Insects and RNA sample preparation

B. mori and M. sexta were purchased as larvae and/or pupae from Educational Science ( www.educationalscience.com) and reared up to emergence of the imago, at which point they were dissected. Total RNA was purified from whole animals, isolated body parts (legs, heads, thoraces), and from flight muscles using Trizol ( www.invitrogen.com) as previously described by Ayme-Southgate et al. (2008).

Degenerate primers and splicing analysis

Degenerate primers have been described elsewhere (Ayme-Southgate et al. 2011). RTPCR reactions were performed as described before with different RNA preparations and primer sets (Southgate and Ayme-Southgate 2001). Annealing for both the RT and PCR reactions were tested using a range of temperatures to optimize each primer set. DNA fragments were isolated after agarose gel electrophoresis and subcloned into the pGEM-T easy shuttle vector (Promega,  www.promega.com), which was then followed by sequencing (Genewiz Inc.,  www.genewiz.com). Sequences from overlapping clones were manually assembled into contigs.

Bioinformatics analysis

For the B. mori projectin, contigs were isolated following tblastn searches of GenBank WGS database using a series of gene fragments from D. melanogaster projectin. The tblastn algorithm compares a query protein sequence to the 6-frame-translation of a DNA sequence; in this case, the contigs available for the B. mori genome (Altschul et al. 1990). The EST database from the two silkworm transcriptome projects, available through SILKBASE and GenBank, was also searched, and resulting EST sequences were aligned on the genomic sequence. For the D. plexippus projectin, a BLAST search of the recently published genome was performed using B. mori projectin cDNA as query. Sequence comparisons were carried out using the CLUSTALW algorithm, and the alignments were viewed in Jalview (Thompson et al. 1994, 1997).

Supplementary Table.

Summary of contigs from the B. mori genome database assembled to generate projectin genomic sequence.

B. mori and D. plexippus projectin sequences

The annotation in the GenBank database reported a B. mori projectin homolog based on genome analysis of the B. mori Z chromosome (Koike et al. 2003). This annotation predicted a series of short proteins with names such as D-titin, kettin, and projectin-like containing immunoglobulin (Ig) and/or fibronectin type III (Fn) domains (GenBank ID: NM_001114995 for projectin-like). None of these entries represented the correct gene for the projectin protein, as they were too short and did not contain the characteristic pattern of Fn and Ig domains expected for projectin. At the start of this project, the B. mori projectin gene was assembled de novo following tblastn searches of B. mori supercontigs (Mita et al. 2004) available in the GenBank database using a series of peptide fragments from the D. melanogaster projectin. Extensive overlap was established for most of the contigs representing the B. mori projectin gene (Supplementary Table 1). Some of the contigs available in GenBank were probably incorrectly assembled, however, as projectin domains known to be adjacent are coded on opposite strands or in reverse order. In other cases, some contigs contained a deletion covering several domains, which were present in other contigs. Many of these issues could have resulted from the presence of repetitive sequences. Despite our efforts, five gaps remain in the genomic sequence, as repeated searches of the GenBank database fail to return contigs overlapping these gaps, and we assume that these sequences were not obtained during the original genome sequencing. We have not attempted to ‘clean’ the original assembly or to sequence the missing genomic DNA (see below).

The exon-intron pattern was predicted over most of the gene by performing translation in all three frames and visual alignment with the D. melanogaster projectin amino acid sequence. The PEVK region could not be entirely predicted by this approach (see below). Ambiguous splice sites were resolved by RT-PCR using B. mori total RNA followed by sequencing of cDNA products. In cases where the assembly of the genomic contigs was ambiguous, the prediction was verified by RTPCR amplification across the gaps/misalignment and cDNA sequencing (see Materials and Methods for details; data not shown). EST sequences from two silkworm transcriptome projects available through SILKBASE and GenBank (Mita et al. 2003) were retrieved. The few EST sequences available were consistent with the exon-intron prediction. Of the five gaps remaining in the genomic sequence, all but one occurred within intron sequences and were bridged by our own cDNA sequencing or EST data, so it is indeed a continuous gene. The one gap falling within a coding region encompassed a segment of the kinase domain. The corresponding cDNA sequence was obtained following RT-PCR, but the exact exon-intron pattern for this part of the gene is uncertain because the genomic sequence was unavailable.

The projectin gene for D. plexippus was retrieved from the recently available genome data (Zhan et al. 2011). The combined sequences from two super-contigs (GenBank ID # AGBW01006173.1 and AGBW01009765.1) cover the entire projectin gene, except for half of the second Ig domain, which is located in the gap between the two super-contigs and for which no genomic data are available.

Figure 1.

Diagram of the conserved projectin domain organization. This pattern is common to all projectin characterized so far, including the lepidopteran proteins from this study. The lg and FnIII domains are represented as barrels to reflect their globular nature. The [Fn-Fn-Ig] module is repeated 14 times within the central core region. The Nhh-terminus is composed of two tracts of eight (N8lg) and six (N6lg) Ig domains separated by the PEVK-NTCS1 region. High quality figures are available online.

Table 1.

