Diversity and evolution of the P450 family in arthropods

The P450 family (CYP genes) of arthropods encodes diverse enzymes involved in the metabolism of foreign compounds and in essential endocrine or ecophysiological functions. The P450 sequences (CYPome) from 40 arthropod species were manually curated, including 31 complete CYPomes, and a maximum likelihood phylogeny of nearly 3000 sequences is presented. Arthropod CYPomes are assembled from members of six CYP clans of variable size, the CYP2, CYP3, CYP4 and mitochondrial clans, as well as the CYP20 and CYP16 clans that are not found in Neoptera. CYPome sizes vary from two dozen genes in some parasitic species to over 200 in species as diverse as collembolans or ticks. CYPomes are comprised of few CYP families with many genes and many CYP families with few genes, and this distribution is the result of dynamic birth and death processes. Lineage-specific expansions or blooms are found throughout the phylogeny and often result in genomic clusters that appear to form a reservoir of catalytic diversity maintained as heritable units. Among the many P450s with physiological functions, six CYP families are involved in ecdysteroid metabolism. However, five so-called Halloween genes are not universally represented and do not constitute the unique pathway of ecdysteroid biosynthesis. The diversity of arthropod CYPomes has only partially been uncovered to date and many P450s with physiological functions regulating the synthesis and degradation of endogenous signal molecules (including ecdysteroids) and semiochemicals (including pheromones and defense chemicals) remain to be discovered. Sequence diversity of arthropod P450s is extreme, and P450 sequences lacking the universally conserved Cys ligand to the heme have evolved several times. A better understanding of P450 evolution is needed to discern the relative contributions of stochastic processes and adaptive processes in shaping the size and diversity of CYPomes

Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites

Short term transcriptional responses of P450s to phytochemicals in insects and mites

Cytochrome P450 monooxygenases (P450s) play a key role in the detoxification of phytochemicals in arthropod herbivores. We present here an overview of recent progress in understanding the breadth and specificity of gene expression plasticity of P450s in response to phytochemicals. We discuss experimental setups and new findings in mechanisms of P450 regulation. Whole genome transcriptomic analysis of arthropod herbivores, either after direct administration of phytochemicals or after host plant shifts, allowed to integrate various levels of chemical complexity and lead to the unbiased identification of responsive P450 genes. However, despite progress in identification of inducible P450s, the link between induction and metabolism is still largely unexplored, and to what extent the overall response is biologically functional should be further investigated. In the near future, such studies will be more straightforward as forward and reverse genetic tools become more readily available

Transcriptomic plasticity in the arthropod generalist Tetranychus urticae upon long-term acclimation to different host plants

The two-spotted spider mite Tetranychus urticae is an important pest with an exceptionally broad host plant range. This generalist rapidly acclimatizes and adapts to a new host, hereby overcoming nutritional challenges and a novel pallet of constitutive and induced plant defenses. Although recent studies reveal that a broad transcriptomic response upon host plant transfer is associated with a generalist life style in arthropod herbivores, it remains uncertain to what extent these transcriptional changes are general stress responses or host-specific. In the present study, we analyzed and compared the transcriptomic changes that occur in a single T. urticae population upon long-term transfer from Phaseolus vulgaris to a similar, but chemically defended, host (cyanogenic Phaseolus lunatus) and to multiple economically important crops (Glycine max, Gossypium hirsutum, Solanum lycopersicum and Zea mays). These long-term host plant transfers were associated with distinct transcriptomic responses with only a limited overlap in both specificity and directionality, suggestive of a fine-tuned transcriptional plasticity. Nonetheless, analysis at the gene family level uncovered overlapping functional processes, recruiting genes from both well-known and newly discovered detoxification families. Of note, our analyses highlighted a possible detoxification role for Tetranychus-specific short-chain dehydrogenases and single PLAT domain proteins, and manual genome annotation showed that both families are expanded in T. urticae. Our results shed new light on the molecular mechanisms underlying the remarkable adaptive potential for host plant use of generalist arthropods and set the stage for functional validation of important players in T. urticae detoxification of plant secondary metabolites

Significance and interpretation of molecular diagnostics for insecticide resistance management of agricultural pests

Insecticide resistant pests become increasingly difficult to control in current day agriculture. Because of environmental and health concerns, the insecticide portfolio to combat agricultural pests is gradually decreasing. It is therefore crucial to make rational decisions on insecticide use to assure effective resistance management. However, resistance monitoring programs that inform on pest susceptibility and resistance are not yet common practice in agriculture. Molecular markers of resistance that are turned into convenient diagnostic tools are urgently needed and will only increase in importance. This review investigates which factors determine the strength, diagnostic value, and success of a diagnostic marker, and in which cases recent technical advances might provide new opportunities for decision making in an operational meaningful way

