Attack of the dark clones the genetics of reproductive and color traits of South African honey bees (Apis mellifera spp.).

The traits of two subspecies of western honey bees, Apis mellifera scutellata and A.m. capensis, endemic to the Republic of South Africa (RSA), are of biological and commercial relevance. Nevertheless, the genetic basis of important phenotypes found in these subspecies remains poorly understood. We performed a genome wide association study on three traits of biological relevance in 234 A.m. capensis, 73 A.m. scutellata and 158 hybrid individuals. Thirteen markers were significantly associated to at least one trait (P ≤ 4.28 × 10-6): one for ovariole number, four for scutellar plate and eight for tergite color. We discovered two possible causative variants associated to the respective phenotypes: a deletion in GB46429 or Ebony (NC_007070.3:g.14101325G>del) (R69Efs*85) and a nonsense on GB54634 (NC_007076.3:g.4492792A>G;p.Tyr128*) causing a premature stop, substantially shortening the predicted protein. The mutant genotypes are significantly associated to phenotypes in A.m. capensis. Loss-of-function of Ebony can cause accumulation of circulating dopamine, and increased dopamine levels correlate to ovary development in queenless workers and pheromone production. Allelic association (P = 1.824 x 10-5) of NC_007076.3:g.4492792A>G;p.Tyr128* to ovariole number warrants further investigation into function and expression of the GB54634 gene. Our results highlight genetic components of relevant production/conservation behavioral phenotypes in honey bees.

The complete mitochondrial genome of Apis mellifera jemenitica (Insecta: Hymenoptera: Apidae), the Arabian honey bee.

The mitochondrial genome of a worker Apis mellifera jemenitica was 16,623 bp. It consisted of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs and a control region. Phylogenetic analyses suggest a close relationship between A. m. jemenitica, A. m. lamarckii and A. m. syriaca.

The mitochondrial genome of the Maltese honey bee, Apis mellifera ruttneri (Insecta: Hymenoptera: Apidae).

The mitochondrial genome of Apis mellifera ruttneri consisted of 13 protein-coding genes, two rRNAs, 22 tRNAs, an AT-rich control region, and was 16,577 bp long. The phylogenetic analyses suggested that A. m. ruttneri was closely related to two North African subspecies: A. m. sahariensis and A. m. intermissa.

An overview on the nomenclatural and phylogenetic problems of native Asian brine shrimps of the genus Artemia Leach, 1819 (Crustacea, Anostraca).

The genus Artemia Leach, 1819 is a cosmopolitan halophilic crustacean, consisting of bisexual species and obligate parthenogenetic populations. Asia is rich in Artemia biodiversity. More than 530 Artemia sites have been recorded from this area and more than 20 species/subspecies/variety names have been used for them. There exist various problems in the nomenclature, identification, and phylogenetic status of Artemia native to Asia, which are discussed in this paper.

Genetic diversity and population structure of two subspecies of western honey bees (Apis mellifera L.) in the Republic of South Africa as revealed by microsatellite genotyping.

Apis mellifera scutellata and Apis mellifera capensis, two native subspecies of western honey bees in the Republic of South Africa (RSA), are important to beekeepers in their native region because beekeepers use these bees for honey production and pollination purposes. Additionally, both bees are important invasive pests outside of their native ranges. Recently, whole mitogenome sequencing and single nucleotide polymorphisms were used to study their genetic diversity. To add to our knowledge of the molecular ecology of both bees, we tested the ability of microsatellites to be used as a tool to discriminate between A.m. capensis and A.m. scutellata. We analyzed the genetic variability and overall population structure of both bee subspecies and hybrids of the two by genotyping individuals collected from RSA (N = 813 bees from 75 apiaries) at 19 microsatellite DNA loci. Overall, populations averaged between 9.2 and 11.3 alleles per locus, with unbiased heterozygosity values ranging from 0.81 to 0.86 per population. Bayesian clustering analyses revealed two distinct evolutionary units, though the results did not match those of earlier morphometric and molecular analyses. This suggests that the microsatellites we tested were not sufficient for subspecies identification purposes, especially for Cape and hybrid bees. Nevertheless, the microsatellite data highlight the considerable genetic diversity within both populations and a larger-than-expected hybridization zone between the natural distributions of A.m. capensis and A.m. scutellata.

The impact of one-decade ecological disturbance on genetic changes: a study on the brine shrimp Artemia urmiana from Urmia Lake, Iran.