Characteristics of the projectin gene in Lepidoptera. The lepidopteran genes are compared to the projectin genes in other insect species. Dmel: Drosophila melanogaster; Amel: Apis melllfera; Tcas: Tribolium castaneum; Api: Acyrtosiphon pisum; Bmo: Bombyx mori, and Dple: Danaus plexippus.

The gene characteristics for B. mori and D. plexippus projectins are summarized in Table 1 and compared with D. melanogaster, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), A. mellifera, and Acyrthosiphum pisum Harris (Hemiptera:Aphididae) (Ayme-Southgate et al. 2008). The overall domain organization of projectin in the two lepidopteran insects was identical to all the projectin proteins characterized so far (Figure 1; Ayme-Southgate et al. 2008, 2011). Even though the full length of the two lepidopteran projectin genes could not be ascertained completely, they were the largest of all the projectin genes characterized, with one of the highest number of exons (Table 1). Similar to the situation found in D. melanogaster, the largest exon in both genes contained approximately half of the domains for the core region (the section of the protein composed of the repeated Fn-Fn-Ig modules). For the remainder of the protein, individual Ig and Fn domains were often split between two exons, a situation more similar to the one found in insects from more basal orders such as A, pisum (Ayme-Southgate et al. 2008).

N-terminus sequence determination in M. sexta

To gain access to the NH₂-terminal sequence of projectin in M. sexta, a series of degenerate primers based on sequence alignment of several Ig domains from the N8Ig and N6Ig tracts was used (Figure 1; Ayme-Southgate et al. 2011), as well as primers based on B. mori sequences. RT-PCR amplifications using M. sexta RNA were performed with these different primer sets, and these products were cloned and sequenced. The NH₂-terminus region for M. sexta followed the standard pattern found in all projectin genes, which is two separate tracts of 8 and 6 Ig domains respectively with small interspersed linker sequences of 5 to 46 amino acids in length, separated by a unique sequence (see Figure 1).

PEVK structure in B. mori, D. plexippus, and M. sexta genes

Species-specific primers from M. sexta Ig8 and Ig9 domains were used to amplify the unique sequence between the N8Ig and N6Ig regions. Internal primers were used in a second stage to try and amplify larger cDNA products for the M. sexta PEVK-NTCS-1 segments. The cDNA sequence was aligned to the corresponding genomic sequences in B. mori and D. plexippus. Using this approach, the PEVK and NTCS-1 exons for both insects, as well as the intron-exon boundaries in all three species, were predicted (Figure 2). Only one of the M. sexta exons could not be identified in either B. mori or D. plexippus, possibly because this exon was very short, with only 24 nucleotides (exon #5 in Figure 2). Several of the splice sites were confirmed in M. sexta through the sequencing of alternate splice products (see below).

Figure 2.

Exon-intron pattern for the PEVK-NTCS-1 segments in lepidopteran projectins. The striped boxes indicate exons that are only predicted by alignment to Manduca sexta. The ? indicates the exon that was not found in Bombyx mori and Danaus plexippus. PEVK exon numbers are indicated above the M. sexta map. Introns are not to scale. High quality figures are available online.

The possibility of additional exons in the PEVK region does exist, but these exons would probably be rarely expressed, as this region was thoroughly amplified in M. sexta, and all resulting cDNAs were sequenced. Also, the available continuous B. mori and D. plexippus PEVK genomic sequences were translated in all three frames and visually scanned for open stretches of at least 10 amino acids with elevated PEVK content. No additional exons were predicted using this approach (data not shown).

Alignment of PEVK-NTCS-1 segments from B. mori, D. plexippus, and M. sexta with those available from some of the other species was performed using CLUSTALW and viewed with Jalview (see Materials and Methods for details). As shown by the alignment presented in Figure 3, the current subdivision of this unique sequence into two segments is supported; there is a highly divergent PEVK region and the conserved NTCS-1 segment, which is positioned just before the second stretch of six Ig domains (solid black line above alignment in Figure 3). Contrary to the PEVK segments found in other proteins (human titin, C. elegans TTN-1, and Drosophila sallimus), the projectin PEVK regions described here did not contain any repeating pattern. This was consistent with all other insect projectin PEVK segments, except for a short repeat found exclusively in A. mellifera projectin (Ayme-Southgate et al. 2011). The NTCS-I region was described as the largest conserved region when comparing projectins from basal (dragonfly) and more derived insects (Ayme-Southgate et al. 2011). In derived insects, including the three lepidopteran sequences described here, this conserved segment was slightly longer (by 28–35 amino acids) and began with a ‘YERP’ motif (boxed residues in the alignment; Figure 3). This conserved block was not present in the sequences of Pediculus humanus L. (Phthiraptera: Pediculidae), A. pisum, or the two dragonfly species, P. longipennis and L. pulchella (Ayme-Southgate et al. 2011). Two YERP motifs were present in the lepidopteran and D. melanogaster PEVK regions (black boxes in Figure 3 and # in Figure 4).

Figure 3.

