Eye Degeneration and Loss of otx5b Expression in the Cavefish Sinocyclocheilus tileihornes.

Cave animals possess remarkable phenotypes associated with existence in their dark environments. The Chinese cavefish Sinocyclocheilus tileihornes shows substantial eye degeneration, a trait shared by most cave species. The extent to which independent evolution of troglomorphic traits uses convergent molecular genetic mechanisms is as yet unknown. We performed transcriptome-wide gene expression profiling in S. tileihornes eyes and compared results with those from the closely related surface species S. angustiporus and an independently derived congeneric cavefish, S. anophthalmus. In total, 52.85 million 100 bp long paired-end clean reads were generated for S. tileihornes, and we identified differentially expressed genes between the three possible pairs of species. Functional analysis of genes differentially expressed between S. tileihornes and S. angustiporus revealed that phototransduction (KEGG id: dre04744) was the most significantly enriched pathway, indicating the obvious differences in response to captured photons between the cavefish S. tileihornes and the surface species S. angustiporus. Analysis of key genes regulating eye development showed complete absence of otx5b (orthodenticle homolog 5) expression in S. tileihornes eyes, probably related to degradation of rods, but normal expression of crx (cone-rod homeobox). The enriched pathways and Otx5 are involved in phototransduction, photoreceptor formation, and regulation of photoreceptor-related gene expression. Unlike the S. tileihornes reported here, S. anophthalmus has reduced crx and otx5 expression. These results show that different species of cavefish within the same genus that independently evolved troglodyte characteristics can have different genetic mechanisms of eye degeneration.

RDL mutations predict multiple insecticide resistance in Anopheles sinensis in Guangxi, China.

BACKGROUND: Anopheles sinensis is a major vector of malaria in China. The gamma-aminobutyric acid (GABA)-gated chloride channel, encoded by the RDL (Resistant to dieldrin) gene, is the important target for insecticides of widely varied structures. The use of various insecticides in agriculture and vector control has inevitably led to the development of insecticide resistance, which may reduce the control effectiveness. Therefore, it is important to investigate the presence and distribution frequency of the resistance related mutation(s) in An. sinensis RDL to predict resistance to both the withdrawn cyclodienes (e.g. dieldrin) and currently used insecticides, such as fipronil. METHODS: Two hundred and forty adults of An. sinensis collected from nine locations across Guangxi Zhuang Autonomous Region were used. Two fragments of An. sinensis RDL (AsRDL) gene, covering the putative insecticide resistance related sites, were sequenced respectively. The haplotypes of each individual were reconstructed by the PHASE2.1 software, and confirmed by clone sequencing. The phylogenetic tree was built using maximum-likelihood and Bayesian inference methods. Genealogical relations among different haplotypes were also analysed using Network 5.0. RESULTS: The coding region of AsRDL gene was 1674 bp long, encoding a protein of 557 amino acids. AsRDL had 98.0% amino acid identity to that from Anopheles funestus, and shared common structural features of Cys-loop ligand-gated ion channels. Three resistance-related amino acid substitutions (A296S, V327I and T345S) were detected in all the nine populations of An. sinensis in Guangxi, with the 296S mutation being the most abundant (77-100%), followed by 345S (22-47%) and 327I (8-60%). 38 AsRDL haplotypes were identified from 240 individuals at frequencies ranging from 0.2 to 34.8%. Genealogical analysis suggested multiple origins of the 345S mutation in AsRDL. CONCLUSIONS: The near fixation of the 296S mutation and the occurrence of the 327I and 345S mutations in addition to 296S, in all the nine tested An. sinensis populations in Guangxi, strongly indicate a risk of multiple insecticide resistance. The haplotype diversity plus genetic heterogeneities in the geographical distribution, and multiple origins of AsRDL alleles call for a location-customized strategy for monitoring and management of insecticide resistance.

Diversity and frequency of kdr mutations within Anopheles sinensis populations from Guangxi, China.

