Candidate Chemosensory Genes Identified in the Procambarus clarkii (Decapoda: Cambaridae) by Antennules Transcriptome Analysis

Zhengfei Wang1*, Chenchen Shen1,2, Yiping Zhang1, Dan Tang1,3, Yaqi Luo1, Yaotong Zhai1, Yayun Guan1, Yue Wang1 and Xinyu Wang1

1Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Synthetic Innovation Center for Coastal Bio-agriculture, Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, School of Wetlands, Yancheng Teachers University, Yancheng 224001, Jiangsu Province, China

2College of Fisheries and Life sciences, Shanghai Ocean University, Shanghai 201306, China

3College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211800, Jiangsu Province, China

ABSTRACT

Olfaction plays a vital role in the survival and reproduction of shrimps. However, very little information is available about Procambarus clarkii, an important aquaculture species. Thus, we conducted the antennules transcriptome of Procambarus clarkii and identified 26 olfactory-related genes, including nine ionotropic receptors, two ionotropic glutamate receptors, ten variant ionotropic glutamate receptors, four cytochrome P450s, and one carboxylesterase. Our study suggested that ionotropic receptors were the main odorant receptors in Procambarus clarkii. Additionally, the key genes that are responsible for olfactory transduction in Procambarus clarkii were identified in the cAMP-mediated olfactory transduction pathway, including Cyclic nucleotide-gated cation channel beta-1, Adenylyl cyclase III, Calcium/calmodulin-dependent protein kinase II and Na+/Ca2+ exchanger. As a whole, our study laid a solid foundation for further functional elucidations of olfactory molecular mechanism in Procambarus clarkii, and provided further insight for a better understanding of olfaction molecular mechanism in crustaceans.

Article Information

Received 26 May 2022

Revised 19 June 2022

Accepted 01 August 2022

Available online 14 November 2022

(early access)

Published 13 January 2024

Authors’ Contribution

All authors contributed to the study conception and design. Investigation: ZFW, YPZ, CCS. Data curation: YPZ, CCS, DT. Funding acquisition: ZFW. Project administration: YPZ, CCS. Resources: ZFW, DT. Software and Visualization: YPZ, CCS. Validation: YQL, YTZ, YYG, YW, XYW. Writing original draft: YPZ. Writing review, and editing: YPZ, CCS. All authors have read and agreed to the published version of the manuscript.

Key words

Procambarus clarkii, Olfactory molecular mechanism, Chemosensory genes, Antennules, cAMP-signaling pathway

DOI: https://dx.doi.org/10.17582/journal.pjz/20220526170507

* Corresponding author: wangzf@yctu.edu.cn

0030-9923/2024/0002-0513 $ 9.00/0

Copyright 2024 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Abbreviations

AC3, adenylyl cyclase III; CAMK2, calcium/calmodulin dependent protein kinase II; CG, creophagy group; CNGB1, cyclic nucleotide-gated cation channel beta-1; CNGs, cyclic nucleotide-gated ion channels; CXEs, carboxylesterases; CYPs, cytochrome P450s; DEGs, differentially expressed genes; GO, gene ontology; GPCRs, G-protein-coupled receptors; HG, herbivory group; IGluRs, ionotropic glutamate receptors; IRs, ionotropic receptors; KEGG, kyoto encyclopedia of genes and genomes; KOG, euKaryotic orthologous groups; ML, maximum-likelihood; NJ, neighbor-joining; NR, non-redundant protein database; NCX, Na+/Ca2+ exchanger; ODEs, odorant-degrading enzymes; ORF, open reading frame; ORNs, olfactory receptor neurons; ORs, odorant receptors; PFAM, protein families database; TMDs, transmembrane domains; VIGluRs, variant ionotropic glutamate receptors; WG, water group.

INTRODUCTION

Red swamp crayfish, Procambarus clarkii (Crustacea: Decapoda), has high production and economic values in China (Zhou et al., 2017). In 2019, the total annual production of P. clarkii reached nearly 2,089,600 tons based on the Ministry of Agriculture data in China (www.yyj.moa.gov.cn). P. clarkii is an omnivorous species in freshwater, and adult crayfish in nature have a wide range of diets: Fish, shrimp, clam, algae, batata, and corn (Gherardi, 2006; Yue et al., 2010). Moreover, olfaction is the main factor affecting the ingestion of crayfish, and the antennules of P. clarkii have previously been reported as the main olfactory organ used to distinguish stimuli in the ingestion process (Kruangkum et al., 2019; Charles et al., 2016). However, the olfaction molecular mechanisms associated with the antennules of P. clarkii remain largely unknown.

