Results
Sequencing and transcriptome
assembly
In total, we obtained over 787 million high-quality paired-end 150-bp
reads, with an average of 39.4 million reads per tissue type
(Supplemental Table 1; accession numbers will become available upon
publication). After filtering, we retained over 766 million reads
(Supplemental Table 2). An average of 88.4% of these reads mapped to
the N. lecontei genome. Our genome-guided de novotranscriptome assembly identified 132,243 contigs. After further
filtering, we retained 58,353 contigs. Across our 77 libraries, average
mapping rate to these contigs was 85.5% (range: 74.9-91.6%). The
provisionally annotated transcriptome contained 16,714 contigs
representing 9,304 predicted insect genes. Using these 16,714 contigs as
the transcriptome, a high mapping rate was maintained across all
libraries (mean: 76.7%; range: 60.5-88.6%).
Decoupling of gene-expression profiles
increases with ecological dissimilarity of N. lecontei life
stages
Consistent with our prediction that differences in gene-expression
traits would be most pronounced between ecologically dissimilar life
stages (Figure 1B), stages separated by major and complete metamorphosis
were clearly distinct as shown in the first principle component (PC1) of
our principle component analysis (Figure 2A). By contrast, there were no
clear distinctions between cryptic and aposematic larvae or between
males and females along either of the first two gene-expression PCs. PC2
primarily separated tissues within each life stage (Figure 2A). Along
this axis, gene expression in larval and adult heads was clearly
distinct from expression in other tissues, as was expression in the
adult antennal tissues. Based on these results, we analyzed
gene-expression decoupling among stages/sexes in two ways: all tissues
combined and heads only (Supplemental Tables 3 and 4, respectively).
Regardless of whether we looked at all tissues or heads only, both the
magnitude of differential expression (log fold-change) and significance
(Benjamini-Hochberg adjusted p-value) increased in accordance with ADH
predictions across metamorphic transitions (Figure 2B for all tissues).
Specifically, the percentage of DEGs increased with ecological
dissimilarity of the life stages compared: 2.2% of genes were
differentially expressed between cryptic and aposematic larvae, 24.7%
between aposematic and dispersing larvae, and 25.1% between dispersing
larvae and adult males (Supplemental Table 3; heads only: 2.9%, 17.6%,
and 31.1%, respectively, Supplemental Table 4). The proportion of DEGs
increased concurrently with the extremity of metamorphosis (Fisher’s
exact tests; P < 1 x 10-20) except
for the all-tissue dispersing larvae vs. male comparison (P =0.59).
Despite males and females being more morphologically dissimilar than the
feeding and dispersing larval stages, the percentage of DEGs between
sexually dimorphic adults (all tissues: 7.0%, Supplemental Table 3;
head only: 6.5%, Supplemental Table 4) was far less than that observed
across both the major and complete metamorphic transitions (Fisher’s
exact tests; P < 1 x 10-208)(Figure
2B). In contrast, we observed a higher percentage of DEGs between the
sexes than between the two feeding larval stages (Fisher’s exact tests;P < 1.0 x 10-20). Taken together with
the PCA, these results indicate that gene-expression decoupling between
the sexes is more or less on par with the minor metamorphic transition
between cryptic and aposematic larvae and far less pronounced than the
decoupling observed between more extreme life-stage transitions.
Overall, these transcriptome-wide analyses support the ADH prediction
that genetic decoupling increases as the ecological demands of the
different life stages become more dissimilar, as well as the prediction
that genetic decoupling tends to be more pronounced between
developmental stages than between the sexes.
Variation in decoupling among different types of
gene-expression traits reflects changes in
ecology
Consistent with our prediction that genes that mediate changing
ecological interactions will exhibit the most pronounced decoupling,
many of the top differentially expressed genes between life stages/sexes
corresponded to candidate genes that were related to their ecological
differences (Supplemental Table 5). Among the top differentially
expressed genes between cryptic and aposematic larvae were genes
potentially involved in immunity, metabolism and detoxification. For
example, esterase FE4-like (elevated in bodies of aposematic
feeding larvae), is a gene thought to be involved in resistance to
organophosphate insecticides and may be essential to ingesting and
sequestering toxic pine resins. The esterase FE4-like gene was
also among the top genes downregulated in the non-feeding dispersing
larvae. The change from an aposematic feeding larva to a dispersing
larva was also accompanied by a pronounced decrease in expression ofCameo2 , a gene that is thought to play a role in carotenoid-based
pigmentation (Y. Li et al., 2014) and has been linked to larval body
color in N. lecontei via QTL mapping (Linnen et al., 2018).
