Inducible responses under joint predation risks
Antagonistic responses would put the prey into a dilemma regarding which
predator to respond to (Fig. S2). In our experiments, size at 16th day
was not significantly expressed under joint predation risks (CH + F vs.
C: P = 0.764). Consistently, unique responses to Chaoboruspredation risk were significantly affected by the presence of fish
predation risk, that is, horn expression in the combination treatment
was greatly impaired compared with that under the Chaoboruspredation risk (maturity: P = 0.012; 16th day: P< 0.001). This effect was highly correlated to individual
development, that is, in the combination treatment, C. cornutahorns were significantly expressed at maturity (CH + F vs. C: P =
0.007) but not at 16th day. Conversely, the effects of joint predation
risks were observed in neither unique responses to fish nor general
responses. As such, the responses of size at maturity (CH + F vs. C:P < 0.001), time to first brood (CH + F vs. C: P= 0.027), and neonate size (CH + F vs. C: P = 0.003) were
significantly altered in the combination treatment but were not
significantly different from those under the fish predation risk
treatment. Thus, various predator-dependent unique responses could be
simultaneously expressed under antagonistic predation risks, although
the prey could not completely avoid the effect of other predation risks,
and the co-expression of unique responses was related to individual
development. These results basically support our hypotheses.
Overview of the assembled
transcriptome
We obtained respectively 78,341,484, 72,493,072, and 69,930,191 clean
reads for C, CH, and F. A total of 37,120 unigenes were assembled using
Trinity, and each sample contained > 66.54% assembled
unigenes (Table S2). Through comparisons against the Nr, KEGG, COG, and
SWISS-PROT databases, 18,343 unigenes (49.4%) were annotated (Fig. S3).
Regarding species distribution in the Nr database, C. cornutashowed the highest comparison rate with D. magna (26.33%),
followed by D. pulex (2.6%) (Fig. S4A). The unigenes enriched in
the KOG database were classified into transcription, ribosomal
structure, gene replication, recombination, and repair (Fig. S4B). The
annotated GO terms were mainly associated with metabolic processes,
cellular processes, cell parts, and binding (Fig. S4C).
DEGs
Paired samples within the same treatment showed high Pearson’s
correlation coefficients (≥0.90) and were clustered in principal
component analysis (Fig. 2), which conformed to the requirements of
biological repetition. Furthermore, the expression patterns of DEGs
showed significant concordance between RT-qPCR and RNA-Seq (Fig. S5),
indicating that our RNA-Seq analysis of the expression data was
reliable.
Compared with the control, Chaoborus and fish predation risks
significantly affected the expression of 1,515 and 846 genes,
respectively (Fig. 3A). Among these, there were 1,399 unique DEGs in CH,
730 unique DEGs in F, 114 general DEGs, and two antagonistic DEGs (Fig.
3B and Table S3). Considering the DEGs triggered by different predators,
we further analyzed the differences in the enriched pathways of C.
cornuta (Supplementary Table S4) and identified the major DEGs and
pathways related to inducible defensive traits (Table 2). The DEGs ofC. cornuta under Chaoborus predation risk, including
cuticle protein, fatty acyl-CoA reductase, and trypsin genes, were
mainly enriched in cutin, suberine, and wax biosynthesis; protein
digestion and absorption; and steroid hormone biosynthesis. Meanwhile,
the DEGs of C. cornuta under fish predation risk, including
ribosomal protein, actin, and short-chain type dehydrogenase genes, were
mainly enriched in cysteine and methionine metabolism, ribosome,
phototransduction, and unsaturated fatty acid biosynthesis. The general
DEGs, including cysteine proteinase, HSP70, actin, and alpha-tubulin
genes, were enriched in apoptosis pathways and antigen processing and
presentation. Therefore, specific unique responses to different
predators were dominant at the transcriptional level: unique DEGs (2,129
genes) > general DEGs (114 genes) >
antagonistic DEGs (2 genes).
Correlation between different inducible
responses
Nine individual traits and 40 representative DEGs (Table S5) revealed
strong Pearson’s correlations within unique responses to fish orChaoborus larvae (Fig. 4). For instance, the response of horn
expression at maturity was significantly correlated with changes in
horns at 16th day (P = 0.05) and total offspring number (P= 0.05). However, the unique responses to fish and Chaoboruslarvae were weakly correlated in both analyses. Only a few significant
correlations appeared on the weak correlation area in transcriptional
expression. For instance, Unigene0008297 in ribonucleoprotein component
protein expression was significantly and positively correlated with
Unigene0027903 (P= 0.049) and Unigene0005204 (P= 0.013) in
aspartokinase and adenosylhomocysteinase expression, respectively (Fig.