Olfaction plays a critical role throughout the life history of most animals (Du et al., 2018). Odor molecules can stimulate the olfactory organ to form an electric signal in the olfactory sensory neurons via various chemoreceptors and proteins, leading to diverse behaviors through a series of responses. The processes of olfactory detection are mediated by several functionally interrelated gene families: Odorant receptors (ORs) and ionotropic receptors (IRs) during chemosensory stimulus reception; odorant-degrading enzymes (ODEs) including antennal-expressed carboxylesterases (CXEs) and cytochrome P450s (CYPs) during degradation of redundant odorant molecules. ORs belonging to the G-protein-coupled receptors (GPCRs) family can form binding sites for odorant to transform binding chemical signals into neural signals (Benton et al., 2006). IRs, which are evolved from ionotropic glutamate receptors (IGluRs) in ancient protostomes, respond to both environmental and cellular signals and function similarly to ORs (Benton et al., 2009; Croset et al., 2010). A kind of special IRs, called IR co-receptors (e.g. IR25a, IR8a, and IR93a), is necessary and highly conserved in insects, and IR co-receptors can bind with IRs to take part in detecting odor (Abuin et al., 2011; Rytz et al., 2013). Furthermore, the extra odor molecules might affect the sensitivity of olfactory organs, ODEs can quickly degrade odor molecules and terminate short odor signals for continuous stimulation (Prestwich, 1987) to maintain the sensitivity of olfactory organs.

The major olfactory receptor of crustaceans was presented as the IRs identified in antennules (Daniel, 1997). Studies on the olfactory systems of crustaceans have been carried out on Decapoda such as Panulirus argus, Pagurus bernhardus, and Coenobita clypeatus (Charles et al., 2016). As confirmed by in situ hybridization, PargIR25a and PargIR93a were expressed in P. argus (Corey et al., 2013). In hermit crab, 18 IRs including IR25a and IR93a were identified in P. bernhardus, and IR25a, IR93a, and 27 divergent IRs including 22 variant ionotropic glutamate receptors (VIGluRs) were identified in C. clypeatus (Groh et al., 2013; Groh-Lunow et al., 2014). Despite the aforementioned studies, the researches on the olfactory receptors of Decapoda are still insufficient.

To get a better understanding of the molecular mechanisms underlying olfactory chemoreception in Decapoda, we selected P. clarkii as a representative economic shrimp and performed transcriptomics on antennules via Illumina sequencing technology. This study could help better understand the mechanisms of olfactory recognition in P. clarkii, and provide a solid foundation for further study on other Decapoda species.

MATERIALS AND METHODS

Animals and sample preparation

The intact and healthy P. clarkii were obtained from Yancheng Sunshine Supermarket, Jiangsu, China. All P. clarkii were cultured in freshwater tanks (150 × 150 × 100 cm) with adequate aeration at 20±1℃. The experimental water quality parameters were monitored daily to maintain conditions of pH with 7.0 - 7.5, salinity at 20 parts per thousand (ppt) and dissolved oxygen > 6 mg/L. In order to achieve the better effect of food induction, we chose a variety of plant foods and meat foods and observed shrimp feeding during the temporary rearing period. In a seven-day temporary rearing, P. clarkii were fed with algae, corn, duckweed, clam, shrimp, and fish at 19:00 daily. By comparing the amount of food left, it’s found that P. clarkii preferred to eat corn (plant food) and clam (meat food). Therefore, corn and clam were selected as the inducer in the formal experiment. After 7 days of rearing in the aquarium, every 3 shrimps were randomly selected as a group, with a total of three groups of different inducers: water group (WG); herbivory group (HG); creophagy group (CG). The inducers (corn filtrate in HG; clam filtrate in CG and pure water in WG) were released 2 cm in front of the forehead of P. clarkii, and the antennules were immediately dissected from the individuals after 2 min. All samples were immediately frozen in liquid nitrogen, and stored at -80℃ until used.

Illumina sequencing and de novo assembly

Total RNA of antennules samples were extracted using Trizol reagent (Invitrogen, CA, USA). The purity and concentration of the RNA were detected by Nanodrop 2000, and the RNA integrity was detected by agarose gel electrophoresis. The libraries of tissues were constructed by TruseqTM RNA sample prep Kit, sequenced on an Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA). To obtain high-quality clean reads, trimmed adaptor sequences and low-quality reads were filtered from the RNA-seq raw reads. The transcriptome de novo assembly was carried out by Trinity with parameters set as default, only the longest transcript of each cluster was considered as a unique gene (unigene) for subsequent annotation.

