Amino acid transporters implicated in endocytosis of Buchnera during symbiont transmission in the pea aphid

Abstract Background Many insects host their obligate, maternally transmitted symbiotic bacteria in specialized cells called bacteriocytes. One of the best-studied insect nutritional endosymbioses is that of the aphid and its endosymbiont, Buchnera aphidicola. Aphids and Buchnera are metabolically and developmentally integrated, but the molecular mechanisms underlying Buchnera transmission and coordination with aphid development remain largely unknown. Previous work using electron microscopy to study aphid asexual embryogenesis has revealed that Buchnera transmission involves exocytosis from a maternal bacteriocyte followed by endocytotic uptake by a blastula. While the importance of exo- and endocytic cellular processes for symbiont transmission is clear, the molecular mechanisms that regulate these processes are not known. Here, we shed light on the molecular mechanisms that regulate Buchnera transmission and developmental integration. Results We present the developmental atlas of ACYPI000536 and ACYPI008904 mRNAs during asexual embryogenesis in the pea aphid, Acyrthosiphon pisum. Immediately before Buchnera invasion, transcripts of both genes were detected by whole-mount in situ hybridization in the posterior syncytial nuclei of late blastula embryos. Following Buchnera invasion, expression of both genes was identified in the region occupied by Buchnera throughout embryogenesis. Notably during Buchnera migration, expression of both genes was not concomitant with the entirety of the bacterial mass but rather expression colocalized with Buchnera in the anterior region of the bacterial mass. In addition, we found that ACYPI000536 was expressed in nuclei at the leading edge of the bacterial mass, joining the bacterial mass in subsequent developmental stages. Finally, quantitative reverse transcription real-time PCR suggested that early in development both transcripts were maternally provisioned to embryos. Conclusions We venture that ACYPI000536 and ACYPI008904 function as nutrient sensors at the site of symbiont invasion to facilitate TOR-pathway-mediated endocytosis of Buchnera by the aphid blastula. Our data support earlier reports of bacteriocyte determination involving a two-step recruitment process but suggest that the second wave of recruitment occurs earlier than previously described. Finally, our work highlights that bacteriocyte-enriched amino acid transporter paralogs have additionally been retained to play novel developmental roles in both symbiont recruitment and bacteriome development

Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference

Background: RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant’s lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata’s dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene. Methodology/Principal Findings: Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for tw

Transcriptomics of the Bed Bug (Cimex lectularius)

BACKGROUND: Bed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance. METHODOLOGY AND PRINCIPAL FINDINGS: Using 454 pyrosequencing, we obtained a total of 216,419 reads with 79,596,412 bp, which were assembled into 35,646 expressed sequence tags (3902 contigs and 31744 singletons). Nearly 85.9% of the C. lectularius sequences showed similarity to insect sequences, but 44.8% of the deduced proteins of C. lectularius did not show similarity with sequences in the GenBank non-redundant database. KEGG analysis revealed putative members of several detoxification pathways involved in pesticide resistance. Lamprin domains, Protein Kinase domains, Protein Tyrosine Kinase domains and cytochrome P450 domains were among the top Pfam domains predicted for the C. lectularius sequences. An initial assessment of putative defense genes, including a cytochrome P450 and a glutathione-S-transferase (GST), revealed high transcript levels for the cytochrome P450 (CYP9) in pesticide-exposed versus pesticide-susceptible C. lectularius populations. A significant number of single nucleotide polymorphisms (296) and microsatellite loci (370) were predicted in the C. lectularius sequences. Furthermore, 59 putative sequences of Wolbachia were retrieved from the database. CONCLUSIONS: To our knowledge this is the first study to elucidate the genetic makeup of C. lectularius. This pyrosequencing effort provides clues to the identification of potential detoxification genes involved in pesticide resistance of C. lectularius and lays the foundation for future functional genomics studies

Genome Sequence of the Pea Aphid Acyrthosiphon pisum

Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems

A venom metalloproteinase from the parasitic wasp Eulophus pennicornis is toxic towards its host, tomato moth (Lacanobia oleracae)

Three genes encoding clan MB metalloproteinases (EpMP1–3) were identified from venom glands of the ectoparasitic wasp Eulophus pennicornis. The derived amino acid sequences predict mature proteins of approximately 46 kDa, with a novel two-domain structure comprising a C-terminal reprolysin domain, and an N-terminal domain of unknown function. EpMP3 expressed as a recombinant protein in Pichia pastoris had gelatinase activity, which was inhibited by EDTA. Injection of recombinant EpMP3 into fifth instar Lacanobia oleracea (host) larvae resulted in partial insect mortality associated with the moult to sixth instar, with surviving insects showing retarded development and growth. EpMP3 is expressed specifically in venom glands. These results suggest that EpMP3 is a functional component of Eulophus venom, which is able to manipulate host development