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\begin{document}

\title{Comparative transcriptome analysis of antennae from male crayfish
Procambarus clarkii during the mating period and non-mating period}



\author[1]{Zihao Zhou}%
\author[1]{Lili Mo}%
\author[1]{Wenlong Zeng}%
\author[1]{Dinghong Li}%
\author[1]{Hongying Wu}%
\author[1]{ZHENGJUN WU}%
\author[1]{Jinlong Huang}%
\affil[1]{Guangxi Normal University}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The red swamp crayfish Procambarus clarkii is one of the global invasive
species and has caused huge damage to aquaculture, biodiversity and
ecology in the world. Antennal-expressed receptors are important for P.
clarkii to detect olfactory cues for mate attraction, et al.. However,
few olfactory and chemosensory-related genes were reported in P. clarkii
until now. In the present study, we used RNA sequencing to investigate
the olfactory and chemosensory-related genes of the antenna of P.
clarkii during the non-mating and mating period. A total of 59218
unigenes with an average length of 1056.41 bp, and 4889 differentially
expressed unigenes (DEGs) with 2128 up-regulated and 2761 down-regulated
unigenes were obtained. Of which, 13 up-regulated and 9 down-regulated
DEGs were identified to associated with olfaction and chemical
reception, including 4 IRs or iGluRs, 8 G-protein coupled receptor, 5
transient receptor potential channels (TRP channels), 1 sodium-calcium
exchanger, 1 olfactory receptor, 1 isomerase and 2 chemosensory proteins
(CSPs). CSPs were preliminarily classified into pheromone receptors in
male red swamp crayfish. The results of quantitative real-time reverse
transcription PCR (RT-qPCR) showed that the expression trends of 8
selected unigenes were consistent, and this result also validated the
RNA-Seq data. Our results provide more comprehensive data of olfactory
and chemical communication mechanism after crayfish entering the mating
period.%
\end{abstract}%



\sloppy


\textbf{Comparative transcriptomics ofantennae from male
crayfish\emph{Procambarus clarkii} during the mating and non-mating
periods}

\textbf{Zihao Zhou\textsuperscript{1,2,3}, Lili
Mo\textsuperscript{1,2,3}, Wenlong Zeng\textsuperscript{3},}

\textbf{Dinghong Li\textsuperscript{3}, Hongying Wu\textsuperscript{3},
Zhengjun Wu\textsuperscript{1,2,3}, Jinlong
Huang\textsuperscript{1,2,3}*}

\textsuperscript{1}Key Laboratory of Ecology of Rare and Endangered
Species and Environmental Protection (Guangxi Normal University),
Ministry of Education, Guilin, Guangxi, 541006, China

\textsuperscript{2}Guangxi Key Laboratory of Rare and Endangered Animal
Ecology, Guangxi Normal University, Guilin, Guangxi, 541006, China

\textsuperscript{3}College of Life Sciences, Guangxi Normal University,
Guilin, Guangxi, 541006, China

\textbf{*Correspondence}

Jin-Long Huang, Key Laboratory of Ecology of Rare and Endangered Species
and Environmental Protection (Guangxi Normal University), Ministry of
Education, Guilin, Guangxi; Guangxi Key Laboratory of Rare and
Endangered Animal Ecology, Guangxi Normal University; College of Life
Sciences, Guangxi Normal University.

E-mail: \emph{jl\_huang@163.com}.

\textbf{Funding information}

This work was supported by the National Natural Science Foundation of
China (31960280), Science and Technology Base and Talent Special Project
of Guangxi Province (AD20159042), Guangxi Natural Science Foundation of
China (No. 2018GXNSFAA294024), Guangxi Universities Young and
Middle-aged Teachers Scientific Research Basic Ability Improvement
Project (2019KY0055), Key Research Program of Guangxi Normal University
(2017ZD009).