Analysis of the olive fruit fly Bactrocera oleae transcriptome and phylogenetic classification of the major detoxification gene families

he olive fruit fly Bactrocera oleae has a unique ability to cope with olive flesh, and is the most destructive pest of olives worldwide. Its control has been largely based on the use of chemical insecticides, however, the selection of insecticide resistance against several insecticides has evolved. The study of detoxification mechanisms, which allow the olive fruit fly to defend against insecticides, and/or phytotoxins possibly present in the mesocarp, has been hampered by the lack of genomic information in this species. In the NCBI database less than 1,000 nucleotide sequences have been deposited, with less than 10 detoxification gene homologues in total. We used 454 pyrosequencing to produce, for the first time, a large transcriptome dataset for B. oleae. A total of 482,790 reads were assembled into 14,204 contigs. More than 60% of those contigs (8,630) were larger than 500 base pairs, and almost half of them matched with genes of the order of the Diptera. Analysis of the Gene Ontology (GO) distribution of unique contigs, suggests that, compared to other insects, the assembly is broadly representative for the B. oleae transcriptome. Furthermore, the transcriptome was found to contain 55 P450, 43 GST-, 15 CCE- and 18 ABC transporter-genes. Several of those detoxification genes, may putatively be involved in the ability of the olive fruit fly to deal with xenobiotics, such as plant phytotoxins and insecticides. In summary, our study has generated new data and genomic resources, which will substantially facilitate molecular studies in B. oleae, including elucidation of detoxification mechanisms of xenobiotic, as well as other important aspects of olive fruit fly biology

The complete mitochondrial genome of the house dust mite Dermatophagoides pteronyssinus (Trouessart): a novel gene arrangement among arthropods

<p>Abstract</p> <p>Background</p> <p>The apparent scarcity of available sequence data has greatly impeded evolutionary studies in Acari (mites and ticks). This subclass encompasses over 48,000 species and forms the largest group within the Arachnida. Although mitochondrial genomes are widely utilised for phylogenetic and population genetic studies, only 20 mitochondrial genomes of Acari have been determined, of which only one belongs to the diverse order of the Sarcoptiformes. In this study, we describe the mitochondrial genome of the European house dust mite <it>Dermatophagoides pteronyssinus</it>, the most important member of this largely neglected group.</p> <p>Results</p> <p>The mitochondrial genome of <it>D. pteronyssinus </it>is a circular DNA molecule of 14,203 bp. It contains the complete set of 37 genes (13 protein coding genes, 2 rRNA genes and 22 tRNA genes), usually present in metazoan mitochondrial genomes. The mitochondrial gene order differs considerably from that of other Acari mitochondrial genomes. Compared to the mitochondrial genome of <it>Limulus polyphemus</it>, considered as the ancestral arthropod pattern, only 11 of the 38 gene boundaries are conserved. The majority strand has a 72.6% AT-content but a GC-skew of 0.194. This skew is the reverse of that normally observed for typical animal mitochondrial genomes. A microsatellite was detected in a large non-coding region (286 bp), which probably functions as the control region. Almost all tRNA genes lack a T-arm, provoking the formation of canonical cloverleaf tRNA-structures, and both rRNA genes are considerably reduced in size. Finally, the genomic sequence was used to perform a phylogenetic study. Both maximum likelihood and Bayesian inference analysis clustered <it>D. pteronyssinus </it>with <it>Steganacarus magnus</it>, forming a sistergroup of the Trombidiformes.</p> <p>Conclusion</p> <p>Although the mitochondrial genome of <it>D. pteronyssinus </it>shares different features with previously characterised Acari mitochondrial genomes, it is unique in many ways. Gene order is extremely rearranged and represents a new pattern within the Acari. Both tRNAs and rRNAs are truncated, corroborating the theory of the functional co-evolution of these molecules. Furthermore, the strong and reversed GC- and AT-skews suggest the inversion of the control region as an evolutionary event. Finally, phylogenetic analysis using concatenated mt gene sequences succeeded in recovering Acari relationships concordant with traditional views of phylogeny of Acari.</p

Transcriptome profiling of a spirodiclofen susceptible and resistant strain of the European red mite Panonychus ulmi using strand-specific RNA-seq

Background: The European red mite, Panonychus ulmi, is among the most important mite pests in fruit orchards, where it is controlled primarily by acaricide application. However, the species rapidly develops pesticide resistance, and the elucidation of resistance mechanisms for P. ulmi has not kept pace with insects or with the closely related spider mite Tetranychus urticae. The main reason for this lack of knowledge has been the absence of genomic resources needed to investigate the molecular biology of resistance mechanisms. Results: Here, we provide a comprehensive strand-specific RNA-seq based transcriptome resource for P. ulmi derived from strains susceptible and resistant to the widely used acaricide spirodiclofen. From a de novo assembly of the P. ulmi transcriptome, we manually annotated detoxification enzyme families, target-sites of commonly used acaricides, and horizontally transferred genes implicated in plant-mite interactions and pesticide resistance. In a comparative analysis that incorporated sequences available for Panonychus citri, T. urticae, and insects, we identified radiations for detoxification gene families following the divergence of Panonychus and Tetranychus genera. Finally, we used the replicated RNA-seq data from the spirodiclofen susceptible and resistant strains to describe gene expression changes associated with resistance. A cytochrome P450 monooxygenase, as well as multiple carboxylcholinesterases, were differentially expressed between the susceptible and resistant strains, and provide a molecular entry point for understanding resistance to spirodiclofen, widely used to control P. ulmi populations. Conclusions: The new genomic resources and data that we present in this study for P. ulmi will substantially facilitate molecular studies of underlying mechanisms involved in acaricide resistance