4). Therefore, the unique responses triggered by different predators are
weakly coupled.
Discussion
Our experiments proved that C. cornuta responds differently to
antagonistic predation risks of fish and Chaoborus larvae. Based
on the classification of inducible responses, our results revealed for
the first time that specific predator-dependent unique responses are
dominant, followed by general responses, whereas the antagonistic
responses are rare. In addition, the unique responses triggered by
different predators were extremely weakly coupled and could be elicited
simultaneously. Overall, the experimental results support our hypothesis
that predator-dependent unique responses are dominant, helping the prey
avoid the dilemma of which predator to respond to. Nonetheless, the
unique responses to Chaoborus larvae cannot completely avoid the
effects of fish predation risk, implying the presence of complex costs
and limitations of unique responses to multiple predators.
In C. cornuta , horn expression and body enlargement are adaptive
inducible traits to Chaoborus larva predation (Gu et al., 2021;
Riessen & Trevett-Smith, 2009). A larger individual size at late
developmental stages requires rapid growth and high food intake
(Gianuca, Pantel, & De Meester, 2016), thereby promoting the brood and
total offspring numbers. The horns are formed by the carapace, which
comprises two layers of dermal cells and is covered by chitin in
combination with cuticle proteins (Charles, 2010). Therefore, the
development of morphological defensive traits involves a series of
changes in the expression of chitin, hormone, and epidermal formation
genes at different times (Christjani, Fink, & Elert, 2016; Miyakawa et
al., 2010) as well as the regulation of epidermal cell growth via
endocrine hormones (Weiss, Leese, Laforsch, & Tollrian, 2015). In the
present study, significant changes in the expression of genes and
pathways involved in cuticle protein, cutin, suberine, and wax
biosynthesis as well as steroid hormone biosynthesis were noted. The
expression of these genes may promote the synthesis of related
substances (Fig. 5) and regulate individual growth (Edgar, 2006).
However, the growth and development of cladocerans requires continuous
molting and formation of a new carapace; thus, the maintenance of horns
requires continuous substance synthesis, which may result in constant
distribution costs (Auld et al., 2010). Furthermore, in C.
cornuta , Chaoborus predation risk altered digestion and
absorption by modulating the expression of the trypsin gene, which may
affect the digestion and resource allocation strategies (Von Elert et
al., 2004).
Smaller size, earlier reproduction, and increased brood number are
adaptive responses to fish predation risks, which are similar to the
typical responses of other cladocerans under fish predation (Diel et
al., 2020). In terms of gene expression, our results showed that genes
encoding actin and ribosomal proteins were down-regulated under fish
predation risks. Since actin plays an important role in cytoskeletal
structure, its inhibition may result in a smaller size. Similar results
have been reported in previous studies on inducible defense responses ofD. magna (Effertz et al., 2015; Pijanowska & Kloc, 2004). On the
contrary, Schwarzenberger, Courts, and Von Elert (2009) revealed that
the actin genes in D. magna were up-regulated under fish
predation risks. Since gene expression is jointly regulated by
transcriptional regulators and related proteins (Stibor, 2002),
differential expression patterns could be observed at different time
points (Effertz & Von Elert, 2014). Ribosomal proteins are responsible
for protein assembly and translation; thus, the down-regulation of
ribosomal protein may inhibit the synthesis of proteins essential for
individual growth and development of C. cornuta (Zhou, Liao,
Liao, Liao, & Lu, 2015). In enrichment analysis, some DEGs were found
to be enriched in multiple pathways. Significantly enriched
phototransduction altered the visual perception of Daphnia(Mahato et al., 2014), which may be an adaptation to behavioral
responses, such as habitat selection (Loose & Dawidowicz, 1994) and
escape behavior (Pietrzak, Pijanowska, & Dawidowicz, 2017). InDaphnia , fish predation reduced unsaturated fatty acid levels in
neonates, rendering them vulnerable to starvation (Stibor & Navarra,
2000); therefore, the significantly enriched unsaturated fatty acid
pathways may alter the distribution of these components. Furthermore,
the longevity regulating pathway was significantly enriched under fish
predation risks, which may incur an opportunity cost, that is, a decline
in lifespan (Dawidowicz, Predki, & Pietrzak, 2010).