BACKGROUND: Anopheles sinensis is a major vector of malaria in China and its control is under great threat as the development of insecticide resistance. Voltage-gated sodium channel (VGSC) is the target of several classes of insecticides. Genetic mutations of VGSC have been documented to confer knockdown resistance (kdr) to dichlorodiphenyltrichloroethane (DDT) and pyrethroids in mosquitoes. To control this vector efficiently, it is important to know the resistance-associated genetic mutations, their distribution frequencies and genealogical relations. METHODS: Three hundreds and thirteen (313) adults of An. sinensis collected from nine locations across Guangxi Zhuang Autonomous Region were used. The partial sequence of the An. sinensis voltage gated sodium channel gene (AS-VGSC) containing codon 1014 was sequenced. PHASE2.1 was used to construct the haplotypes of each individual, and the accuracy of haplotypes was further confirmed by clone sequencing. The genealogical relations of kdr mutations in AS-VGSC was analysed using TCS 2.1 and Network 5.0. RESULTS: Sixteen AS-VGSC haplotypes including seven haplotypes carrying non-synonymous mutations at codon 1014, and fifty-five AS-VGSC genotypes were identified from 313 mosquitoes collected from nine geographical locations across Guangxi. The number of haplotypes in each of the nine populations ranged from 5 to 13. The frequency of haplotypes carrying kdr mutations ranged from 2.7 to 80.0 % within the nine populations, of which 1014C was unexpectedly high in the northeast of Guangxi. Genealogical analysis suggested multiple origins of kdr mutations in An. sinensis. CONCLUSION: Diverse haplotypes of AS-VGSC are distributed in Guangxi. The presence of haplotypes carrying mutations at codon 1014 indicates a risk of pyrethroid and DDT resistance. The kdr mutations show differential distribution geographically, with high frequencies occurred in the northeast of Guangxi. Genealogical analysis suggests multiple origins of kdr mutations in An. sinensis populations in Guangxi. These findings have important practical implications for the sustainability of An. sinensis control programmes.

Phylogenetic analysis and expression differences of eye-related genes in cavefish genus Sinocyclocheilus.

The adaptive evolution of visual systems has been observed in many cavefish. However, little is known about the molecular mechanisms underlying these adaptations, which include regressive changes such as eye degeneration. Here, we analyzed phylogenetic and expression patterns of 6 eye-related genes (crx, foxg1b, opn1sw2, otx2, rho and sox2) in 12 Sinocyclocheilus species from China, including 8 stygobionts and 4 stygophiles, and examined photoreceptor cell morphology of these species. Those eye-degenerated species of Sinocyclocheilus were polyphyletic and showed different degrees of photoreceptor defects in responses to cave environments. The eye loss and degeneration are the result of convergent evolution. Although S. anophthalmus grouped with the eye-normal species, it displayed not only a high degree of eye degeneration but also significant expression differences in eye-related genes compared with the eye-normal species. The gene foxg1b, which was determined to be under positive selection, might play an important role in the process of eye degeneration in S. anophthalmus based on differential expression. Eye-related gene expression and selection may have contributed to the polyphyly of the cave species. We examined gene expression and duplication in 6 eye-related genes and revealed that these genes displayed considerable diversity in relative expression in Sinocyclocheilus fishes. Otx2 and sox2 were significantly up-regulated in individual cave species, while the other 4 genes (crx, foxg1b, opn1sw2 and rho) were significantly down-regulated. These findings provide a valuable resource for elucidating molecular mechanisms associated with visual system evolution in cavefish.

Unexpected relationships of substructured populations in Chinese Locusta migratoria.

BACKGROUND: Highly migratory species are usually expected to have minimal population substructure because strong gene flow has the effect of homogenizing genetic variation over geographical populations, counteracting random drift, selection and mutation. The migratory locust Locusta migratoria belongs to a monotypic genus, and is an infamous pest insect with exceptional migratory ability - with dispersal documented over a thousand kilometers. Its distributional area is greater than that of any other locust or grasshopper, occurring in practically all the temperate and tropical regions of the eastern hemisphere. Consequently, minimal population substructuring is expected. However, in marked contrast to its high dispersal ability, three geographical subspecies have been distinguished in China, with more than nine being biologically and morphologically identified in the world. Such subspecies status has been under considerable debate. RESULTS: By multilocus microsatellite genotyping analysis, we provide ample genetic evidence for strong population substructure in this highly migratory insect that conforms to geography. More importantly, our genetic data identified an unexpected cryptic subdivision and demonstrated a strong affiliation of the East China locusts to those in Northwest/Northern China. The migratory locusts in China formed three distinct groups, viz. (1) the Tibetan group, comprising locusts from Tibet and nearby West China high mountain regions; this is congruent with the previously recognized Tibetan subspecies, L. m. tibetensis; (2) the South China group, containing locusts from the Hainan islands; this corresponds to the Southeast Asia oriental tropical subspecies L. m. manilensis; (3) the North China group, including locusts from the Northwest and Northern China (the Asiatic subspecies L. m. migratoria), Central China and Eastern China regions. Therefore, the traditional concept on Locusta subspecies status established from Uvarov in 1930s needs to be revised. The three groups of locusts probably have separate evolutionary histories that were most likely linked to Quaternary glaciations events, and derived from different ancestral refugial populations following postglacial expansions. CONCLUSION: The migratory locust populations in China have differentiated into three genetically distinct groups despite high dispersal capability. While this clarified long-standing suspicions on the subspecific diversification of this species in China, it also revealed that the locusts in the vast area of East China are not the oriental subspecies but the Asiatic subspecies, an unexpected substructuring pattern. The distribution pattern of the three locust groups in China may be primarily defined by adaptive differentiation coupled to Quaternary glaciations events. Our results are of general significance both for locust research and for phylogeographical study of flora and fauna in China, illustrating the potential importance of phylogeographical history in shaping the divergence and distribution patterns of widespread species with strong dispersal ability.

