both species. A high level of gene introgression was evident through hybridization between both species. All groups declared previously as ' H. molifrix ' were actually confirmed biochemically to be H. molitrix . However, all groups declared as ' A. nobilis ' were proved to be a mixture of A. nobilis and its hybrids of different level with H. molitrix . This suggests it is impossible to distinguish between A. nobilis and hybrids using their external morphology only.     

Molecular data and phylogenetic analysis of Myxobolus pseudonobilis n. sp. infecting the gill filaments of silver carp,Hypophthalmichthys molitrix Valenciennes, 1844

In the present study, we combined morphological and phylogenetic methods to characterize Myxobolus pseudonobilis n. sp. infecting Hypophthalmichthys molitrix Valenciennes, 1844 from Chongqing, China. The morphology and molecular characteristics of M. pseudonobilis n. sp. were distinct from those of other previously described Myxobolus species. Mature myxospores were ovoid in frontal view with spore dimensions of 10.0 ± 0.4 (9.3–10.9) μm in length and 8.5 ± 0.2 (7.9–9.0) μm in width. Two polar capsules occupying approximately half of the myxospore length were unequal in size. The larger polar capsule containing 6 to 7 filament coils measured 5.2 ± 0.3 (4.5–5.8) μm in length and 3.6 ± 0.2 (3.2–3.9) μm in width, while the smaller capsule with 4 to 5 filament coils measured 3.9 ± 0.3 (3.0–4.4) μm in length and 2.5 ± 0.3 (2.1–3.6) μm in width. The comparison of molecular characteristics demonstrated similarities and genetic distances of 18S rDNA sequences of 95.19% - 98.20% and 1.82% - 5.46%, respectively, between M. pseudonobilis n. sp. and its morphologically similar species, and secondary structures were also distinctly different. Moreover, phylogenetic analysis showed that M. pseudonobilis n. sp. was clustered with other myxobolids possessing spores with a blunt anterior end and branched independently. In addition, the morphology of myxosporeans as an important indicator was discussed.     

Native bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix populations in the Pearl River are threatened by Yangtze River introductions as revealed by mitochondrial DNA

Bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix have been two economically important aquaculture species in China for centuries. In the past decades, bighead and silver carp have been introduced from the Yangtze River to many river systems in China, including the Pearl River, in annual, large-scale, stocking activities to enhance wild fisheries. Nonetheless, few studies have assessed the ecological or genetic impacts of such introductions on native conspecific fish populations. An mtDNA D-loop segment of 978 bp from 213 bighead carp samples from 9 populations and a 975 bp segment from 204 silver carp samples from 10 populations were obtained to evaluate genetic diversity and population integrity. Results from a haplotype network analysis revealed that most haplotypes of the Pearl River clustered with those of Yangtze River origin and only a small proportion were distinct, suggesting that both the native Pearl River bighead and silver carp populations are currently dominated by genetic material from the Yangtze River. The genetic diversity of Pearl River populations is high in both species because of this inter-population gene flow, but the diversity of native Pearl River populations is low. To preserve the native genetic diversity, stocking of non-native fingerlings should cease immediately and native Pearl River bighead and silver carp fish farms should be established. This research demonstrates the danger to native biodiversity across China because of the substantial, ongoing stock-enhancement activities without prior genetic assessment.     

Human health risks of metalloids and metals in muscle tissue of silver carp Hypophthalmichthys molitrix (Valenciennes, 1844) from Lake Flag Boshielo,South Africa

The Olifants River, a major tributary of the Limpopo River, is one of the most polluted rivers in South Africa. Consequently, concerns regarding the human health impact of long-term consumption of fish from the Olifants River have been raised in recent studies. Nevertheless, Lake Flag Boshielo situated on the main stem of the Olifants River has been proposed as a site for an inland fishery. Planktivorous silver carp Hypophthalmichthys molitrix (Valenciennes, 1844) is among the potential target species for such a fishery. Therefore, a desk-top human health risk assessment was conducted for silver carp from Lake Flag Boshielo. From January to November 2013, muscle samples from 50 specimens were collected and analysed for metals and metalloids. The hazard quotient based on a weekly meal of 150 g exceeded the acceptable level for As, Cd, Cr, Co, Pb, Hg, Se, V and Zn. Compared with previous studies from Lake Flag Boshielo, muscle tissue concentrations of As, Cr, Pb, Hg, Se, V and Zn for H. molitrix were higher. Based on the metal and metalloid concentrations reported in this study, long-term consumption of silver carp from Lake Flag Boshielo might pose a health risk to impoverished rural communities.     

A note on the growth of two exotics in England; the silver carp (Hypophthalmichthys molitrix Val.) and the bighead (Aristichthys nobilis Rich.)

Observations were made on the growth of microphagous silver carp and bigheads in a tank housed under a horticultural polythene tunnel. It is suggested that the species may be of interest for cultivation and might also be useful in removing nutrients from eutrophic waters in the United Kingdom.     

Construction of a genetic linkage map for silver carp (Hypophthalmichthys molitrix)

For silver carp (Hypophthalmichthys molitrix), a combined microsatellite (or simple sequence repeat) and amplified fragment length polymorphism (AFLP) sex average linkage map was constructed. A total of 483 markers (245 microsatellites and 238 AFLPs) were assigned to 33 linkage groups. The map spanned 1352.2 cM, covering 86.4% of the estimated genome size of silver carp. The maximum and average spaces between 420 loci were 21.5 cM and 3.2 cM, respectively. The length of linkage groups ranged from 3.6 cM to 98.5 cM with an average of 41.0 cM. The number of markers per group varied from 2 to 44 with an average of 14.6. The AFLP markers significantly improved the integrity of microsatellite-based linkage groups and increased the genome coverage and marker evenness. A genome-wide recombination suppression was observed in male. In an extreme case, six microsatellites co-segregated in male, but spanned a 45.1 cM region in female.