Under different predation risks, C. cornuta showed general
responses, such as the expression of cysteine protease, heat shock
protein, actin, and tubulin genes. The cDNA sequence of crustacean
cysteine is similar to that of insect cathepsin L, which regulates the
molting cycle and promotes cell death during development (Agrawal,
Bagchi, & Bagchi, 2005). Thus, cysteine protease likely affected
molting and increased brood number in the present study. The
up-regulation of heat shock proteins is an adaptive response to various
environmental stresses, including predation risks (Pijanowska & Kloc,
2004). As this response is rapid and returns to the previous state after
long-term treatment (Pauwels, Stoks, & De Meester, 2005; Pauwels,
Stoks, Decaestecker, & De Meester, 2007), the down-regulation of heat
shock protein genes likely promoted the recovery of heat shock proteins
in the present study. Similarly, general responses of the actin and
tubulin genes, which are involved in cytoskeleton formation and other
life activities have been observed in Daphnia (Pijanowska &
Kloc, 2004), although their specific functions warrant further research
(Chen et al., 2018).
Based on the classification of inducible responses, we further analyzed
the early transcriptional data of Daphnia magna in response to
vertebrate (sticklebacks) and invertebrate (Triops ) predation
risks (Orsini et al., 2016). The results are consistent with our finding
that unique responses to different predators are dominant, while
antagonistic responses are rare (Fig. 6 and Table S6). Moreover,
previous weighted gene co-expression network analysis revealed that the
most significantly co-expressed gene networks under vertebrate and
invertebrate predation risks were unique (Orsini et al., 2018). Thus,
diverse predator-dependent unique responses are favored by cladocerans
during their co-evolution with multiple predators. For successful
evolution of diverse predator-dependent unique responses, genotype,
selection, and cost are important. First, the genotypes of cladocerans
in ponds or lakes are highly diverse and the inducible traits of
different clones are uncoupled (Boersma, Spaak, & De Meester, 1998;
Decaestecker, De Meester, & Mergeay, 2009; Stoks, Govaert, Pauwels,
Jansen, & De Meester, 2016). Second, multiple predators produce
variable selection effects, which contribute to predator-dependent
unique defensive traits (Herzog & Laforsch, 2013; Heynen, Bunnefeld, &
Borcherding, 2017). Finally, environmental costs, such as altered
predator regimes, may exceed maintenance costs (Decaestecker et al.,
2002; Tollrian, 1995; Yin, Laforsch, Lohr, & Wolinska, 2011).
Therefore, diverse predator-dependent unique responses are favored by
prey.
In the present study, prey exhibited coupled responses to the same
predator but extremely weakly coupled predator-dependent unique
responses to antagonistic predation risks. Evidently, prey can alter
resource allocation strategies under a single type of predation risk,
resulting in an array of adaptive responses (Reede, 1995). In addition,
the co-expression of uncoupled unique inducible responses can help prey
avoid the dilemma of which predator to respond to, thus improving their
survival rate under antagonistic predation risks. For instance, smallerC. cornuta individuals are less likely to be found by fish
(O’Brien, 1987). Simultaneously, horn expression renders C.
cornuta less vulnerable to predation by Chaoborus larvae (Gu et
al., 2021). However, the effects of joint predation risks on the
expression of predator-dependent unique responses are prominent and
could be influenced by development, indicating a complex trade-off
underlying adaptation to multiple predation risks (Riessen & Gilbert,
2019). Therefore, further studies are warranted to elucidate the
mechanisms of the phenomenon that diverse inducible responses of prey
can be elicited simultaneously under joint predation risks.
Conclusions
Through responses of individual traits and transcriptome, the present
study revealed the inducible responses of C. cornuta to predation
by Chaoborus larvae and fish. To cope with such antagonistic
predation risks, C. cornuta mainly altered cuticle gene
expression and developed horns under Chaoborus predation risk,
while it altered ribosome gene expression and reduced body size under
fish predation risk. Our analysis of these inducible responses revealed
for the first time that predator-dependent unique responses are dominant
and antagonistic responses are rare, implying that predator-dependent
unique responses are favored by cladocerans during their long-term
co-evolution with multiple predators, thereby lowering the environmental
cost of inducible defenses. However, unique responses to one predator
cannot completely avoid their dilemma of responding to other predators,
although this dilemma is relatively limited. The present study expands
our understanding of the evolution and expression of inducible defenses
under multiple predation risks.