Putative pigmentation and detoxification genes were also among the top
differentially expressed genes between larvae and adult males
(Supplemental Table 5).
The top differentially expressed genes between males and females for
each tissue appear to be related to the sex-specific tasks of the adults
(Supplemental Table 5). For example, females, which must properly
provision their eggs, had elevated expression for avitellogenin-like gene, which is associated with egg yolk
formation, hormone regulation, lifespan, and foraging behavior (Ihle,
Page, Frederick, Fondrk, & Amdam, 2010; Munch, Ihle, Salmela, & Amdam,
2015; Nunes, Ihle, Mutti, Simoes, & Amdam, 2013; Seehuus, Norberg,
Gimsa, Krekling, & Amdam, 2006; Wheeler, Ament, Rodriguez-Zas, &
Robinson, 2013) There was also increased expression for many major
royal jelly protein family genes, known to play a role reproductive
maturation and tending to young (Buttstedt, Moritz, & Erler, 2013;
Dobritzsch, Aumer, Fuszard, Erler, & Buttstedt, 2019; Drapeau, Albert,
Kucharski, Prusko, & Maleszka, 2006). Among the genes with unusually
high expression in males were three chemosensory genes (OR54 ,OR22 , and OBP9 ) that were particularly high in the
antennae and may play a role in mate finding.
Chemosensory genes vary in decoupling across life stages
Consistent with our a priori predictions, the magnitude of decoupling
for manually curated chemosensory genes was highly variable (Figure 3A).
While several chemosensory genes had similar levels of expression across
life stages (e.g. those falling along the dotted lines), others were
among the most decoupled genes in the transcriptome (e.g., those falling
at the edges of the transcriptome-wide cloud of points). This contrasted
with a family of housekeeping genes that have a similar family
size—the ribosomal protein L genes (RPLs). As expected, these
genes were highly coupled across development and had nearly identical
expression levels in comparisons between the all sexes or stages (e.g.,
those that fall on the dotted line). Figure 3B directly compares
variation in decoupling for these two categories of genes. Consistent
with the ADH, gene-expression decoupling for chemosensory genes was both
more variable (i.e., wider distribution) and significantly higher than
RPLs in 3 out of 4 comparisons (Figure 3B and Supplemental Table 4;
Mann-Whitney U test P < 1.0 x 10-3).
The exception was the aposematic vs. dispersing larvae comparison
representing the major metamorphic transition (P = 0.90).
Overall, these results are consistent with a scenario in which selection
has favored coupling for some chemosensory genes and stage-specific
decoupling for others.
To gain additional insight into how chemosensory gene expression changes
across the life cycle, we created a heat map of stage-specific
expression for each gene (Figure 3C). Whereas some chemosensory genes
were expressed only during a single life stage, others were expressed at
moderate to high levels in multiple life stages. We further analyzed
these genes by identifying those that were in the highest 10% of
expression for each stage/sex. This analysis revealed several
chemosensory genes that were highly expressed in a single stage/sex
while others were highly expressed in multiple stages and/or both sexes
(Figure 3D and Supplemental Table 6). Together, these patterns provide
clues into chemosensory gene function. For example, chemosensory genes
that were highly expressed by both feeding larvae and females
(OBP5 and OR25 ) could be involved in detecting host-plant
cues. Likewise, genes highly expressed in both sexes of reproductive
adults (OBP2 , OBP8 , and OBP10 ) may play roles in
conspecific mate recognition. Genes expressed by all three stages
(OBP4 , OBP9 , and OR55 ) may be important for
detecting conspecific or host cues that are critically important
throughout the life span. In contrast, genes with high expression in
only a single stage may only be essential for a single task or
ecological pressure. For example, OBP13 is expressed at high
levels in feeding larvae with low levels of expression in dispersing
larvae an almost no expression in adults. Each adult sex also has
specific chemosensory genes with OR20 and OR41 expressed
almost exclusively in female antennae while OR22 , OR51 ,
and OR54 expressed almost exclusively in male antennae. Notably,
although all larvae were males, we did not identify any genes that were
highly expressed in larval and adult males to the exclusion of adult
females, suggesting once again that ecology is a better predictor of
gene-expression patterns than sex in this species.