\textbf{Abstract}

The red swamp crayfish, \emph{Procambarus clarkii,} is globally an
invasive species and has caused huge damage to aquaculture,
biodiversity, and ecology worldwide. Antennae-expressed receptors are
important for \emph{P. clarkii} to detect olfactory and chemosensory
cues for mate attraction. However, only a few olfactory and
chemosensory-related genes have been reported in \emph{P. clarkii} to
date. In the present study, we performed RNA sequencing to investigate
the olfactory and chemosensory-related genes of the antenna of \emph{P.
clarkii} during the non-mating and mating periods. A total of 59218
unigenes with an average length of 1056.41 bp, and 4889 differentially
expressed unigenes (DEGs), among which 2128 were upregulated, while 2761
were downregulated were obtained. Further, 13 upregulated and 9
downregulated DEGs were associated with olfaction and chemical
reception, including 4 IRs or iGluRs, 8 G-protein coupled receptors, 5
transient receptor potential channels (TRP channels), 1 sodium-calcium
exchanger, 1 olfactory receptor, 1 isomerase, and 2 chemosensory
proteins (CSPs). CSPs were preliminarily classified as pheromone
receptors in male red swamp crayfish. The results of quantitative
real-time reverse transcriptase PCR (RT-qPCR) showed that the trends of
expression of 8 selected unigenes were consistent with RNA-Seq results.
Our results provide more comprehensive data for olfactory and chemical
communication mechanisms after crayfish enter the mating period.

\textbf{Keywords:} \emph{Procambarus clarkii} , antenna transcriptome,
RNA-Seq, olfactory, chemical communication

\textbf{INTRODUCTION}

Olfaction, as a major sensory stimulus, plays an important role in
almost vertebrates and invertebrates. For most invertebrates, olfaction
generally takes a more principal account than other sensory modalities
(Krieger and Breer, 1999). Olfaction can provide essential information
for crustaceans in finding food, avoiding predators, and performing
other social behaviors (Breithaupt, 2011; Derby and Zimmer, 2012; Hay,
2011; Kamio and Derby, 2017; Schmidt and Mellon, 2011). Olfaction
mediates the reproductive processes in crustaceans. For example, in the
marine shrimp, \emph{Lysmata wurdemanni} , the males with more olfactory
sensilla search or approach the females earlier, thereby increasing
their rate of mating success (Zhang et al., 2009).

Previous studies suggest that the antenna is the predominant olfactory
organ of most crustaceans (Harzsch et al., 2011; Urbschat and Scholtz,
2019; Waldrop, 2013). Additionally, crustaceans receive chemical signals
through several parallel channels, which can be summarized into two
modes of ``olfaction'' and ``distributed chemoreception'' (Schmidt and
Mellon, 2011). ``Olfaction'' is mediated by unimodal olfactory sensilla
called ``aesthetasc'', which are only present on the first antenna of
the crustaceans (Harzsch and Krieger, 2018). Aesthetascs are innervated
by olfactory receptor neurons (ORNs) (Derby et al., 2016). ``Distributed
chemoreception'' is mediated by various bimodal sensillae in the first
antenna (Solari et al., 2017), mouthparts (Garm et al., 2004; Garm et
al., 2003), chelipeds, and walking appendages (Altner et al., 1983;
Schmidt and Gnatzy, 1984). It is innervated by both chemoreceptor
neurons (CRNs) and mechanoreceptor neurons (MRNs). However, there is a
gap in the data for olfactory and chemo-receptors of crustaceans. So
far, the acknowledged olfactory-related proteins in crustaceans include
ionotropic receptors (IRs), ionotropic glutamate receptors (iGluRs),
G-protein coupled receptors (GPCRs), and transient receptor potential
channel (TRP channel), while proteins such as gustatory receptor-like
receptors (Grls) and gustatory receptors (GRs) are only found in a few
crustacean species (Derby et al., 2016; Kozma et al., 2020a; Kozma et
al., 2020b).

The red swamp crayfish, \emph{Procambarus clarkii} (Girard, 1852), has
its origins in North-eastern Mexico and South America (Shen et al.,
2014), and was introduced to China from Japan during the 1930s (Shen et
al., 2020). At present, it is an indispensable aquatic-economic animal
(Liu et al., 2021). However, wild crayfish in the natural ecosystem of
China have caused huge losses to agriculture, biodiversity, and
aquaculture. Moreover, the red swamp crayfish is a carrier of the white
spot syndrome virus (WSSV) and parasites, whereby it may cause infection
and death of other commercial shrimps in case of spread without
effective control (Zhu et al., 2009). Therefore, effectively and
specifically controlling wild crayfish requires more attention. In
recent years, control of invasive species using sex pheromone has been
adopted for other species of insects (Gherardi et al., 2011; Johnson et
al., 2015; Keller-Costa et al., 2014). Previous studies confirm that the
males can detect sex pheromones through their olfactory organs
uniaxially, and then search for the signal source or perform courtship
behaviors (Oyama et al., 2020). Moreover, the male red swamp crayfish
recognizes female mating receptivity by detecting the urinary components
of females (Kubec et al., 2019). However, little is known about the
chemosensory mechanism of action of these essential chemicals (such as
sex pheromone components) in this species.