Gene functional annotation

The unigenes were annotated by searching against the NCBI Non-Redundant protein database (NR), String, Swiss-Prot database, and EuKaryotic Orthologous Groups (KOG) databases, with a threshold of E-value ≤ 10-5. Based on the results of NR database annotation, the Blast 2GO program was employed to perform Gene Ontology (GO) annotation. The relationships among the unigenes and construct pathways were predicted by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Differential expression analysis and enrichment analysis

The expression level of each unigene was measured by using the fragments per kilobase of exon model per million mapped reads (FPKM) method using RSEM (Mortazavi et al., 2008). Significantly differentially expressed genes (DEGs) were selected with multiple check corrections based on the standard FDR < 0.05, |logFC| >= 1. To ensure the high quality of differentially expressed gene data, we eliminated the unigenes with obsessively low levels of expression (RPKM < 1). GO term enrichment and KEGG pathway functional enrichment analysis of DEGs was performed using Goatools (https://github.com/tanghaibao/GOatools) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do), respectively. We defined the corrected p-value <= 0.05 to indicate significantly enriched (Minoru et al., 2008).

Identification of chemosensory genes

The annotation results were used to select putative olfactory receptors, including IRs, IGluRs, VIGluRs, CXEs, and CYPs during iterative searches. An open reading frame (ORF) finder (https://www.ncbi.nlm.nih.gov/orffinder/) was used to predict the complete ORF and TMHMM Server 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) was used to predict the transmembrane domains (TMDs) (Touhara et al., 2009). Putative olfactory receptors need at least one TMD (Sheng et al., 2017; Shen et al., 2021). According to previous researches, ionotropic receptors (IRs, IGluRs, VIGluRs) should contain at least one characteristic structural domain, ligand channel (LCD-domain: PF00060) or ligand-binding (LBD-domain: PF10613).

Construction of phylogenetic trees

To explore the relationships about olfactory receptors in Decapoda, olfactory receptors from different species (Litopenaeus vannamei, Eriocheir japonica sinensis, P. argus, C. clypeatus, P. bernhardus, and Palaemon serratus) were used for a comparison with our data. Amino acid sequence alignments were performed using MEGA5, with phylogenetic trees constructed using the neighbor-joining (NJ) method with 10,000 bootstrap replications. Additionally, to ensure the accuracy of phylogenetic analysis, the phylogenetic trees of P. clarkii chemosensory genes were also constructed in IQ-TREE using the best-fitting substitution-model automatically with maximum-likelihood (ML) with 1,000 bootstrap replications. FigTree v 1.4.4, Adobe Illustrator CS4 and Photoshop C36 were used to visualize the phylogenetic trees.

RESULTS

Sequencing and assembly

The transcriptomic characteristics of P. clarkii were analyzed with high throughput Illumina sequencing technology. A total of 56,902,942, 51,010,266 and 49,809,984 raw reads were, respectively obtained from WG, HG, CG. After quality checks, 56,875,944, 50,939,572 and 49,744,810 clean reads were severally found for WG, HG, CG (Table Ⅰ). Further assembly analysis showed that 755,807 transcripts contributed to 582,398 unigenes with a mean length of 479.89 bp (Supplementary Fig. S1), the N50 length of 512 bp, and a GC content of 45.02% (Supplementary Table SⅠ).

 

Table I. Statistics for Illumina reads from the Procambarus clarkii antennules transcriptome.

Sample	Raw reads	Clean reads	Q20 (%)	Q30 (%)	GC (%)
WG	56,902,942	56,875,944	96.72	92.03	48.93
HG	51,010,266	50,939,572	97.4	93.52	44.54
CG	49,809,984	49,744,810	97.35	93.46	46.19

 

Table II. Summary of Procambarus clarkii annotation information.

Category	Unigenes	Percentage (%)
GO	35,628	20.07
KEGG	24,640	13.88
NR	45,459	25.61
PFAM	22,629	12.75
STRING	12,731	7.17
SWISS-PROT	36,436	20.52

 

Functional annotation

According to the results of annotation, 45,459 (25.61%) and 36,436 (20.52%) unigenes showed significant matches in the NR and Swiss-Prot protein databases, while 35,628 (20.07%) and 24,640 (13.88%) unigenes could be classified by GO and KEGG databases, respectively (Table II). GO annotation analysis suggested that the unigenes could be classified into three parts: “biological process”, “cellular component” and “molecular function” (Supplementary Fig. S2). Most of the unigenes were in the terms “cellular process”, “cell”, and “binding”. KOG is a tool to identify orthologous and paralogous proteins in eukaryotes and assign them to functional categories. In P. clarkii, the most enriched terms were in “General function prediction only” (Supplementary Fig. S3). In the KEGG database, the most of unigenes were assigned to the “Global and overview maps” pathways (Supplementary Fig. S4).