Urmia Lake, the largest natural habitat of the brine shrimp Artemia urmiana, has progressively desiccated over the last two decades, resulting in a loss of 80% of its surface area and producing thousands of hectares of arid salty land. This ecological crisis has seriously affected the lake's native biodiversity. Artemia urmiana has lost more than 90% of its population during the decade from 1994 (rainy period) to 2004 (drought period) due to salinity increasing to saturation levels (∼300 g/l). We studied the influence of this ecological crisis on the genetic diversity of A. urmiana in Urmia Lake, based on one cyst collections in 1994 and 2004. AMOVA analysis on ISSR data demonstrated a 21% genetic variation and there was a 5.5% reduction of polymorphic loci between samples. PCoA showed that 77.42% and 68.75% of specimens clustered separately in 1994 and 2004, respectively. Our analyses of four marker genes revealed different genetic diversity patterns with a decrease of diversity at ITS1 and an increase for Na+/K+ ATPase. There was no notable difference in genetic variation detected for COI and 16S genes between the two periods. However, they represented distinctly different haplotypes. ITS1 and COI followed a population expansion model, whereas Na+/K+ ATPase and 16S were under demographic equilibrium without selective pressure in the 1994 samples. Neutrality tests confirmed the excess of rare historical and recent mutations present in COI and ITS1 in both samples. It is evident that a short-term ecological disturbance has impacted the genetic diversity and structure of A. urmiana.

Development of an in vitro diagnostic method to determine the genotypic sex of Xenopus laevis.

A genotypic sex determination assay provides accurate gender information of individuals with well-developed phenotypic characters as well as those with poorly developed or absent of phenotypic characters. Determination of genetic sex for Xenopus laevis can be used to validate the outcomes of Tier 2 amphibian assays, and is a requirement for conducting the larval amphibian growth and development assay (LAGDA), in the endocrine disruptor screening program (EDSP), test guidelines. The assay we developed uses a dual-labeled TaqMan probe-based real-time polymerase chain reaction (real-time PCR) method to determine the genotypic sex. The reliability of the assay was tested on 37 adult specimens of X. laevis collected from in-house cultures in Eurofins EAG Agroscience, Easton. The newly designed X. laevis-specific primer pair and probe targets the DM domain gene linked-chromosome W as a master female-determining gene. Accuracy of the molecular method was assessed by comparing with phenotypic sex, determined by necropsy and histological examination of gonads for all examined specimens. Genotypic sex assignments were strongly concordant with observed phenotypic sex, confirming that the 19 specimens were male and 18 were female. The results indicate that the TaqMan® assay could be practically used to determine the genetic sex of animals with poorly developed or no phenotypic sex characteristics with 100% precision. Therefore, the TaqMan® assay is confirmed as an efficient and feasible method, providing a diagnostic molecular sex determination approach to be used in the amphibian endocrine disrupting screening programs conducted by regulatory industries. The strength of an EDSP is dependent on a reliable method to determine genetic sex in order to identify reversals of phenotypic sex in animals exposed to endocrine active compounds.

The complete mitochondrial genome of Apis mellifera unicolor (Insecta: Hymenoptera: Apidae), the Malagasy honey bee.

The complete mitochondrial genome of the endemic Malagasy honey bee Apis mellifera unicolor is 16,373 bp and comprises 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. The mitochondrial genome closely resembles mitogenomes of other published Apis mellifera subspecies, and the phylogenetic analysis suggests that A. m. unicolor is distinct from other African (A) lineage honey bees but is most closely related to the honey bees from southern African: A. m. scutellata and A. m. capensis.

The mitochondrial genome of the Carniolan honey bee, Apis mellifera carnica (Insecta: Hymenoptera: Apidae).

Sequencing the mitochondrial genome of the Carniolan honey bee, Apis mellifera carnica, revealed 16,358 bp, consisting of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a control region. Phylogenetic analysis supported a close relationship to another south-eastern European (C-lineage) honey bee, A. m. ligustica.

The mitochondrial genome of Apis mellifera simensis (Hymenoptera: Apidae), an Ethiopian honey bee.

The complete mitochondrial genome of Apis mellifera simensis was 16,523 bp long. The 13 protein-coding genes, two rRNAs, and 22 tRNAs resembled other Apis mitogenomes. The location of this Apis subspecies in our phylogenetic tree supported the hypothesis that this subspecies is distinct, and is most closely related to A. m. scutellata and A. m. monticola.

The complete mitochondrial genome of the West African honey bee Apis mellifera adansonii (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome of the West African honey bee Apis mellifera adansonii consisted of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. It was 16,466 bp and consisted of 84.7% AT nucleotides. This subspecies had a similar mitogenome to those of other southern African honey bees, namely A. m. scutellata, A. m. capensis, and A. m. monticola.

The mitochondrial genome of the Spanish honey bee, Apis mellifera iberiensis (Insecta: Hymenoptera: Apidae), from Portugal.

