A first annotated genome sequence for Haliotis midae with genomic insights into abalone evolution and traits of economic importance.

Haliotis midae or "perlemoen" is one of five abalone species endemic to South Africa, and being palatable, the only commercially important abalone species with a high international demand. The higher demand for this abalone species has resulted in the decrease of natural stocks due to overexploitation by capture fisheries and poaching. Facilitating aquaculture production of H. midae should assist in minimising the pressure on the wild populations. Here, the draft genome of H. midae has been sequenced, assembled, and annotated. The draft assembly resulted in a total length of 1.5 Gb, contig N50 of 0.238 Mb, scaffold N50 of 0. 238 Mb and GC level of 40%. Gene annotation, combining ab initio and evidence-based pipelines identified 52,280 genes with protein coding potential. The genes identified were used to predict orthologous genes shared among the four other abalone species (H. laevigata, H. rubra, H. discus hannai and H. rufescens) and 4702 orthologous genes were shared across the five species. Among the orthologous genes in abalones, single copy genes were further analysed for signatures of selection and several molecular regulatory proteins involved in developmental functions were found to be under positive selection in specific abalone lineages. Furthermore, whole genome SNP-based phylogenomic assessment was performed to confirm the evolutionary relationship among the considered abalone species with draft genomes, reaffirming that H. midae is closely related to the Australian Greenlip (H. laevigata) and Blacklip (H. rubra). The study assists in the understanding of genes related to various biological systems underscoring the evolution and development of abalones, with potential applications for genetic improvement of commercial stocks.

Gene expression differentials driven by mass rearing and artificial selection in black soldier fly colonies.

The micro-evolutionary forces that shape genetic diversity during domestication have been assessed in many plant and animal systems. However, the impact of these processes on gene expression, and consequent functional adaptation to artificial environments, remains under-investigated. In this study, whole-transcriptome dynamics associated with the early stages of domestication of the black soldier fly (BSF), Hermetia illucens, were assessed. Differential gene expression (DGE) was evaluated in relation to (i) generational time within the cultured environment (F2 vs. F3), and (ii) two selection strategies [no artificial selective pressure (NS); and selection for greater larval mass (SEL)]. RNA-seq was conducted on 5th instar BSF larvae (n = 36), representing equal proportions of the NS (F2 = 9; F3 = 9) and SEL (F2 = 9; F3 = 9) groups. A multidimensional scaling plot revealed greater gene expression variability within the NS and F2 subgroups, while the SEL group clustered separately with lower levels of variation. Comparisons between generations revealed 898 differentially expressed genes (DEGs; FDR-corrected p < 0.05), while between selection strategies, 213 DEGs were observed (FDR-corrected p < 0.05). Enrichment analyses revealed that metabolic, developmental, and defence response processes were over-expressed in the comparison between F2 and F3 larvae, while metabolic processes were the main differentiating factor between NS and SEL lines. This illustrates the functional adaptations that occur in BSF colonies across generations due to mass rearing; as well as highlighting genic dynamics associated with artificial selection for production traits that might inform future selective breeding strategies.

Using SPAdes, AUGUSTUS, and BLAST in an Automated Pipeline for Clustering Homologous Exome Sequences.

Cross-species exome sequencing approaches provide unprecedented avenues for obtaining genetic diversity, evolutionary relationships, and functional information from a variety of organisms including non-model species. These approaches offer cost-effective opportunities to study multiple individuals or species in parallel, but also create bioinformatics challenges in the application of multiple but powerful bioinformatics tools for the identification of homologous gene families across individual or species boundaries. Popular tools of this kind include SPAdes for sequence assembly, AUGUSTUS for ab initio gene prediction, and BLAST for building homologous sequence families. These tools can also be sophisticated in terms of installation and usage. Here, we present detailed steps on how to run these tools for the recovery and clustering of exon sequences from cross-species raw exome-capture data into homologous sequence families. We also present a utility pipeline, CODSEQCP, that automates these steps to cluster exon sequences, facilitating population genomics and evolutionary studies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Reads assembly using SPAdes Basic Protocol 2: Coding sequence extraction using AUGUSTUS Basic Protocol 3: Sequence clustering using BLAST Alternate Protocol: How to run CODSEQCP.

