Figure 8 Relative levels of selected DEG expression by RT-qPCR and
RNA-Seq. Levels are measured using the 2-ΔΔCT method;
β-actin is the internal normalization control. Data are shown as mean ±
SD; * P < 0.05, ** P < 0.01.
DISCUSSION
Numerous behavioral studies indicate that the male red swamp crayfish
have “unidirectional receptors” for the sex pheromones released by
females during the MP (Monteclaro et al., 2010; Oyama et al., 2020;
Peddio et al., 2019). Male individuals can recognize different phases of
females by detecting the pheromones and then performing courtship
behaviors after checking the sex pheromone levels (Kubec et al., 2019).
However, previous research suggests that the frequencies of copulation
and oviposition in red swamp crayfish are significantly reduced when the
temperature goes below 15 °C (Egly et al., 2019). Before RNA-Seq, we
found that male red swamp crayfish were attracted to female conditioned
water in MP but not in NMP. We speculated that the expression of
olfactory and chemosensory genes in antennae may be altered due to
different temperatures and periods. In this study, we identified the
olfactory or chemosensory genes and pathways in antennae of P.
clarkii between MP vs. NMP by RNA-Seq for the first time. However, the
information on olfactory and chemosensory genes was as sparse as
expected, presumably since genetic annotation of species closely related
to P. clarkii is scarce in public genetic databases; in general,
the genetic information on olfaction in crustaceans is lacking. This
study is a necessary supplement to the genetic information library for
red swamp crayfish.
In this study, we identified a total of 22 DEGs related to “olfactory”
or “chemosensory”, including 4 “IRs or iGluRs”, 8 “G-protein
coupled receptor”, 5 “transient receptor potential”, 1
“sodium-calcium exchanger”, and 2 “chemosensory proteins (CSPs)” but
no insect olfactory proteins such as “odorant-binding proteins (OBPs)”
or “odorant receptors (ORs)”, consistent with results for other
crustaceans (Groh et al., 2014; Kozma et al., 2020b; Kozma et al.,
2018). Crustaceans and insects share a common ancestor; the latter
evolved from ancestors and migrated from the sea to land, which required
olfactory receptors for detecting signals in the air (Missbach et al.,
2014). OBPs have been reported in insects and vertebrates (Pelosi et
al., 2006; Sanchez-Gracia et al., 2009), and are suggested to adapt to
the volatile signaling molecules and pheromones more effectively (Derby
et al., 2016; Missbach et al., 2015). ORs are considered an adaptation
to the terrestrial life of insects (Krang et al., 2012). Thus far, no
study on crustaceans reports ORs, suggesting that these may be
insect-specific (Missbach et al., 2014).
iGluRs mediate neuronal communication by forming glutamate ion channels
in various animals (Benton et al., 2009). Generally, iGluRs assemble as
heterotetramers, and each monomer of iGluRs has 4 major domains, namely
amino-terminal domain (ATD), ligand-binding domain (LBD), common
pore-forming transmembrane domain (TMD), and an intracellular C-terminal
domain (CTD) (Reiner and Levitz, 2018). iGluRs can be divided into four
well-known subfamilies as follows: kainate, delta,
a-amino-3-hydroxy-5-methyl-4-isoxazo-lepropionic acid receptor (AMPARs),
and N-methyl-D-aspartate receptors (NMDARs), which perform various
functions (Regan and Furukawa, 2016). Nevertheless, iGluRs are
reportedly expressed in different parts of several crustaceans (Kozma et
al., 2020a) but changes in their expressions are rarely reported. In the
present study, several iGluRs were found to be downregulated after the
crayfish entered the MP, which warrants further investigation into the
phenomenon.
Ionotropic receptors (IRs) are a subset of ion channels that are gated
by ligands (Harzsch and Krieger, 2018). These have been reported in
crustaceans, chelicerates, nematodes, annelids, and gastropods (Derby et
al., 2016). Corset et al., (2010) suggest that IRs may have evolved from
non-NMDAR iGluRs in ancient protostomes (Croset et al., 2010). However,
unlike iGluRs, only co-receptors, IR8a and IR25a, have 4 major domains
while other IRs only have 3 domains (Derby et al., 2016). In this study,
we found 6 types of IRs or IR-like, including IR93a, IR4, IR7, IR8a,
IR21a-like, and IR25a-like. Three co-receptors (IR25a, IR8a, and IR93a)
were identified in our study but not co-receptor IR76b, which is present
in different organs of red swamp crayfish (Kozma et al., 2020a).
Furthermore, we focused on changes in the gene expressions. Notably, of
these related genes, all genes annotated to IR93a were downregulated
when the crayfish entered the MP. IR93a is one of the co-receptors of
the IR family, which is involved in the regulation of humidity and
thermal perception in insects (Enjin et al., 2016; Knecht et al., 2017).
IR93a in Drosophilid interacts with IR25a and exhibits distinct
functions when bound to different IRs (Knecht et al., 2017). A colder
aquatic environment may be a dominant factor that forces the crayfish to
enter into NMP with less movement. During this period, survival is
essential rather thanreproduction. Therefore, perception of humidity and
temperature is more important as the crayfish will then transfer to
subterranean burrows rather than the water bottom (Yoder et al., 2016).