The Spanish honey bee Apis mellifera iberiensis, had a mitochondrial genome of 16,560 bp. It consisted of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and an AT-rich control region. The sample was from Portugal and its mitogenome resembled those of the African (A)-lineage honey bee subspecies. It was most closely related to other North African honey bees, namely A. m. sahariensis and A. m. intermissa.

Population genomics and morphometric assignment of western honey bees (Apis mellifera L.) in the Republic of South Africa.

BACKGROUNDS: Apis mellifera scutellata and A.m. capensis (the Cape honey bee) are western honey bee subspecies indigenous to the Republic of South Africa (RSA). Both bees are important for biological and economic reasons. First, A.m. scutellata is the invasive "African honey bee" of the Americas and exhibits a number of traits that beekeepers consider undesirable. They swarm excessively, are prone to absconding (vacating the nest entirely), usurp other honey bee colonies, and exhibit heightened defensiveness. Second, Cape honey bees are socially parasitic bees; the workers can reproduce thelytokously. Both bees are indistinguishable visually. Therefore, we employed Genotyping-by-Sequencing (GBS), wing geometry and standard morphometric approaches to assess the genetic diversity and population structure of these bees to search for diagnostic markers that can be employed to distinguish between the two subspecies. RESULTS: Apis mellifera scutellata possessed the highest mean number of polymorphic SNPs (among 2449 informative SNPs) with minor allele frequencies > 0.05 (Np = 88%). The RSA honey bees generated a high level of expected heterozygosity (Hexp = 0.24). The mean genetic differentiation (FST; 6.5%) among the RSA honey bees revealed that approximately 93% of the genetic variation was accounted for within individuals of these subspecies. Two genetically distinct clusters (K = 2) corresponding to both subspecies were detected by Model-based Bayesian clustering and supported by Principal Coordinates Analysis (PCoA) inferences. Selected highly divergent loci (n = 83) further reinforced a distinctive clustering of two subspecies across geographical origins, accounting for approximately 83% of the total variation in the PCoA plot. The significant correlation of allele frequencies at divergent loci with environmental variables suggested that these populations are adapted to local conditions. Only 17 of 48 wing geometry and standard morphometric parameters were useful for clustering A.m. capensis, A.m. scutellata, and hybrid individuals. CONCLUSIONS: We produced a minimal set of 83 SNP loci and 17 wing geometry and standard morphometric parameters useful for identifying the two RSA honey bee subspecies by genotype and phenotype. We found that genes involved in neurology/behavior and development/growth are the most prominent heritable traits evolved in the functional evolution of honey bee populations in RSA. These findings provide a starting point for understanding the functional basis of morphological differentiations and ecological adaptations of the two honey bee subspecies in RSA.

Mitochondrial genome diversity and population structure of two western honey bee subspecies in the Republic of South Africa.

Apis mellifera capensis Eschscholtz and A.m. scutellata Lepeletier are subspecies of western honey bees that are indigenous to the Republic of South Africa (RSA). Both subspecies have invasive potential and are organisms of concern for areas outside their native range, though they are important bees to beekeepers, agriculture, and the environment where they are native. The aim of the present study was to examine genetic differentiation among these subspecies and estimate their phylogenetic relationships using complete mitochondrial genomes sequences. We used 25 individuals that were either assigned to one of the subspecies or designated hybrids using morphometric analyses. Phylogenetic analyses of mitogenome sequences by maximum likelihood (ML) and Bayesian inference identified a monophyletic RSA clade, subdivided into two clades. A haplotype network was consistent with the phylogenetic trees. However, members of both subspecies occurred in both clades, indicating that A.m. capensis and A.m. scutellata are neither reciprocally monophyletic nor do they exhibit paraphyly with one subspecies nested within the other subspecies. Furthermore, no mitogenomic features were diagnostic to either subspecies. All bees analyzed from the RSA expressed a substantial level of haplotype diversity (most samples had unique haplotypes) but limited nucleotide diversity. The number of variable codons across protein-coding genes (PCGs) differed among loci, with CO3 exhibiting the most variation and ATP6 the least.

The complete mitochondrial genome and phylogenetic placement of Apis nigrocincta Smith (Insecta: Hymenoptera: Apidae), an Asian, cavity-nesting honey bee.

The complete mitochondrial genome of Apis nigrocincta was sequenced. The mitochondrial genome is a circular molecule of 15,855 bp, including 37 classical eukaryotic mitochondrial regions and an A + T-rich region. Gene directions and arrangements are similar to those of other Apis mitogenomes. Most genes initiated with ATT, though ATG and ATA were also used as start codons. Twelve of 13 protein-coding genes terminated with TAA, though ND2 terminated with TAG. Four PCG genes, eight tRNAs and both rRNAs were encoded on the heavy strand while all others were encoded on the light strand (9 PCGs and 14 tRNAs). Overall, the GC content composed 15.6% of the mitogenome. All of the 22 tRNA genes, ranging from 66 to 114 bp, have a typical cloverleaf structure. A phylogenetic tree showed that A. nigrocincta clustered closest to A. cerana. The complete mitogenome of A. nigrocincta provides essential information on the biogeography and evolution of this Asian honey bee species.

