Mitochondrial DNA haplotype diversity and origin of captive sand tiger sharks (Carcharias taurus)

The sand tiger shark (Carcharias taurus) is listed as globally vulnerable by the International Union for Conservation of Nature (IUCN) with geographically isolated and separated global populations with little or no gene flow between them. Captive-breeding of these sharks in aquaria would reduce the need to populate displays with wild-caught individuals; however, sand tigers are notoriously difficult to breed in captivity. In this study we analysed 520bp of the mitochondrial D-loop to assess the haplotype diversity of 19 captive sand tiger sharks from aquaria in the UK and US. Genetic material was sampled in a non-invasive fashion through DNA extracted from shed teeth. Data obtained were compared to known, geographically mapped wild haplotypes to establish whether individuals from different global populations are being housed together. Results identified the haplotype of a minimum of 10 of the 19 sharks, detecting four different haplotypes, and identifying a previously undescribed haplotype (haplotype K). A major genetic subdivision between the haplotypes of the North West Atlantic and those of other global populations has been previously shown from population genetic analyses. Our results indicate that captive sharks can be from either side of this subdivision and occasionally these can be co-housed in the same aquarium. Since sharks with highly divergent genetic ancestry are being kept together, these findings have implications for conservation efforts regarding the individual needs of sand tiger shark populations and for captive-breeding program success rates

A GTT microsatellite repeat motif and differentiation between morphological forms of Littorina saxatilis: speciation in progress?

A size (and sequence) variable microsatellite has been identified in the gastropod Littorina saxatilis following sequencing of products obtained through randomly amplified polymorphic DNA amplification. Size frequency distributions for this GTT repeat motif have been produced from a total of 439 L. saxatilis. Although there is evidence for a high prevalence of null alleles, consistent, largely significant, differences are found between the average size of this repeat in high shore, thin shelled morphs (L. saxatilis H) compared to mid shore, thick shelled animals (L. saxatilis M), with the former having consistently larger allele sizes. These findings are evident on both a large scale (around the coast of Britain) and a microgeographic scale (replicated sampling of a single shore). Unusually large alleles (>400 bp) are also considerably more prevalent in L. saxatilis H, and sequencing of these, including DNA flanking the GTT repeat, indicates that they do not represent independent expansion events. The significant difference in GTT repeat size, and increased prevalence of such related alleles in the H form compared to the M morph suggests that either gene flow is greater within, than between, morphs or that selection is acting on this locus or a closely linked locus. We argue for the latter scenario and discuss why this indicates that these forms are diverging across their range in the British Isles

Susceptibility of Chironomus plumosus larvae (Diptera: Chironomidae) to entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae): potential for control

Chironomidae, or non-biting midges, are found worldwide in a wide variety of aquatic habitats. During periods of mass adult eclosion they can become a nuisance and health hazard. Current control methods target the aquatic larval stage and include the use of insect growth regulators or insecticides, which may be prohibited in certain environments or affect non-target organisms. The aim of this study was to investigate whether entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae, currently employed for control of terrestrial crop pests, could be used as a viable biocontrol for the aquatic larval stages of the Chrionomidae, offering an alternative to current chemical methods. We demonstrate that Steinernema feltiae (Filipjev, 1934), Steinernema carpocapsae (Weiser, 1955), Steinernema kraussei (Steiner, 1923) and Heterorhabditis bacteriophora (Poinar, 1975) are able to survive in water up to 96 h and are able to parasitize and kill Chironomus plumosus (Linnaeus, 1758) larvae, with mortality observed after just 24 h exposure and with < 20% survival after 4 days. We also show that following application to the water column, EPNs sink to the bottom of the lentic water body and can remain alive for more than 96 h. Taken together, we believe that several EPN species could be developed as a valid form of biocontrol for Chironomidae

Identification of the main malaria vectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay

Background: The Anopheles gambiae sensu lato species complex comprises seven sibling species of mosquitoes that are morphologically indistinguishable. Rapid identification of the two main species which vector malaria, Anopheles arabiensis and An. gambiae sensu stricto, from the non-vector species Anopheles quadriannulatus is often required as part of vector control programmes. Currently the most widely used method for species identification is a multiplex PCR protocol that targets species specific differences in ribosomal DNA sequences. While this assay has proved to be reasonably robust in many studies, additional steps are required post-PCR making it time consuming. Recently, a high-throughput assay based on TaqMan single nucleotide polymorphism genotyping that detects and discriminates An. gambiae s.s and An. arabiensis has been reported. Methods: A new TaqMan assay was developed that distinguishes between the main malaria vectors (An. Arabiensis and An. gambiae s.s.) and the non-vector An. quadriannulatus after it was found that the existing TaqMan assay incorrectly identified An. quadriannulatus, An. merus and An. melas as An. gambiae s.s. The performance of this new TaqMan assay was compared against the existing TaqMan assay and the standard PCR method in a blind species identification trial of over 450 samples using field collected specimens from a total of 13 countries in Sub-Saharan Africa. Results: The standard PCR method was found to be specific with a low number of incorrect scores (<1%), however when compared to the TaqMan assays it showed a significantly higher number of failed reactions (15%). Both the new vector-specific TaqMan assay and the exisiting TaqMan showed a very low number of incorrectly identified samples (0 and 0.54%) and failed reactions (1.25% and 2.96%). In tests of analytical sensitivity the new TaqMan assay showed a very low detection threshold and can consequently be used on a single leg from a fresh or silica-dried mosquito without the need to first extract DNA. Conclusion: This study describes a rapid and sensitive assay that very effectively identifies the two main malaria vectors of the An. gambiae species complex from the non-vector sibling species. The method is based on TaqMan SNP genotyping and can be used to screen single legs from dried specimens. In regions where An. merus/melas/ bwambae, vectors with restricted distributions, are not present it can be used alone to discriminate vector from non-vector or in combination with the Walker TaqMan assay to distinguish An. arabiensis and An. gambiae s.s