Like other crustaceans, the red swamp crayfish's antenna is the most
dominating sensory organ that perceives and locates chemical signals or
pheromones released by their mates (Breithaupt, 2011). Therefore, to
further understand the communication mechanism through olfaction and
chemosensation in male red swamp crayfish, we collected the antenna of
the male crayfish in the mating period (MP) as the experimental group
and the non-mating period (NMP) as the control group. Subsequently,
next-generation sequencing (NGS) was performed to sequence the antenna
transcriptome of red swamp crayfish in MP vs. NMP. Our findings may help
better understand the mechanisms of olfactory and chemosensory responses
in red swamp crayfish, and provide a solid foundation for further
studies on the relationship between olfactory-related genes and
semiochemicals.

\textbf{MATERIALS AND METHODS}

\textbf{Animal collection and preparation}

\emph{P. clarkii} were purchased (300 in total) from Guilin city,
Guangxi province, China. The average weight of the mature male crayfish
was 16.22 \selectlanguage{ngerman}± 1.82 g. The length of the antenna was longer than 5 cm
(Figure 1). A total of 20 crayfish were cultured in one water tank and
fed twice a day on artificial food throughout the experimental period
(Li et al., 2012). All experimental animals were male crayfish and had
no contact with female crayfish or pheromones during the feeding
process. Other conditions such as water temperature and illumination
time etc., were changed naturally according to the season. We divided
the crayfish into two following groups: 100 individuals were randomly
selected as the experimental group and 100 as the control group. The
former were fed until April (the initial stage of the MP) and the latter
until January (NMP). Antennae were collected, frozen in liquid nitrogen,
and stored at \selectlanguage{english}-80 degC until the extraction of total RNA.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1 The red swamp crayfish, \emph{Procambarus clarkii} .

\textbf{Total RNA extraction and detection}

The library construction and sequencing of transcriptomes were performed
by Majob Technology Co. Ltd (Shanghai, China). Total RNA extraction was
performed using the Trizol reagent (Invitrogen, Shanghai, China). Both
concentration and purity were measured using a Nanodrop 2000
spectrophotometer (Invitrogen, Massachusetts, USA). The degradation of
extracted RNA was detected on a 1\% agarose gels and RNA integrity
number (RIN) was assessed using Agilent Bioanalyzer 2100 (Agilent
Technologies, USA).

\textbf{Library construction and sequencing}

mRNAs were enriched using magnetic beads with Oligo (dT) and randomly
fragmented using fragmentation buffer. Under the action of reverse
transcriptase, the fragmented mRNAs were used as templates for
first-strand cDNA synthesis using random hexamer primers. Subsequently,
second-strand cDNA was synthesized using DNA polymerase I and RNase H.
We added an end-repair mix (including the end-repair enzyme mix and
end-repair buffer) to patch the cohesive ends of the double-strand cDNA,
followed by the addition of tail and sequencing adapters. Subsequently,
cDNA was amplified by PCR. The cDNA library was obtained after purifying
the amplification products with AMPure XP beads. Thereafter, QuantiFluor
dsDNA System and Quantus Fluorometer (Promega, Madison, Wisconsin, USA)
were used to detect the concentration and inter size of the library,
respectively. The effective concentration of the library was accurately
quantified by q-PCR (quantitative polymerase chain reaction). Finally,
the processed samples were sequenced on the Illumina HiSeqXten/NovaSeq
6000 Sequencing Platform.

\textbf{de novo assembly and annotation}

Using SeqPrep (\emph{https://github.com/jstjohn/SeqPrep}) and Sickle
(\emph{https://github.com/najoshi/sickle}), clean reads were obtained by
filtering the sequencing adapters, primer sequences, and the low-quality
sequences from raw reads. Using the Trinity software (Grabherr et al.,
2011), obtained clean reads were assembled de novo into unigenes, which
were the encoding sequences. The corresponding amino acid sequences were
detected by TransRate (\emph{http://hibberdlab.com/transrate/}) to
obtain information on the comprehensive gene function.

The unigene sequences were compared using six databases, including the
National Center for Biotechnology Information (NCBI) Non-Redundant
Protein Sequence Database (Nr)
(\emph{ftp://ftp.ncbi.nlm.nih.gov/blast/db/}), Protein family (Pfam)
(\emph{http://pfam.xfam.org/}), Gene Ontology (GO)
(\emph{http://www.geneontology.org}) Swiss-Prot
(\emph{http://web.expasy.org/docs/swiss-prot\_guideline.html}), Cluster
of Orthologous Groups of proteins (COG)
(\emph{http://www.ncbi.nlm.nih.gov/COG/}), and Kyoto Encyclopedia of
Genes and Genomes (KEGG) (\emph{http://www.genome.jp/kegg/}). Functional
annotation was processed using the Blast2GO software (Conesa et al.,
2005).

