Figure 1.
Illustration of bracovirus production and integration.
In part 1 specific portions of the wasp genome are targeted, replicated, and circularized into bracovirus DNA circles. These circles are produced in the wasp ovary then packaged in a viral capsid and lipid membrane. In Part 2 the bracoviruses are injected into the secondary host. In part 3 the bracovirus enters the secondary host cell, shedding the capsid. This typically occurs in haemocytes, with the proteins produced by the transferred DNA destroying or disabling the haemocyte's ability to defend the secondary host against wasp larvae. Occasionally, bracovirus DNA will integrate into the secondary host nuclear DNA as shown in part 4. If steps 3 and 4 occur in a germ line cell the bracovirus DNA may be passed on to the secondary host's offspring. Subsequent genetic drift or selection can result in the HTS becoming fixed in the population.
Table 1.
Summary information for candidate horizontally transferred sequences.
Figure 2.
Representation of highly conserved sequences in insect genomes.
Phylogenetic tree generated from sequence data using PHYML. Coding regions from Nasonia vittripennis were used as tblastx queries against the Bombyx mori genome assembly to identify highly conserved regions. 300 conserved regions were selected at random and searched for in a broad range of insect genome assemblies using tblastx. In each insect species, 290–299 of these highly conserved regions were found (a rate of 96–99%). Missing sequences are an unknown combination of genomic deletions and incomplete assemblies. With a divergence time of ∼300 MYA between Bombyx and Nasonia, there has been sufficient time for genomic deletions to occur. Branch lengths are based on amino acid divergence between species.
Figure 3.
Two examples of HTS found in Bombyx (the same two HTS targeted for PCR in Figure 4) aligned to bracovirus sequences from Cotesia congregata.
The top portion shows a HTS (PDV101) aligned to Cotesia congregata bracovirus circle 12 (NC_006644.1). Predicted protein in the donor sequence shown (YP_184814.1). The bottom portion show a HTS (PDV32) aligned to Cotesia congregata bracovirus circle 5 (NC_006637.1). PDV32 is part of homology group 1, which is shown aligned to PDV32. In both examples the HTS have undergone point mutations and insertion/deletions that are too numerous to accurately represent here so example mutations have been drawn. Primers used in the PCR reaction shown in Figure 4 are shown with green triangles.
Figure 4.
Gels displaying PCR amplification of HTS in Bombyx mori.
Gels were ethidium bromide stained and run with a 100 bp ladder (brighter bands at 500 bp and 1000 bp). Primers used are shown in fig 3. As a further test, additional PCR reactions were run with alternative primers for each HTS with the same results (see Figure S2). A) PCR results showing amplification of a product spanning the junction of the transferred wasp DNA and native Bombyx sequence. Lanes 2, 4, and 6 have primers targeting PDV32 (expected band size: 1037) and lanes 1, 3, and 5 have primers targeting PDV101(expected band size 1090). Lanes 5 and 6 use Bombyx mori strain 418(Chinese), lanes 3 and 4 Bombyx mori strain 214(Japanese), lanes 1 and 2 wild type Drosophila melanogaster as a negative control. B) We tested the same primer set above targeting PDV32 against a diverse panel of insect genomic DNA (all lanes tested with the same primers). DNA used in the reactions was as follows. Lane 1: Chlosyne lacinia (Lepidoptera). Lane 2: Apis mellifera (honeybee, Hymenoptera). Lane 3: wild type Drosophila melanogaster (Diptera). Lane 4: Bombyx mori strain 555 (European). Lane 5: Bombyx mori strain Nistari (Indian). Lane 6: Bombyx mori strain 418 (Chinese). Lane 7: Bombyx mori strain 214 (Japanese).