Feed and Host Genetics Drive Microbiome Diversity with Resultant Consequences for Production Traits in Mass-Reared Black Soldier Fly (Hermetia illucens) Larvae.

Mass rearing the black soldier fly, Hermetia illucens, for waste bioremediation and valorisation is gaining traction on a global scale. While the health and productivity of this species are underpinned by associations with microbial taxa, little is known about the factors that govern gut microbiome assembly, function, and contributions towards host phenotypic development in actively feeding larvae. In the present study, a 16S rDNA gene sequencing approach applied to a study system incorporating both feed substrate and genetic variation is used to address this knowledge gap. It is determined that the alpha diversity of larval gut bacterial communities is driven primarily by features of the larval feed substrate, including the diversity of exogenous bacterial populations. Microbiome beta diversity, however, demonstrated patterns of differentiation consistent with an influence of diet, larval genetic background, and a potential interaction between these factors. Moreover, evidence for an association between microbiome structure and the rate of larval fat accumulation was uncovered. Taxonomic enrichment analysis and clustering of putative functional gut profiles further suggested that feed-dependent turnover in microbiome communities is most likely to impact larval characteristics. Taken together, these findings indicate that host-microbiome interactions in this species are complex yet relevant to larval trait emergence.

Patterns of Genetic Diversity and Mating Systems in a Mass-Reared Black Soldier Fly Colony.

The black soldier fly (BSF), Hermetia illucens, is a promising candidate for the emerging insect farming industry with favourable characteristics for both bioremediation and production of animal delivered nutritive and industrial compounds. The genetic management of commercial colonies will become increasingly important for the sustainability of the industry. However, r-selected life history traits of insects pose challenges to conventional animal husbandry and breeding approaches. In this study, the long-term genetic effects of mass-rearing were evaluated as well as mating systems in the species to establish factors that might influence genetic diversity, and by implication fitness and productivity in commercial colonies. Population genetic parameters, based on microsatellite markers, were estimated and compared amongst two temporal wild sampling populations and four generations (F28, F48, F52, and F62) of a mass-reared colony. Furthermore, genetic relationships amongst mate pairs were evaluated and parentage analysis was performed to determine the oc-currence of preferential mate choice and multiple paternity. The mass-reared colony showed a reduction in genetic diversity and evidence for inbreeding with significant successive generational genetic differentiation from the wild progenitor population. Population-level analysis also gave the first tentative evidence of positive assortative mating and genetic polyandry in BSF. The homoge-neity of the mass-reared colony seems to result from a dual action caused by small effective popu-lation size and increased homozygosity due to positive assortative mating. However, the high ge-netic diversity in the wild and a polyandrous mating system might suggest the possible restoration of diversity in mass-reared colonies through augmentation with the wild population.

Global population genetic structure and demographic trajectories of the black soldier fly, Hermetia illucens.

BACKGROUND: The black soldier fly (Hermetia illucens) is the most promising insect candidate for nutrient-recycling through bioconversion of organic waste into biomass, thereby improving sustainability of protein supplies for animal feed and facilitating transition to a circular economy. Contrary to conventional livestock, genetic resources of farmed insects remain poorly characterised. We present the first comprehensive population genetic characterisation of H. illucens. Based on 15 novel microsatellite markers, we genotyped and analysed 2862 individuals from 150 wild and captive populations originating from 57 countries on seven subcontinents. RESULTS: We identified 16 well-distinguished genetic clusters indicating substantial global population structure. The data revealed genetic hotspots in central South America and successive northwards range expansions within the indigenous ranges of the Americas. Colonisations and naturalisations of largely unique genetic profiles occurred on all non-native continents, either preceded by demographically independent founder events from various single sources or involving admixture scenarios. A decisive primarily admixed Polynesian bridgehead population serially colonised the entire Australasian region and its secondarily admixed descendants successively mediated invasions into Africa and Europe. Conversely, captive populations from several continents traced back to a single North American origin and exhibit considerably reduced genetic diversity, although some farmed strains carry distinct genetic signatures. We highlight genetic footprints characteristic of progressing domestication due to increasing socio-economic importance of H. illucens, and ongoing introgression between domesticated strains globally traded for large-scale farming and wild populations in some regions. CONCLUSIONS: We document the dynamic population genetic history of a cosmopolitan dipteran of South American origin shaped by striking geographic patterns. These reflect both ancient dispersal routes, and stochastic and heterogeneous anthropogenic introductions during the last century leading to pronounced diversification of worldwide structure of H. illucens. Upon the recent advent of its agronomic commercialisation, however, current human-mediated translocations of the black soldier fly largely involve genetically highly uniform domesticated strains, which meanwhile threaten the genetic integrity of differentiated unique local resources through introgression. Our in-depth reconstruction of the contemporary and historical demographic trajectories of H. illucens emphasises benchmarking potential for applied future research on this emerging model of the prospering insect-livestock sector.