As a co-receptor, the interactions of IR93a and other IRs are rarely
reported, and little information is available on controlling IR
expression. No evidence suggests that IRs convey negative signals to
inhibit the expression of other IRs and we speculated that the
expression of IR may be controlled by the association of specific
combinations of transcription factors with these sequences (Rytz et al.,
2013).
Notably, DEGs related to the TRP channels were typically upregulated.
TRP channels are homotetramers or heterotetramers with six transmembrane
segments. There are eight subfamilies of TRP channels, including TRPC,
TRPA, TRPP, TRPN, TRPV, TRPM, TRPM, and TRPT (Van den Eynde et al.,
2021; Venkatachalam et al., 2014; Venkatachalam and Montell, 2007). In
the present study, six types of TRP channels (TRPA1, Pyrexia TRPA,
TRPM2, TRPM3, TRPC4, and TRPV5) were identified. TRP channels
participate in many sensory processes, such as vision, olfactory,
audition, and temperature sensation, which affect the behaviors of
creatures profoundly (Fowler and Montell, 2013). As the climate becomes
warmer, more movements are generated by benthos and crayfish (Johnson et
al., 2014; Larson and Magoulick, 2011; Wittwer et al., 2018). We
speculated that the crayfish could enhance self-abilities for various
senses instinctively for environmental adaptation.
A total of 3 and 5 GPCRs were up and downregulated, respectively. GPCRs
are the most abundant cell surface receptors in the mammalian genome,
accounting for more than 1 % of the human genome (Lander et al., 2001).
In crustaceans, GPCRs are widely known to regulate physiological
processes such as neuromodulation (Rump et al., 2021), reproduction (Tu
et al., 2021), and neuropeptide conduction (Bao et al., 2018). Rump et
al. (2021) inferred that GPCRs mediate olfactory sensation in four
decapod crustaceans and suggested their function as putative
chemosensory receptors (Rump et al., 2021). Based on our results, we
reasonably suggest GPCRs may be a fundamental class of signaling
receptors rather than pheromone-specific receptors. In addition, only
one GR was identified in our study, and its expression was higher
relative to other unigenes even though there was no significant
difference between NMP and MP groups. GRs are reportedly widely
expressed in legs and mouthparts of insects but are rarely found in
crustaceans. Only the water flea, Daphnia pulex, a branchiopod
crustacean, has 58 GRs, while other studies show that only a few GRs or
GRLs are expressed in the antennae of crustaceans Panulirus argus(1 GR) (Kozma et al., 2020b), Homarus americanus (4 GRLs), andCallinectes sapidus (1 GR) (Kozma et al., 2020a). Our results
further show the scarcity of GRs in decapod crustaceans, which indicated
that these may not be the dominant chemosensory receptors in decapod
antennae.
Surprisingly, two CSPs were up-regulated and annotated in the Pfam
database to “insect pheromone-binding family, A10/OS-D”. Their
expressions were significantly higher in the MP group than in the NMP
group. In addition, the expressions of OS-D proteins were also much
higher relative to other unigenes. The OS-D gene family in insects are
chemosensory proteins (CSPs), which possess a group of hydrophobic
binding pockets (Wanner et al., 2004). The size, solubility, and overall
structure of CSPs are similar to those of OBPs (Rothemund et al., 1999)
but CSPs are less specific and more highly conserved (Jacquin-Joly et
al., 2001). Previous studies support that CSPs are sensory-related
proteins with pheromone binding function in insects (Bohbot et al.,
1998; Jacquin-Joly et al., 2001). Unlike insect-specific proteins
(OBPs), CSPs are found in crustaceans such as Daphnia carinata ,
and are expressed in ovaries, thoracic limbs, rectum, and second
antennae in both sexual and asexual females (Li et al., 2016). This
suggests that the CSPs may respond to environmental signals and control
the reproductive switch from sexual to asexual reproduction in D.
carinata (Li et al., 2016). Generally, owing to the lack of OBPs,
crustaceans only have 0-2 CSPs. (Derby et al., 2016). Our results were
consistent. Even though CSPs are broadly reported in insects,
crustaceans, and myriapods (Chipman et al., 2014; Pelosi et al., 2014;
Zhou et al., 2006), their functions in crustaceans remain unknown. Based
on our results, we speculated that
CSPs may be one of the dominant
signaling receptors of pheromone binding in male red swamp crayfish.
More attention to the expressions and changes of CSPs is needed in
further studies.
CONCLUSION
In this study, RNA-Seq technology was used to analyze the antennae
transcriptome of male red swamp crayfish between MP and NMP. A total of
13 upregulated and 9 downregulated DEGs were associated with olfactory-
and chemosensory-related functions, of which 2 related to CSPs were
remarkably upregulated after the red swamp crayfish entered the MP.
Based on the levels of CSP-related DEG expression, we suggest that CSP
may be the key receptor for chemical signals or sex pheromone reception
in the antennae of red swamp crayfish. The results presented herein will
be fundamental for future functional studies on olfactory and
chemosensory genes in P. clarkii . The findings are expected to
clarify the olfactory and chemical communication mechanisms in P.
clarkii and provide new targets for invasion management in the future.