Identification of candidate ionotropic receptors (IRs, IGluRs and VIGluRs)

Twenty-one candidate ionotropic receptors were identified including 9 PcIRs, 2 PcIGluRs, and 10 PcVIGluRs in P. clarkii antennules transcriptome. All PcIRs contained at least one characteristic structural domain (LCD-domain, LBD-domain) and 1-2 TMDs except PcIR93a with 4 TMDs (Table Ⅲ). To understand the phylogenetic relationships of IRs in Decapoda, NJ tree and ML tree were constructed for 9 PcIRs and IRs of additional Decapoda species (Fig. 1). Although two different methods were adopted, the results tended to be consistent in this study. The phylogenetic trees (NJ tree and ML tree) indicated that PcIR93a was clustered with highly-conserved IR93a subfamilies. Then, IR co-receptors (IR25a and IR8a) of Decapoda formed other branch. Moreover, the majority of PcIRs and PargIR7 were clustered into one branch implying the closely orthologous relationships with P. argus.

 

 

Table III. Statistics of Procambarus clarkii chemosensory genes information.

Gene name	TMD (No.)	Complete ORF	Species	Identity (%)	Pfam
PcIR1	2	No	Panulirus argus	76.9	PF10613.6; PF00497.17
PcIR2	1	No	Panulirus argus	70.3	PF10613.6
PcIR3	2	No	Panulirus argus	59.8	PF00060.23
PcIR4	1	No	Panulirus argus	65.4	PF00060.23
PcIR5	1	No	Panulirus argus	81.8	PF00060.23
PcIR6	1	No	Panulirus argus	63.1	PF00060.23
PcIR7	1	No	Panulirus argus	68.3	PF00060.23
PcIR8	2	No	Panulirus argus	66.7	PF10613.6; PF00060.23; PF00497.17
PcIR93a	4	No	Panulirus argus	82.4	PF00060.23; PF10613.6; PF00497.17
PcIGluR1	2	Yes	Cancer borealis	75.4	PF10613.6; PF00060.23; PF00497.17
PcIGluR2	3	Yes	Homarus americanus	89.1	PF00060.23; PF10613.6; PF00497.17; PF01094.25
PcVIGluR1	1	No	Coenobita clypeatus	79.4	PF00060.23
PcVIGluR2	2	No	Coenobita clypeatus	76.7	PF00060.23
PcVIGluR3	1	No	Coenobita clypeatus	57	PF00060.23
PcVIGluR4	2	No	Coenobita clypeatus	70.2	PF00060.23
PcVIGluR5	1	No	Coenobita clypeatus	74.7	PF00060.23
PcVIGluR6	2	Yes	Coenobita clypeatus	56.4	PF00060.23
PcVIGluR7	4	No	Coenobita clypeatus	54.7	PF00060.23
PcVIGluR8	1	Yes	Coenobita clypeatus	46.9	PF00060.23
PcVIGluR9	2	No	Coenobita clypeatus	71.8	PF00060.23
PcVIGluR10	3	No	Coenobita clypeatus	62	PF00060.23
PcCXE1	1	Yes	Eriocheir japonica sinensis	63.8	PF00135.25; PF07859.10
PcCYP1	1	Yes	Panulirus argus	61.8	PF00067.19
PcCYP2	1	Yes	Hyalella azteca	68.3	PF00067.19
PcCYP3	1	Yes	Faxonius limosus	94.3	PF00067.19
PcCYP4	2	Yes	Penaeus vannamei	72.9	PF00067.19

 