Mitochondrial DNA mutations in individuals occupationally exposed to ionizing radiation

Mutations in a 443-bp amplicon of the hypervariable region HVR1 of the D-loop of mitochondrial DNA (mtDNA) were quantified in DNA extracted from peripheral blood samples of 10 retired radiation workers who had accumulated external radiation doses of .0.9 Sv over the course of their working life and were compared to the levels of mutations in 10 control individuals matched for age and smoking status. The mutation rate in the 10 exposed individuals was 9.92 3 1025 mutations/ nucleotide, and for the controls it was 8.65 3 1025 mutations/ nucleotide, with a procedural error rate of 2.65 3 1025 mutations/ nucleotide. No increase in mtDNA mutations due to radiation exposure was detectable (P 5 0.640). In contrast, chromosomal translocation frequencies, a validated radiobiological technique for retrospective dosimetric purposes, were significantly elevated in the exposed individuals. This suggests that mutations identified through sequencing of mtDNA in peripheral blood lymphocytes do not represent a promising genetic marker of DNA damage after low-dose or low-doserate exposures to ionizing radiation. There was an increase in singleton mutations above that attributable to procedural error in both exposed and control groups that is likely to reflect age-related somatic mutation

Contemporary gene flow between wild An. gambiae s.s. and An. arabiensis

Background In areas where the morphologically indistinguishable malaria mosquitoes Anopheles gambiae Giles and An. arabiensis Patton are sympatric, hybrids are detected occasionally via species-diagnostic molecular assays. An. gambiae and An. arabiensis exhibit both pre- and post-reproductive mating barriers, with swarms largely species-specific and male F1 (first-generation) hybrids sterile. Consequently advanced-stage hybrids (back-crosses to parental species), which would represent a route for potentially-adaptive introgression, are expected to be very rare in natural populations. Yet the use of one or two physically linked single-locus diagnostic assays renders them indistinguishable from F1 hybrids and levels of interspecific gene flow are unknown. Methods We used data from over 350 polymorphic autosomal SNPs to investigate post F1 gene flow via patterns of genomic admixture between An. gambiae and An. arabiensis from eastern Uganda. Simulations were used to investigate the statistical power to detect hybrids with different levels of crossing and to identify the hybrid category significantly admixed genotypes could represent. Results A range of admixture proportions were detected for 11 field-collected hybrids identified via single-locus species-diagnostic PCRs. Comparison of admixture data with simulations indicated that at least seven of these hybrids were advanced generation crosses, with backcrosses to each species identified. In addition, of 36 individuals typing as An. gambiae or An. arabiensis that exhibited outlying admixture proportions, ten were identified as significantly mixed backcrosses, and at least four of these were second or third generation crosses. Conclusions Our results show that hybrids detected using standard diagnostics will often be hybrid generations beyond F1, and that in our study area around 5% (95% confidence intervals 3%-9%) of apparently ‘pure’ species samples may also be backcrosses. This is likely an underestimate because of rapidly-declining detection power beyond the first two backcross generations. Post-F1 gene flow occurs at a far from inconsequential rate between An. gambiae and An. arabiensis, and, especially for traits under strong selection, could readily lead to adaptive introgression of genetic variants relevant for vector control

Limited genomic divergence between intraspecific forms of Culex pipiens under different ecological pressures

Abstract Background: Divergent selection can be a major driver of ecological speciation. In insects of medical importance, understanding the speciation process is both of academic interest and public health importance. In the West Nile virus vector Culex pipiens, intraspecific pipiens and molestus forms vary in ecological and physiological traits. Populations of each form appear to share recent common ancestry but patterns of genetic differentiation across the genome remain unknown. Here, we undertook an AFLP genome scan on samples collected from both sympatric and allopatric populations from Europe and the USA to quantify the extent of genomic differentiation between the two forms. Results: The forms were clearly differentiated but each exhibited major population sub-structuring between continents. Divergence between pipiens and molestus forms from USA was higher than in both inter- and intra-continental comparisons with European samples. The proportion of outlier loci between pipiens and molestus (≈3 %) was low but consistent in both continents, and similar to those observed between sibling species of other mosquito species which exhibit contemporary gene flow. Only two of the outlier loci were shared between inter-form comparisons made within Europe and USA. Conclusion: This study supports the molestus and pipiens status as distinct evolutionary entities with low genomic divergence. The low number of shared divergent loci between continents suggests a relatively limited number of genomic regions determining key typological traits likely to be driving incipient speciation and/or adaptation of molestus to anthropogenic habitats