New Mitochondrial Gene Rearrangement in Psyttalia concolor, P. humilis and P. lounsburyi (Hymenoptera: Braconidae), Three Parasitoid Species of Economic Interest.

The family Braconidae consists mostly of specialized parasitoids, some of which hold potential in biocontrol of agricultural pests. Psyttalia concolor, Psyttalia humilis and Psyttalia lounsburyi are parasitoids associated with Bactrocera oleae, a major pest of cultivated olives. The native range of Psyttalia concolor is the Mediterranean, and P. humilis and P. lounsburyi are native to sub-Saharan Africa. This study reports the mitochondrial genomes of the three species, thus laying the foundation for mitogenomic analyses in the genus Psyttalia. Comparative mitogenomics within Braconidae showed a novel gene arrangement in Psyttalia in involving translocation and inversion of transfer RNA genes. The placement of Psyttalia in the subfamily Opiinae was well-supported, and the divergence between Psyttalia and its closest relative (Diachasmimorpha longicaudata) was at ~55 MYA [95% highest posterior density (HPD): 34-83 MYA]. Psyttalia lounsburyi occupied the most basal position among the three Psyttalia, having diverged from the other two species ~11 MYA (95% HPD: 6-17 MYA). Psyttalia concolor and P. humilis were recovered as sister species diverged at ~2 MYA (95% HPD: 1.1-3.6 MYA). This phylogeny combining new sequences and a set of 31 other cyclostomes and non-cyclostomes highlights the importance of a comprehensive taxonomic coverage of Braconidae mitogenomes to overcome the lack of robustness in the placement of several subfamilies.

The development of genome-wide single nucleotide polymorphisms in blue wildebeest using the DArTseq platform.

Blue wildebeest (Connochaetes taurinus taurinus) are economically important antelope that are widely utilised in the South African wildlife industry. However, very few genomic resources are available for blue wildebeest that can assist in breeding management and facilitate research. This study aimed to develop a set of genome-wide single nucleotide polymorphism (SNP) markers for blue wildebeest. The DArTseq genotyping platform, commonly used in polyploid plant species, was selected for SNP discovery. A limited number of published articles have described the use of the DArTseq platform in animals and, therefore, this study also provided a unique opportunity to assess the performance of the DArTseq platform in an animal species. A total of 20,563 SNPs, each located within a 69 bp sequence, were generated. The developed SNP markers had a high average scoring reproducibility (>99%) and a low percentage missing data (~9.21%) compared to other reduced representation sequencing approaches that have been used in animal studies. Furthermore, the number of candidate SNPs per nucleotide position decreased towards the 3' end of sequence reads, and the ratio of transitions (Ts) to transversions (Tv) remained similar for each read position. These observations indicate that there was no read position bias, such as the identification of false SNPs due to low sequencing quality, towards the tail-end of sequencing reads. The DArTseq platform was also successful in identifying a large number of informative SNPs with desirable polymorphism parameters such as a high minor allele frequency (MAF). The Bos taurus genome was used for the in silico mapping of the marker sequences and a total of 6020 (29.28%) sequences were successfully mapped against the bovine genome. The marker sequences mapped to all of the bovine chromosomes establishing the genome-wide distribution of the SNPs. Moreover, the high observed Ts:Tv ratio (2.84:1) indicate that the DArTseq platform targeted gene-rich regions of the blue wildebeest genome. Finally, functional annotation of the marker sequences revealed a wide range of different putative functions indicating that these SNP markers can be useful in functional gene studies. The DArTseq platform, therefore, represents a high-throughput, robust and cost-effective genotyping platform, which may find adoption in several other African antelope and animal species.