The complete mitochondrial genome of Apis mellifera meda (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome of the western honey bee subspecies Apis mellifera meda was sequenced. This mitochondrial genome is 16,248 bp in length, with 37 classical eukaryotic mitochondrial genes and an A + T-rich region. Gene direction and arrangement are similar to those of other Apis mitogenomes. All genes initiate with ATT (six genes), ATG (four genes), ATA (two genes), and ATC (one gene) start codons and terminate with a TAA stop codon. Four genes are encoded on the heavy and nine on the light strands, respectively. All of the 22 tRNA genes, ranging from 66 to 78 bp, have a typical cloverleaf structure. The complete mitogenome of A.m. meda provides information on the biogeography and evolution of A. mellifera subspecies.

The complete mitochondrial genome of the Egyptian honey bee, Apis mellifera lamarckii (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome of the western honey bee subspecies Apis mellifera lamarckii was sequenced. This mitochondrial genome is 16,589 bp in length with 37 classical eukaryotic mitochondrial genes and an A + T-rich region. Gene directions and arrangements are similar to those of other Apis mitogenomes. Seven genes begin with ATT, four with ATG, and two with ATA (none with ATC) and all genes terminate with TAA. Four genes are encoded on the heavy strand and nine are encoded on light strand. All of the 22 tRNA genes, ranging from 66 to 80 bp, have a typical cloverleaf structure. A phylogenetic tree showed that A.m. lamarckii clusters with other A. mellifera subspecies, as expected.

The complete mitochondrial genome of Apis nuluensis Tingek, an Asian honey bee (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome of Apis nuluensis Tingek was sequenced. The mitochondrial genome was 15,843 bp in length, with 37 classical eukaryotic mitochondrial genes and an A + T-rich region. Gene directions and arrangements were similar to those of other Apis mitogenomes. Most genes initiate with ATT (though ATG and ATC also were used) and all genes terminated with TAA. Nine genes were encoded on the light strand while four were encoded on the heavy strand. All 22 tRNA genes have a typical cloverleaf structure. The most likely phylogenetic tree showed A. nuluensis clustering with A. cerana. The complete mitogenome of A. nuluensis completes the sequencing of all mitogenomes of the currently accepted species of Apis.

The complete mitochondrial genome of an east African honey bee, Apis mellifera monticola Smith (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome of Apis mellifera monticola was sequenced and annotated. The genome is 16,343 bp in length and encodes all 37 mitochondrial genes with an A + T content of 84.8%. Gene directions and arrangements are identical to those of other sequenced mitogenomes in Apis. Most genes initiated with ATT, though ATG, ATA, and ATC also were used as start codons. All genes terminated with TAA. Four PCG genes, eight tRNAs and both rRNAs are encoded on the heavy strand while all others are coded on the light strand (nine PCGs and 14 tRNAs). Overall, the GC content composed 15.2% of the mitogenome. All of the 22 tRNA genes, ranging from 63 to 78 bp, have a typical cloverleaf structure. A phylogenetic tree showed that A.m. monticola clusters with other African subspecies.

A note on the biogeographical origin of the brine shrimp Artemia urmiana Günther, 1899 from Urmia Lake, Iran.

The brine shrimp Artemia urmiana, an abundant inhabitant of the hypersaline Urmia Lake in northwestern Iran, has recently been described from Lake Koyashskoe, also a shallow hypersaline lake that is located on the Black Sea coast of the Crimean Peninsula (Ukraine). This discovery has questioned the endemicity of A. urmiana in Urmia Lake and has also brought into question the biogeographical origin of this species. In the present study, we combined recent genetic divergence data (mtDNA-COI) with palaeoecological evidence to address the biogeographical origin of A. urmiana. Calibration of the molecular clock of the COI region was set by assigning the age of the micro-crustacean Daphnia pulex minimally at 145 Mya. The divergence age of A. urmiana in Urmia Lake dates back to 383,000 years, whereas Ukrainian Artemia reflects a very young populations that diverged about 196,000 years ago. Palaeoecological evidence suggests that the age of the major habitat of A. urmiana i.e. Urmia Lake goes back to the Tertiary Period while the Ukranian habitats of the species are very young, by virtue of geological features of the Holocene age. We conclude that the biogeographical origin of A. urmiana is outside of Europe and the current state of knowledge strongly suggests that Urmia Lake has been the major source of its expansion into its modern habitats in Europe.