Insecticide resistance in Anopheles arabiensis in Sudan: temporal trends and underlying mechanisms

Background: Malaria vector control in Sudan relies mainly on indoor residual spraying (IRS) and the use of long lasting insecticide treated bed nets (LLINs). Monitoring insecticide resistance in the main Sudanese malaria vector, Anopheles arabiensis, is essential for planning and implementing an effective vector control program in this country. Methods: WHO susceptibility tests were used to monitor resistance to insecticides from all four WHO-approved classes of insecticide at four sentinel sites in Gezira state over a three year period. Insecticide resistance mechanisms were studied using PCR and microarray analyses. Results: WHO susceptibility tests showed that Anopheles arabiensis from all sites were fully susceptible to bendiocarb and fenitrothion for the duration of the study (2008–2011). However, resistance to DDT and pyrethroids was detected at three sites, with strong seasonal variations evident at all sites. The 1014 F kdr allele was significantly associated with resistance to pyrethroids and DDT (P 7 in allelic tests). The 1014S allele was not detected in any of the populations tested. Microarray analysis of the permethrinresistant population of An. arabiensis from Wad Medani identified a number of metabolic genes that were significantly over-transcribed in the field-collected resistant samples when compared to the susceptible Sudanese An. arabiensis Dongola strain. These included CYP6M2 and CYP6P3, two genes previously implicated in pyrethroid resistance in Anopheles gambiae s.s, and the epsilon-class glutathione-S-transferase, GSTe4. Conclusions: These data suggest that both target-site mechanisms and metabolic mechanisms play an important role in conferring pyrethroid resistance in An. arabiensis from Sudan. Identification in An. arabiensis of candidate loci that have been implicated in the resistance phenotype in An. gambiae requires further investigation to confirm the role of these genes

The genetic basis of size in pet dogs: The study of quantitative genetic variation in an undergraduate laboratory practical.

The teaching of quantitative genetic variation in the undergraduate laboratory practical environment can be difficult as, for quantitative phenotypes that are under the control of multiple loci, detection of phenotypic differences caused by individual variants is problematical without large samples, impractical in such classes. Pet dogs provide a clear example of quantitative genetic variation with individual breeds ranging in size from 1 to 70 kg weight yet with little intrabreed variability. In contrast to humans where there are few identified genetic variants known to be involved in the genetically controlled size phenotype, in dogs, seven single nucleotide polymorphisms (SNPs) in six genes have been demonstrated to explain half of the phenotypic variance. In the practical described here, a single G-A SNP (within intron 2 of the insulin-like growth factor 1 gene) is studied through PCR, sequencing, and bioinformatics. Average breed weight of dogs of different genotypes at this SNP show significant differences in size (median [IQR] of AA = 10 kg [6-15 kg], AG = 23.75 kg [14-30 kg], GG = 30 kg [24.5-37 kg] from our class data) with an estimate of just ≈N = 16 dogs needing to be genotyped to demonstrate a significant difference in size between dogs harboring the two homozygous genotypes. In the practical described herein, from a single laboratory and a single computer session, students are able to see the clear effect of genotype on a quantitative trait. Examination of the variant in the Ensembl browser (www.ensembl.org) allows students to understand the genomic basis of this variant and appreciate the wealth of data and information publicly available in genome browsers. © 2018 International Union of Biochemistry and Molecular Biology, 46(6):623-629, 2018

Candidate-gene based GWAS identifies reproducible DNA markers for metabolic pyrethroid resistance from standing genetic variation in East African Anopheles gambiae.

Metabolic resistance to pyrethroid insecticides is widespread in Anopheles mosquitoes and is a major threat to malaria control. DNA markers would aid predictive monitoring of resistance, but few mutations have been discovered outside of insecticide-targeted genes. Isofemale family pools from a wild Ugandan Anopheles gambiae population, from an area where operational pyrethroid failure is suspected, were genotyped using a candidate-gene enriched SNP array. Resistance-associated SNPs were detected in three genes from detoxification superfamilies, in addition to the insecticide target site (the Voltage Gated Sodium Channel gene, Vgsc). The putative associations were confirmed for two of the marker SNPs, in the P450 Cyp4j5 and the esterase Coeae1d by reproducible association with pyrethroid resistance in multiple field collections from Uganda and Kenya, and together with the Vgsc-1014S (kdr) mutation these SNPs explained around 20% of variation in resistance. Moreover, the >20 Mb 2La inversion also showed evidence of association with resistance as did environmental humidity. Sequencing of Cyp4j5 and Coeae1d detected no resistance-linked loss of diversity, suggesting selection from standing variation. Our study provides novel, regionally-validated DNA assays for resistance to the most important insecticide class, and establishes both 2La karyotype variation and humidity as common factors impacting the resistance phenotype