\textbf{DEG enrichment analysis}

The Bowtie software (Langmead et al., 2009) was used to compare clean
reads with the unigene library and on combining with RSEM (Li and Dewey,
2011), the expression levels were estimated. The expression of unigenes
was calculated using FPKM (fragments per kilobase per million)
(Mortazavi et al., 2008) between two libraries. Then, the DEGs were
analyzed using the DESeq2 software (Varet et al., 2016). The
resulting\emph{P} -values were adjusted to control the false discovery
rate (FDR). The DEGs were filtered using the threshold of FDR
\textless{} 0.01 and \textbar{} log2 (Fold Change) \textbar{}[?] 1.
Subsequently, GO and KEGG pathway enrichment analyses were conducted
using the Goatools software (Klopfenstein et al., 2018).

\textbf{Real-time qPCR (RT-qPCR)}

To verify the results of our sequencing analyses, we selected 8
olfactory-related genes from the male antennae of red swamp crayfish for
RT-qPCR analysis. The total RNA was reversely transcribed into
first-strand cDNA using the PrimeScript TM 1st stand cDNA Synthesis Kit
(TaKaRa, Shanghai, China), following which the newly synthesized cDNA
was used as the template for RT-qPCR. Specific primers were designed
using the Primer 5.0 software. The \selectlanguage{greek}β-\selectlanguage{english}actin gene was considered an
internal normalization control. The primer sequences of 8 DEGs used for
RT-qPCR are listed in Table S1. The RT-qPCR reactions (20 \selectlanguage{greek}μ\selectlanguage{english}l) contained
2 \selectlanguage{greek}μ\selectlanguage{english}l cDNA, 1 \selectlanguage{greek}μ\selectlanguage{english}l of each primer, 1 \selectlanguage{greek}μ\selectlanguage{english}l dNTP Mix, 10 \selectlanguage{greek}μ\selectlanguage{english}l 2\selectlanguage{ngerman}×SYBR real-time
PCR premixture, and 6 \selectlanguage{greek}μ\selectlanguage{english}l RNase free sterilized ultrapure water. The
reaction procedure was as follows: 95 \selectlanguage{ngerman}°C for 5 min, followed by 40
cycles with 95 °C for 15 s, and 60 °C for 30 s. To confirm
reproducibility, the RT-qPCR reaction for each sample was performed in
three technical replicates and three biological replicates. The level of
expression of selected genes was calculated using the
2\selectlanguage{greek}\textsuperscript{-ΔΔCt}\selectlanguage{english} method (Livak and Schmittgen, 2001).
Comparative analyses for each target gene among different samples were
analyzed using independent sample\emph{t} -tests (SPSS, version 25.0).

\textbf{RESULTS}

\textbf{Assembly and splicing}

A total of 133,462,976 and 125017008 raw reads were obtained from the
NMP and MP groups, respectively. After quality control, we obtained at
least 5.98 Gb reads per sample. A total of 130343776 clean reads were
obtained from the NMP group and 122633780 from the MP group with a Q30
score \textgreater{} 92.04 \%, and the G + C content ranged from 47.83\%
- 49.05\% in NMP group and 44.38 \%- 47.45\% in MP group (Table S2).
Moreover, clean reads were randomly assembled into 78138 transcripts
with an average length of 1147.85 bp and an N50 length of 2379 bp. The
clean reads were further assembled into 59218 unigenes with an average
length of 1056.41 bp and an N50 length of 2229 bp. The length
distribution of transcripts and unigenes is shown in Table 1 and Figure
S1. These results showed that the data were of high quality and the
unigenes qualified for further annotation analysis. The raw data files
have been uploaded to the NCBI sequence read archive (BioProject
accession number: PRJNA508983).

