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.