Two PcIGluRs (PcIGluR1 and PcIGluR2) exhibited four structural domains (LCD-domain, LBD-domain, ATD-domain: PF01094, SBD-domain: PF00497) were identified (Table Ⅲ). According to the structural analyses, complete ORFs and 2-3 TMDs were observed in PcIGluRs. Furthermore, we identified 10 PcVIGluRs denoted as PcVIGluR1-10. All PcVIGluRs contained 1-4 TMDs and LCD-domain. To explore the relationships between IRs, IGluRs, and VIGluRs, the phylogenetic trees for the analysis of ionotropic receptors in P. clarkii, L. vannamei, E. j. sinensis, and other Decapoda were constructed with ML and NJ methods (Fig. 2). Due to diverse algorithms, litter differences in locations in the NJ tree and ML tree were found, but the taxa are generally consistent. Results showed that co-receptors (IR8a and IR25a) were clustered into one branch, and IR93a clustered together alone. In addition, all co-receptors formed clusters together with IGluRs suggesting that these co-receptors had distinct orthologous relationships with IGluRs (Fig. 2). VIGluRs were divided into two branches which clustered with IRs in one large branch demonstrating a closer relationship between the VIGluRs and IRs (Fig. 2).

Identification of CXEs and CYPs

One putative PcCXE and four PcCYPs were selected from the P. clarkii antennules transcriptomes (Table Ⅲ). PcCXE and PcCYPs had 1-2 TMDs and complete ORFs. PcCXE had the two structural domains (Carboxylesterase family: PF00135.25 and Abhydrolase-3 family: PF07859.10), and PcCYPs had the characteristic structural domain (P450 family: PF00067.19).

Analysis of differential gene expression

To identify the key genes that are responsible for olfactory perception in P. clarkii, the unigenes of different libraries were subjected to a comparative analysis. A total of 4,224 genes were significantly differentially expressed (2,393 up-regulated and 1,831 down-regulated) in the HG vs. CG; 7,520 DEGs (3,683 up-regulated and 3,837 down-regulated) in the HG vs. WG; and 6,114 DEGs (2,331 up-regulated and 3,783 down-regulated) in the CG vs. WG (Fig. 3). Remarkably, The KEGG functional enrichment analysis performed in the DEGs revealed variations in the “olfactory transduction” pathway (ko04740) mechanisms for the HG, CG, and WG (Fig. 4). In the CG vs. WG, Adenylyl cyclase Ⅲ (AC3) was down-regulated in the olfactory transduction pathway, which might have adverse effects on the cAMP signaling pathway. In the HG vs. WG, three DEGs, Cyclic nucleotide-gated cation channel beta-1 (CNGB1), Calcium/calmodulin-dependent protein kinase II (CAMK2), and Na+/Ca2+ exchanger (NCX), were up-regulated in the olfactory pathway. These data indicated the key genes that were responsible for the response of P. clarkii in olfactory transduction.

 

DISCUSSION

Olfactory perception is crucial for shrimps. However, the molecular basis of olfactory perception in shrimps is still relatively few. Therefore, we focused on the antennules of P. clarkii to analyze its internal olfactory perception mechanism. A suite of chemosensory genes from the P. clarkii antennules were identified by transcriptome analysis, including nine PcIRs, two PcIGluRs, ten PcVIGluRs, four PcCYPs, and one PcCXE.

IRs play a vital role in the olfactory transduction of odor molecules in crustaceans (Charles et al., 2016). Nine PcIRs were found in the antennules of P. clarkii, including IR93a. The co-receptor IR93a is an essential component of the olfactory detection process, assisting other IRs to detect odor (Rytz et al., 2013). In phylogenetic trees (NJ tree and ML tree), PcIR93a and other Decapoda IR93a were all clustered in the same branch, and it may play a key role in the P. clarkii olfactory perception. Then other IR co-receptors (IR25a and IR8a) of Decapoda species formed one branch. The results showed that co-receptor families were highly conserved in Decapoda. Additionally, PcIRs had relatively closely orthologous relationships with PargIR7, which suggested the olfactory mechanisms correlation between P. clarkii and P. argus. Unfortunately, the number of IRs in P. clarkii was limited. We speculated that the reasons can be attributed to two aspects: (i) the time of odor stimulation was too short to respond; (ii) P. clarkii was not hungry enough to enhance their desire for ingestion.

 

In previous studies, the IR evolutionary process was indistinct. It was reported that IRs are a new family of IGluRs related to excitatory neurotransmission (Wang et al., 2018). According to the analysis of the phylogenetic tree, we found most IR co-receptors had distinct orthologous relationships with IGluRs. This phenomenon proved that IR co-receptors were probably most closely resemble the ancestral IRs derived from IGluRs (Benton et al., 2009; Rytz et al., 2013). However, IRs are different from IGluRs in the olfactory perception of crustaceans. The majority of IRs were clustered in one branch and formed a large branch with VIGluRs. It indicated that IRs have distinct orthologous relationships with VIGluRs, and they might play a role together in the Decapoda olfactory mechanism.