Haplotype reconstruction for scnp DNA: a consensus vote approach with extensive sequence data from populations of the migratory locust (Locusta migratoria).

Single copy nuclear polymorphic (scnp) DNA is potentially a powerful molecular marker for evolutionary studies of populations. However, a practical obstacle to its employment is the general problem of haplotype determination due to the common occurrence of heterozygosity in diploid organisms. We explore here a 'consensus vote' (CV) approach to this question, combining statistical haplotype reconstruction and experimental verification using as an example an indel-free scnp DNA marker from the flanking region of a microsatellite locus of the migratory locust. The raw data comprise 251-bp sequences from 526 locust individuals (1052 chromosomes), with 71 (28.3%) polymorphic nucleotide sites (including seven triallelic sites) and 141 distinct genotypes (with frequencies ranging from 0.2 to 25.5%). Six representative statistical haplotype reconstruction algorithms are employed in our CV approach, including one parsimony method, two expectation-maximization (EM) methods and three Bayesian methods. The phases of 116 ambiguous individuals inferred by this approach are verified by molecular cloning experiments. We demonstrate the effectiveness of the CV approach compared to inferences based on individual statistical algorithms. First, it has the unique power to partition the inferrals into a reliable group and an uncertain group, thereby allowing the identification of the inferrals with greater uncertainty (12.7% of the total sample in this case). This considerably reduces subsequent efforts of experimental verification. Second, this approach is capable of handling genotype data pooled from many geographical populations, thus tolerating heterogeneity of genetic diversity among populations. Third, the performance of the CV approach is not influenced by the number of heterozygous sites in the ambiguous genotypes. Therefore, the CV approach is potentially a reliable strategy for effective haplotype determination of nuclear DNA markers. Our results also show that rare variations and rare inferrals tend to be more vulnerable to inference error, and hence deserve extra surveillance.

Phylogeographical structure and demographic expansion in the endemic alpine stream salamander (Hynobiidae: Batrachuperus) of the Qinling Mountains.

The Qinling Mountains of China provide an excellent study area for assessing the effect of Pleistocene climatic oscillations and paleogeological events on intraspecific diversification. To assess genetic diversity of an endemic stream salamander, Batrachuperus tibetanus, for its conservation, a phylogeographical survey was performed based on mitochondrial DNA and morphological data. The mitochondrial data revealed three lineages of B. tibetanus in the Qinling Mountains. A lineage present in the northwestern Qinling Mountains groups with the Tibet lineage of B. tibetanus, and the remaining Qinling populations are eastern and western lineages that separated ~3-4 million years ago (Ma). The eastern and western Qinling lineage delineation is supported by three morphological variables (snout length, eye diameter and axilla-groin length). The divergence of the two major lineages was likely caused by orogenesis of the Qinling Mountains during the late Cenozoic, and the two lineages were subsequently affected at different levels by Pleistocene climatic oscillations showing different signals of demographic expansion. A large suitable area of B. tibetanus through the Qinling Mountains since the last glacial maximum (LGM) indicated the adaptation of this species to the climatic changes. However, low genetic diversity within populations indicate the urgency of preserving the vulnerable populations and endemic lineages.

CVhaplot: a consensus tool for statistical haplotyping.

Haplotypes contain genealogical information and play a prominent part in population genetic and evolutionary studies. However, haplotype inference is a complex statistical problem, showing considerable internal algorithm variability and among-algorithm discordance. Thus, haplotypes inferred by statistical algorithms often contain hidden uncertainties, which may complicate and even mislead downstream analysis. Consensus strategy is one of the effective means to increase the confidence of inferred haplotypes. Here, we present a consensus tool, the CVhaplot package, to automate consensus techniques for haplotype inference. It generates consensus haplotypes from inferrals of competing algorithms to increase the confidence of haplotype inference results, while improving the performance of individual algorithms by considering their internal variability. It can effectively identify uncertain haplotypes potentially associated with inference errors. In addition, this tool allows file format conversion for several popular algorithms and extends the applicability of some algorithms to complex data containing triallelic polymorphic sites. CVhaplot is written in PERL and freely available at http://www.ioz.ac.cn/department/agripest/group/zhangdx/CVhaplot.htm.