Phylogeography, genetic diversity, and population structure of Nile crocodile populations at the fringes of the southern African distribution.

Nile crocodiles are apex predators widely distributed in sub-Saharan Africa that have been viewed and managed as a single species. A complex picture of broad and fine-scale phylogeographic patterns that includes the recognition of two species (Crocodylus niloticus and Crocodylus suchus), and the structuring of populations according to river basins has started to emerge. However, previous studies surveyed a limited number of samples and geographical regions, and large areas of the continent remained unstudied. This work aimed at a fine scale portrait of Nile crocodile populations at the fringes of their geographic distribution in southern Africa. Wild and captive individuals were sampled across four major river systems (Okavango, Lower Kunene, Lower Shire and Limpopo) and the KwaZulu-Natal region. A multi-marker approach was used to infer phylogeographic and genetic diversity patterns, including new and public mitochondrial data, and a panel of 11 nuclear microsatellites. All individuals belonged to a phylogenetic clade previously associated with the C. niloticus species, thus suggesting the absence of C. suchus in southern Africa. The distribution of mitochondrial haplotypes indicated ancestral genetic connectivity across large areas, with loss of diversity along the north-south axis. Genetic variation partitioned the populations primarily into western and eastern regions of southern Africa, and secondarily into the major river systems. Populations were partitioned into five main groups corresponding to the Lower Kunene, the Okavango, the Lower Shire, and the Limpopo rivers, and the KwaZulu-Natal coastal region. All groups show evidence of recent bottlenecks and small effective population sizes. Long-term genetic diversity is likely to be compromised, raising conservation concern. These results emphasize the need for local genetic assessment of wild populations of Nile crocodiles to inform strategies for management of the species in southern Africa.

The complete mitochondrial genome of Bactrocera biguttula (Bezzi) (Diptera: Tephritidae) and phylogenetic relationships with other Dacini.

Bactrocera biguttula is an African olive fruit fly that does not attack cultivated olives but rather develops in the fruits of wild species of Olea and Noronhia. The complete mitochondrial genome of an individual specimen was characterized in comparison to other Bactrocera. The phylogenetic relationships of B. biguttula with other Dacini were investigated, with special focus on B. oleae, an agricultural pest known to attack cultivated and wild olives. The sequence had a total length of 15,829 bp, and included the typical features of insect mitogenomes, similarly to the other Bactrocera analysed. Start codons included ATG, ATC, ATT, and TCG (in COI). The majority of stop codons (TAA) were fully encoded, whereas in some cases only TA or T were present. The complete sequence was biased towards A + T, with a positive AT-skew and a negative GC-skew. The predicted cloverleaf structure of tRNASer1 showed absence of the DHU arm, a common feature in insects and other Metazoans. Phylogenetic reconstruction showed that B. biguttula and B. oleae are sister species, having diverged from a common ancestor < 10 Myr ago. This result warrants future genomic comparisons between these two closely related species for investigating the specific adaptations to the different hosts.

Barcoding of parasitoid wasps (Braconidae and Chalcidoidea) associated with wild and cultivated olives in the Western Cape of South Africa 1.

Wild and cultivated olives harbor and share a diversity of insects, some of which are considered agricultural pests, such as the olive fruit fly. The assemblage of olive-associated parasitoids and seed wasps is rich and specialized in sub-Saharan Africa, with native species possibly coevolving with their hosts. Although historical entomological surveys reported on the diversity of olive wasp species in the Western Cape Province of South Africa, no comprehensive study has been performed in the region in the molecular era. In this study, a dual approach combining morphological and DNA-based methods was used for the identification of adult specimens reared from olive fruits. Four species of Braconidae and six species of Chalcidoidea were identified, and DNA barcoding methodologies were used to investigate conspecificity among individuals, based on randomly selected representative specimens. Morphological identifications were congruent with DNA data, as NJ and ML trees correctly placed the sequences for each species either at the genus or species level, depending on the available taxa coverage, and genetic distances strongly supported conspecificity. No clear evidence of cryptic diversity was found. Overall seed infestation and parasitism rates were higher in wild olives compared to cultivated olives, and highest for Eupelmus spermophilus and Utetes africanus. These results can be used for early DNA-based detection of wasp larvae in olives and to further investigate the biology and ecology of these species.