Rapid catabolism of redundant odorant molecules by ODEs including CXEs and CYPs might regulate odorant/pheromone concentration, participating in signal termination to maintain the sensitivity of olfactory organs (Vosshall, 2008). SlCXE7 works in pheromone signal termination and reduction of surplus odorant in Spodoptera littoralis (Durand et al., 2011). It was reported that treatment of the sensilla by a P450 inhibitor caused anosmia, suggesting that the P450-mediated metabolic clearance of odor is required for olfactory detection (Pottier et al., 2012). In our study, PcCXE and PcCYPs may protect P. clarkii olfactory receptor neurons (ORNs) against disturbance from unnecessary odors.

The transduction of odorant signal was reported to occur primarily through a cAMP-mediated pathway in most olfactory neurons (Juilfs et al., 1997), and Cyclic nucleotide-gated ion channels (CNGs) can be directly activated by cAMP (Gutièrrez-Mecinas et al., 2008). In this study, CNGB1 was present in the CNGs of the olfactory sensory neurons and was up-regulated in the HG, suggesting CNGB1 was involved in the cAMP-mediated olfactory transduction pathway. Furthermore, AC3 is an important component of the cAMP-signaling pathway (Bakalyar and Reed, 1990; Monneron and D’Alayer, 1980) and can transform ATP into cAMP causing cellular signal response (Taussig and Gilman, 1995). AC3 was significantly differentially expressed in the CG, implying that the importance of the cAMP-mediated olfactory transduction pathway in the olfactory perception of P. clarkii. Then Calcium ion balance was beneficial to olfactory signal transduction (Chen et al., 2006). In our study, NCX and CAMK2 were up-regulated in the HG. NCX plays an important quantitative role in the Ca2+ transport of neurons as a relatively new family of transporters (Hassan and Lytton, 2019), and the up-regulation of NCX revealed that NCX promoted the dynamic adaptation of Ca2+ signaling in sensory conduction neurons of the olfactory system to maintain calcium ion balance in P. clarkii. CAMK2 is a multifunctional serine/threonine-protein kinase that delivers calcium signals in a variety of cellular processes (Wong et al., 2019), and it was reported that calmodulin can inhibit G protein-coupled receptor kinases to indirectly activate the cAMP signaling pathway (Jones and Reed, 1989). These results suggested that the cAMP-mediated pathway might play an important role in the olfactory transduction of P. clarkii.

CONCLUSION

In summary, our study provided the first report on antennal transcriptome analysis in P. clarkii. A suite of chemosensory genes was identified including nine PcIRs, two PcIGluRs, ten PcVIGluRs, four PcCYPs, and one PcCXE. IRs originated from IGluRs are the main olfactory receptors of P. clarkii, and closely related to VIGluRs. IRs and VIGluRs might play a key role in the olfactory transduction of P. clarkii. Moreover, we found one type of olfactory transduction pathway in P. clarkii: cAMP-signaling pathway. In the olfactory transduction pathway, we report four genes (CNGB1, AC3, NCX, and CAMK2) related to olfactory detection in P. clarkii. This study will facilitate functional research on olfactory mechanisms in P. clarkii and other Decapoda.

Acknowledgement

This study was supported by grants from National Natural Science Foundation of China to ZFW (No. 31702014), Jiangsu Provincial Key Laboratory for Bioresources of Saline Soils Open Foundation to ZFW (Grant no. JKLBS2019006), and Doctoral Scientific Research Foundation of Yancheng Teachers University to ZFW.

DECLARATION

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animal were followed.

Consent to publish

All authors have read and approved the submission of this manuscript.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20220526170507

Statement of conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Abuin, L., Bargeton, B., Ulbrich, M.H., Isacoff, E.Y., Kellenberger, S., and Benton, R., 2011. Functional architecture of olfactory ionotropic glutamate receptors. Neuron, 69: 44-60. https://doi.org/10.1016/j.neuron.2010.11.042

Bakalyar, H., and Reed, R., 1990. Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science, 250: 1403-1406. https://doi.org/10.1126/science.2255909

Benton, R., Sachse, S., Michnick, S.W., and Vosshall, L.B., 2006. Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. PLoS Biol., 4: 240-257. https://doi.org/10.1371/journal.pbio.0040020

Benton, R., Vannice, K.S., Gomez-Diaz, C., and Vosshall, L.B., 2009. Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell, 136: 149-162. https://doi.org/10.1016/j.cell.2008.12.001