CLUSTAL-W-generated amino acid sequence alignment between PEVK-NTCS-I regions in different insects. The intensity of the shading indicates the level of amino acid identity. Black boxes indicate the position of the YERP motifs. The solid black line corresponds to the NTCS-1 segment. Dmel: Drosophila melanogaster; Amel: Apis melllfera; Tcas: Tribolium castaneum; Api: Acyrtosliphon pisum; Bmo: Bombyx mori, and Dple: Danaus plexippus. High quality figures are available online.

Alternative isoforms of the PEVK-NTCS-1 segments in different muscle types

The alternative splicing pattern for the PEVKNTCS-1 region was also ascertained, and the analysis indicated that the PEVK region was the site of several alternative splicing combinations. As shown in Figure 4, there were only two exons that could be alternatively spliced to generate the shortest form (exons # 3 and 6), compared to 11 exons in D. melanogaster and T. castaneum and up to 21 in A. mellifera. Only 4 PEVK variants were detected in M. sexta PEVK, compared to at least 10 in D. melanogaster (Southgate and Ayme-Southgate 2001). The longest splice variant identified in M. sexta would encode a 377 amino acid-long PEVK region, and the shortest form would be 205 amino acids. The length difference between the longest and shortest variants was therefore not as ‘striking’ in M. sexta as it is in other insects, for example 75 and 530 amino acids for the shortest and longest variants respectively in D. melanogaster (Table 2). Also, the YERP motifs were included in all of the M. sexta PEVK variants, whereas they were excluded from the short variant in other insects (Figure 4). The characteristics of the PEVK and NTCS-1 segments for all three lepidopteran genes are summarized in Table 2 together with the corresponding regions in other insect projectin proteins.

Table 2.

Characteristics of the PEVK-NTCS-1 regions in Lepidoptera as compared to other insects. L.pul: Libellula pulchella, A.mel: Apis mellifera; T.cas: Tribolium castaneum; D.mel: Drosophila melanogaster; M.sex: Manduca sexta; B.mor: Bombyx mori; and D.ple: Danaus plexippus.

Figure 4.

Diagram representation of the PEVK exons in different insect species. A) Exon pattern in Lepidoptera, together with the isoforms detected in Manduca sexta. Exon composition for the alternative variants is presented below the map together with their expected sizes following amplification with primers Ms1–6R. B) Pattern in other insects (Ayme-Southgate et al 2011). The exons used in the shortest variants are shaded in gray. Intron sizes are not to scale. High quality figures are available online.

The long form of the PEVK segment in all lepidopteran genes was most similar in length and PEVK content to the T. castaneum sequence. In contrast, the PEVK content of the short form, but not its length, was closer to the short variant of D. melanogaster. The short isoform in M. sexta also had a lower P, E, V, and K content than the longest isoform; this was in sharp contrast to the situation in all other studied insects, where the P, E, V, and K, content of the short variant can be as high as 100 percent (see Table 2).

The presence and specificity of alternative isoforms in the PEVK-NTCS-1 region were ascertained by performing RT-PCR amplification using RNAs extracted from several body parts, as well as from isolated flight muscles from M. sexta (see Materials and Methods for details). Primers were designed from exons flanking and internal to the PEVK-NTCS-1 segments.

The variant composition was qualitatively identical across muscle types irrespective of the primer pair used for the amplification. As presented in Figure 5A in the Ms1–6R reaction, the isoforms A and B (1,373 and 1,321 bp respectively; see Figure 4) were present in all muscles, and the C/D isoforms (926 and 893 bp respectively) were present in thorax and flight samples, as well as faintly in leg and head samples. The Ms1-6R primer set also faintly amplified two products around 500 bp, which were present in all muscle types. These products were sequenced and correspond to M. sexta hemocytin gene as identified by a BLAST homology search (Kotani et al. 1995; Tanaka et al. 2008). The reason for this reproducible amplification is unknown.

The Ms1-6R primer set amplified the entire PEVK-NTCS-1 region. Because short PCR products are favored in PCR amplification reactions, the absence of projectin products shorter than 800 bp in the Ms1–6R reaction was unlikely due to the low abundance of any short isoform. For the same reason, relative contribution of short and longer PEVK variants could not be ascertained completely from these data, even though isoforms A/B seemed to be the most abundant variants in both head and leg RNA samples.

Isoforms A, B, and C were also detected in all muscle types using primers Ms1–2R (Figure 5B). The shortest 480 bp (corresponding to isoform D) product in the Ms1–2R reaction was not detectable in the leg RNA sample. This product only differed from isoform C (corresponding to the 500 bp product) by the exclusion of exon 6 (see Figure 4), which was only 33 bp in length. So, isoform D could be considered a flight muscle-specific isoform.

Figure 5.

The PEVK variants are identical in all muscle types in Manduca sexta. Products of RT-PCR reactions were analyzed by gel electrophoresis. A): primers Ms1–6R and B): primers Msl2R (see Figure 4). The muscle types are as follows: L: leg, T: whole thorax, F: flight muscle, and H: head. St: I kb ladder from New England Biolabs ( www.neb.com). Sizes are indicated on the right. A/B and C/D refer to the isoforms depicted in Figure 4. * is a contamination product corresponding to M. sexta hemocytin gene as identified by sequencing and BLAST homology search. High quality figures are available online.