The complete mitochondrial genome and phylogenetic position of the leopard catshark, Poroderma pantherinum.

We present the first mitochondrial genome of a South African endemic catshark, Poroderma pantherinum. The complete mitogenome is 16,686 bp in length, comprising 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and one non-coding control region. Similar to other shark mitogenomes, it is AT rich (61.1%), with a GC content of 38.9%. Protein-coding genes used one of two start codons (ATG and GTG) and one stop codon (TAA/TA-/T-). Phylogenetic analysis of the leopard catshark and 34 carcharhinid species showed that it clusters with two other scyliorhinid species (Cephaloscyllium umbratile and Scyliorhinus canicula) with 100% support.

Identification and characterization of miRNAs transcriptome in the South African abalone, Haliotis midae.

Aquatic animal diseases are one of the most important limitations to the growth of aquaculture. miRNAs represent an important class of small ncRNAs able to modulate host immune and stress responses. In Mollusca, a large phylum of invertebrates, miRNAs have been identified in several species. The current preliminary study identified known miRNAs from the South African abalone, Haliotis midae. The economic and ecological importance of abalone makes this species a suitable model for studying and understanding stress response in marine gastropods. Furthermore, the identification of miRNA, represents an alternative and powerful tool to combat infectious disease.

Evaluation of de novo assembly technique in the South African abalone Haliotis midae transcriptome: A comparison from Illumina and 454 systems.

Next generation sequencing platforms have recently been used to rapidly characterize transcriptome sequences from a number of non-model organisms. The present study compares two of the most frequently used platforms, the Roche 454-pyrosequencing and the Illumina sequencing-by-synthesis (SBS), on the same RNA sample obtained from an intertidal gastropod mollusc species, Haliotis midae. All the sequencing reads were deposited in the Short Read Archive (SRA) database are retrievable under the accession number [SRR071314 (Illumina Genome Analyzer II)] and [SRR1737738, SRR1737737, SRR1737735, SRR1737734 (454 GS FLX)] in the SRA database of NCBI. Three transcriptomes, composed of either pure 454 or Illumina reads or a mixture of read types (Hybrid), were assembled using CLC Genomics Workbench software. Illumina assemblies performed the best de novo transcriptome characterization in terms of contig length, whereas the 454 assemblies tended to improve the complete assembly of gene transcripts. Both the Hybrid and Illumina assemblies produced longer contigs covering more of the transcriptome than 454 assemblies. However, the addition of 454 significantly increased the number of genes annotated.

Estimates of effective population size and inbreeding in South African indigenous chicken populations: implications for the conservation of unique genetic resources.

Conservation of locally adapted indigenous livestock breeds has become an important objective in sustainable animal breeding, as these breeds represent a unique genetic resource. Therefore, the Agricultural Research Council of South Africa initiated a conservation programme for four South African indigenous chicken breeds. The evaluation and monitoring of the genetic constitution of these conservation flocks is important for proper management of the conservation programme. Using molecular genetic analyses, the effective population sizes and relatedness of these conservation flocks were compared to village (field) chicken populations from which they were derived. Genetic diversity within and between these populations are further discussed within the context of population size. The conservation flocks for the respective breeds had relatively small effective population sizes (point estimate range 38.6-78.6) in comparison to the field populations (point estimate range 118.9-580.0). Furthermore, evidence supports a transient heterozygous excess, generally associated with the occurrence of a recent population bottleneck. Genetic diversity, as measured by the number of alleles, heterozygosity and information index, was also significantly reduced in the conservation flocks. The average relatedness amongst the conservation flocks was high, whilst it remained low for the field populations. There was also significant evidence for population differentiation between field and conservation populations. F st estimates for conservation flocks were moderate to high with a maximum reached between VD_C and VD_F (0.285). However, F st estimates for field population were excessively low between the NN_C and EC_F (0.007) and between EC_F and OV_F (0.009). The significant population differentiation of the conservation flocks from their geographically correlated field populations of origin is further supported by the analysis of molecular variance (AMOVA), with 10.51 % of genetic diversity ascribed to population differences within groups (F SC = 0.106). The results suggest that significant genetic erosion has occurred within the conservation flocks due to inbreeding, pronounced effects of random drift and selection. It might be necessary to introduce new breeding individuals from the respective field populations in order to increase the effective population sizes of the conservation flocks and counter the effects of genetic erosion.