Charles, D., Derby, Mihika, T., Kozma, Adriano, Senatore, Manfred, and Schmidt, 2016. Molecular mechanisms of reception and perireception in crustacean chemoreception: A comparative review. Chem. Senses., 41: 381-398. https://doi.org/10.1093/chemse/bjw057

Chen, T., Takeuchi, H., and Kurahashi, T., 2006. Odorant inhibition of the olfactory cyclic nucleotide-gated channel with a native molecular assembly. J. Gen. Physiol., 128: 365-371. https://doi.org/10.1085/jgp.200609577

Corey, E.A., Bobkov, Y., Ukhanov, K., Ache, B.W., and Matsunami, H., 2013. Ionotropic crustacean olfactory receptors. PLoS One, 8: e60551. https://doi.org/10.1371/journal.pone.0060551

Croset, V., Rytz, R., Cummins, S.F., Budd, A., Brawand, D., Kaessmann, H., Gibson, T.J., Benton, R., and Stern, D.L., 2010. Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction. J. Neurogenet., 24: 30-31. https://doi.org/10.1371/journal.pgen.1001064

Daniel, B., 1997. Chemosensory activation of an antennular grooming behavior in the spiny lobster, Panulirus argus, is tuned narrowly to l-glutamate. Biol. Bull. US, 193: 107-115. https://doi.org/10.2307/1542756

Du, L., Zhao, X., Liang, X., Gao, X., Liu, Y., and Wang, G., 2018. Identification of candidate chemosensory genes in Mythimna separata by transcriptomic analysis. BMC Genomics, 19: 518. https://doi.org/10.1186/s12864-018-4898-0

Durand, N., Carot-Sans, G., Bozzolan, F., Rosell, G., Siaussat, D., Debernard, S., Chertemps, T., and Maïbèche-Coisne, M., 2011. Degradation of pheromone and plant volatile components by a same odorant-degrading enzyme in the cotton leafworm, Spodoptera littoralis. PLoS One, 6: e29147. https://doi.org/10.1371/journal.pone.0029147

Gherardi, F., 2006. Crayfish invading europe: The case study of Procambarus clarkii. Mar. Freshw. Behav. Physiol., 39: 175-191. https://doi.org/10.1080/10236240600869702

Groh, K.C., Heiko, V., Stensmyr, M.C., Ewald, G.W., and Hansson, B.S., 2013. The hermit crab’s nose antennal transcriptomics. Front. Neurosci. Switz., 7: 266. https://doi.org/10.3389/fnins.2013.00266

Groh-Lunow, K.C., Getahun, M.N., Ewald, G.W., and Hansson, B.S., 2014. Expression of ionotropic receptors in terrestrial hermit crab’s olfactory sensory neurons. Front. Neurosci. Switz., 8: 448. https://doi.org/10.3389/fncel.2014.00448

Gutièrrez-Mecinas, M., Blasco-Ibáez, J.M., Nàcher, J., Varea, E., and Crespo, C., 2008. Distribution of the a3 subunit of the cyclic nucleotide-gated ion channels in the main olfactory bulb of the rat. Neuroscience, 153: 1164-1176. https://doi.org/10.1016/j.neuroscience.2008.03.012

Hassan, M.T., and Lytton, J., 2019. Potassium-dependent sodium-calcium exchanger (nckx) isoforms and neuronal function. Cell Calcium, 86: 102135. https://doi.org/10.1016/j.ceca.2019.102135

Jones, D.T., and Reed, R.R., 1989. Golf: An olfactory neuron specific--g protein involved in odorant signal transduction. Science, 244: 790-795. https://doi.org/10.1126/science.2499043

Juilfs, D.M., Fulle, H.J., and Zhao, A.Z., 1997. A subset of olfactory neurons that selectively express cgmp-stimulated phosphodiesterase (pde2) and guanylyl cyclase-d define a unique olfactory signal transduction pathway. P. natl. Acad. Sci. USA, 94: 3388-3395. https://doi.org/10.1073/pnas.94.7.3388

Kruangkum, T., Saetan, J., Chotwiwatthanakun, C., Vanichviriyakit, R., Thongrod, S., Thintharua, P., Tulyananda, T., and Sobhon, P., 2019. Co-culture of males with late premolt to early postmolt female giant freshwater prawns, Macrobrachium rosenbergii resulted in greater abundances of insulin-like androgenic gland hormone and gonad maturation in male prawns as a result of olfactory receptors. Anim. Reprod. Sci., 210: 106198. https://doi.org/10.1016/j.anireprosci.2019.106198

Minoru, K., Michihiro, A., Susumu, G., Masahiro, H., Mika, H., Masumi, I., Toshiaki, K., Shuichi, K., Shujiro, O., Toshiaki, T., and Yoshihiro, T., 2008. Kegg for linking genomes to life and the environment. Nucl. Acids Res., 36: 480-484. https://doi.org/10.1093/nar/gkm882

Monneron, A., and D’Alayer, J., 1980. Subcellular localization of adenylate cyclase in thymocytes. BBA-Gen. Subjects., 629: 50-60. https://doi.org/10.1016/0304-4165(80)90263-9

Mortazavi, A., Williams, B.A., Mccue, K., Schaeffer, L., and Wold, B., 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods, 5: 621-628. https://doi.org/10.1038/nmeth.1226

Pottier, M.A., Bozzolan, F., Chertemps, T., Jacquin-Joly, E., and Mabèche-Coisne, M., 2012. Cytochrome p450s and cytochrome p450 reductase in the olfactory organ of the cotton leafworm Spodoptera littoralis. Insect Mol. Biol., 21: 568-580. https://doi.org/10.1111/j.1365-2583.2012.01160.x

Prestwich, D.G., 1987. Chemistry of pheromone and hormone metabolism in insects. Science, 237: 999-1006. https://doi.org/10.1126/science.3616631

Rytz, R., Croset, V., and Benton, R., 2013. Ionotropic receptors (irs): Chemosensory ionotropic glutamate receptors in Drosophila and beyond. Insect Biochem. Mol., 43: 888-897. https://doi.org/10.1016/j.ibmb.2013.02.007

Shen, C., Tang, D., Zhang, Y., Wu, L., Luo, Y., Tang, B., and Wang, Z., 2021. Identification of putative ingestion-related olfactory receptor genes in the Chinese mitten crab (Eriocheir japonica sinensis). Genes Genom., 43: 479-490. https://doi.org/10.1007/s13258-021-01065-4

Sheng, S., Liao, C.W., Zheng, Y., Zhou, Y., Xu, Y., Song, W.M., He, P., Zhang, J., and Wu, F.A., 2017. Candidate chemosensory genes identified in the endoparasitoid Meteorus pulchricornis (Hymenoptera: Braconidae) by antennal transcriptome analysis. Comp. Biochem. Physiol. D., 22: 20-31. https://doi.org/10.1016/j.cbd.2017.01.002

Taussig, R., and Gilman, A.G., 1995. Mammalian membrane-bound adenylyl cyclases. J. biol. Chem., 270: 1. https://doi.org/10.1074/jbc.270.1.1

Touhara, K., and Vosshall, L.B., 2009. Sensing odorants and pheromones with chemosensory receptors. Annu. Rev. Physiol., 71: 307-332. https://doi.org/10.1146/annurev.physiol.010908.163209

Vosshall, L.B., 2008. Scent of a fly. Neuron, 59: 685–689. https://doi.org/10.1016/j.neuron.2008.08.014

Wang, T.T., Si, F.L., He, Z.B., and Chen, B., 2018. Genome-wide identification, characterization and classification of ionotropic glutamate receptor genes (iglurs) in the malaria vector Anopheles sinensis (Diptera: Culicidae). Parasite Vector, 11: 34. https://doi.org/10.1186/s13071-017-2610-x

Wong, M., Samal, A.B., Lee, M., Vlach, J., Novikov, N., Niedziela-Majka, A., Feng, J.Y., Koltun, D.O, Brendza, K.M., Kwon, H.J., Schultz, B.E., Sakowicz, R., Saad, J.S., and Papalia, G.A., 2019. The kn-93 molecule inhibits calcium/ calmodulin-dependent protein kinase II (camkII) activity by binding to ca2+ /cam. J. mol. Biol., 431: 1440-1459. https://doi.org/10.1016/j.jmb.2019.02.001

Yue, G.H., Li, J., Bai, Z., Wang, C.M., and Feng, F., 2010. Genetic diversity and population structure of the invasive alien red swamp crayfish. Biol. Invasions., 12: 2697-2706. https://doi.org/10.1007/s10530-009-9675-1

Zhou, M., Abbas, M.N., Kausar, S., Jiang, C.X., and Dai, L.S., 2017. Transcriptome profiling of red swamp crayfish (Procambarus clarkii) hepatopancreas in response to lipolysaccharide (lps) infection. Fish Shellfish Immunol., 71: 423-433. https://doi.org/10.1016/j.fsi.2017.10.030