Domain repeats related to innate immunity in the South African abalone, Haliotis midae.

Molluscs predominately use the cellular defence system as the primary mechanism of defence against pathogenic infection, in which haemocytes play a pivotal role. Haliotis midae is a commercially important South African species that it is susceptible to bacterial pathogens, fungal and yeast infections in the farming environment. The current study aims to enrich the current knowledge regarding H. midae innate immunity by investigating the presence and evolution of domain repeats. The bioinformatics approach used in this study, detected five repeat families in the H. midae transcriptome. These repeats families include mixed alpha and beta (leucine-rich and ankyrin), spectrin repeats, beta-propellers (WD40) and alfa-structure repeat (TPR-like). The expansion of key gene families related to host defence may be important to abalone adaptation to life in a pathogen-rich environment.

Transcriptome profiles of wild and cultured South African abalone, Haliotis midae.

This report describes the use of pyrosequencing technologies to generate the first comparative analysis of de novo assembled transcriptome data from cultured and wild specimens of the South African abalone. The transcriptome data and database described here provide a significant genomic resource for abalone research. The data set annotated 11,240 genes, which matched genes with known functions in other species. A large number of transmembrane protein domains (4087) that may indicate a high portion of undiscovered gene receptors were identified. Further, we detected an interesting set of transcription factors (516) that are valuable candidates for participating in regulatory events in developmental (such as cell proliferation and differentiation) and reproductive processes.

Comparison of population genetic estimates amongst wild, F1 and F2 cultured abalone (Haliotis midae).

Haliotis midae is South Africa's most important aquaculture species. The reproduction cycle is currently not closed as many farms rely on wild-caught broodstock for seed production. However, there is an increasing interest in genetic improvement in commercial stocks, with a growing number of producers implementing selective breeding strategies. High throughput commercial production and mass spawning make it difficult to maintain breeding records; therefore, mostly mass selection is practised. The high fecundity and unequal parental contributions also often lead to increased levels of inbreeding. This study therefore aimed to assess the genetic effects of such breeding practices on commercial populations of H. midae. Using microsatellite loci, the genetic properties of a wild, an F1 and an F2 population were estimated and compared. Although there was no significant loss of genetic diversity amongst the cultured populations in comparison with the wild progenitor population, there was low-to-moderate genetic differentiation between populations. Relatedness amongst the F2 population was significant, and the rate of inbreeding was high. The effective population size for the F2 (±50) was also comparatively small with respect to the wild (∞) and F1 (±470) populations. These results suggest that farms need to give caution to breeding practices beyond the first (F1) generation and aim to increase effective population sizes and minimise inbreeding to ensure long-term genetic gain and productivity. This study also confirms the usefulness of population genetic analyses for commercial breeding and stock management in the absence of extensive pedigree records.

Detection of molecular signatures of selection at microsatellite loci in the South African abalone (Haliotis midae) using a population genomic approach.

Identifying genomic regions that may be under selection is important for elucidating the genetic architecture of complex phenotypes underlying adaptation to heterogeneous environments. A population genomic approach, using a classical neutrality test and various Fst-outlier detection methods was employed to evaluate genome-wide polymorphism data in order to identify loci that may be candidates for selection amongst six populations (three cultured and three wild) of the South African abalone, Haliotis midae. Approximately 9% of the genome-wide microsatellite markers were putatively subject to directional selection, whilst 6-18% of the genome is thought to be influenced by balancing selection. Genetic diversity estimates for candidate loci under directional selection was significantly reduced in comparison to candidate neutral loci, whilst candidate balancing selection loci demonstrated significantly higher levels of genetic diversity (Kruskal-Wallis test, P<0.05). Pairwise Fst estimates based on candidate directional selection loci also demonstrated increased levels of differentiation between study populations. Various candidate loci under selection showed significant inter-chromosomal linkage disequilibrium, suggesting possible gene-networks underling adaptive phenotypes. Furthermore, several loci had significant hits to known genes when performing BLAST searches to NCBI's non-redundant databases, whilst others are known to be derived from expressed sequences even though homology to a known gene could not be established. A number of loci also demonstrated relatively high similarity to transposable elements. The association of these loci to functional and genomically active sequences could in part explain the observed signatures of selection.