Table 1 The length distribution of assembled transcripts and unigenes.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Length (bp)\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Transcripts\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Unigenes\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Total Number\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
78138\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
59218\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Largest Length\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
34133\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
34133\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Smallest Length\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
201\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
201\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Mean Length\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1147.85\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1059.41\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
N50 Length\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
2379\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
2229\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
200-500\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
38002 (49\%)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
30731 (52\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
501-1000\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
16142 (21\%)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
12097 (20\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1001-2000\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
11054 (15\%)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
7661 (13\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
\textgreater{}2000\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
12940 (16\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
8729 (15\%)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Functional annotation and classification of unigenes}

To investigate information on gene functions more comprehensively, a
total of 57812 unigenes were annotated using six databases including Nr,
Pfam, Swiss-prot, COG, GO, and KEGG. The results of annotation of the
unigenes are as follows: 18388 (31.81\%) unigenes were annotated in Nr;
14039 (24.28\%) in Pfam; 11986 (20.73\%) in Swiss-prot; 14479 (25.04\%)
in COG; 14129 (24.44\%) in GO, and 9176 (15.87\%) in KEGG (Figure 2 and
Table S3).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2 Number of annotated unigenes in 6 databases.

In the Nr database, the \emph{E} -value distribution showed that 62.71\%
of unigenes exhibited homology (\textless{} 1e-30) with previously
reported sequences, and the similarity distribution showed that 82.65\%
of the unigenes were similar (\textgreater{} 60\%) to previous sequences
(Figure 3A, B). The unigenes (12105, 65.20\%) matched best matches to
crustaceans, primarily including \emph{Penaeus vannamei}
(59.17\%),\emph{Hyalella azteca} (4.15\%), and \emph{Procambarus
clarkii} (1.88\%) (Figure 3C).

A total of 14,129 unigenes were classified into three categories,
including biological processes (BP), cellular components (CC), and
molecular function (MF) (Figure 4). Among the BPs, ``cellular process'',
``metabolic process'', and ``biological regulation'' were the top three
functions. In CCs, ``cell part'', ``membrane part'', ``organelle'',
``protein-containing complex'', and ``organelle part'' were the dominant
functions. Most unigenes were assigned to ``binding'' and ``catalytic
activity'' among MFs. Moreover, we identified 83 unigenes related to
olfactory and chemosensation by GO analysis (Table S4).

In the COG database, a total of 14,479 unigenes were assigned to 23 COG
categories (Figure 5). As shown in Figure 5, category S formed the
largest group which represented ``function unknown'' (8329 unigenes,
57.52\%), followed by category O, representing ``posttranslational
modification, protein turnover, and chaperones'' (1192 unigenes,
8.23\%), and category J, representing ``translation, ribosomal
structure, and biogenesis'' (1150 unigenes, 7.94\%).

In our study, 9176 unigenes were assigned to 334 KEGG pathways and
classified into six specific pathway groups (Figure 6). Among the six
groups, the largest was ``translation'' (1,346 unigenes), followed by
``signal transduction'' (1,181), and ``transport and catabolism'' (761).
These results can help elucidate the olfactory-related gene expression
profile during the MP of crayfish and provide theoretical data for gene
mining in crayfish.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 3 Pie charts for E-value (A), similarity (B), and species (C)
distribution of unigenes based on the Nr database.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 4 Gene Ontology (GO) classification of unigenes. All unigenes are
annotated into 3 categories, namely biological process (BP), cellular
component (CC), and molecular function (MF).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 5 COG classification for the assembled unigenes. The X-axis
indicates 23 COG categories and their detailed descriptions are
presented on the right.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 6 KEGG enrichment analysis for unigenes. The concrete description
for the KEGG pathways (level 2) is listed on the left; the number at the
end of each bar chart indicates the number of annotated unigenes. The
descriptions of six major categories of KEEG terms (level 1) are listed
on the right. The lines at the top of the figure show the KEGG level as
well.

\textbf{Identification and annotation analysis of DEGs}

Based on the DESeq2 software, a total of 4889 DEGs with the FDR
\textless{} 0.01 and \textbar{} log2 (Fold Change) \textbar{}[?] 2 were
identified between MP and NMP groups, including 2128 upregulated and
2761 downregulated unigenes (Figure S2). Furthermore, the annotations
from the GO analysis of DEGs were classified into 44 GO categories,
including the BP (18), CC (14), and MF (12). The top 3 GO terms in CC
were cell part (GO:0044464), membrane part (GO:0044425), and
protein-containing complex (GO:0032991); in BP were, cellular process
(GO:0009987), metabolic process (GO:0008152), and biological regulation
(GO:0065007); in MF were, binding (GO:0005488), catalytic activity
(GO:0003824), and structural molecule activity (GO:0005198) (Figure 7).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Figure 7 Classification of DEGs by GO annotation. The 3 categories were
BP, CC, and MF.

\textbf{Olfactory and chemosensory-related DEGs and olfactory
transduction pathway}

As shown in Table 2, 22 identified DEGs were related to olfactory and
chemosensory functions. On comparing the MP and NMP groups, 9 DEGs were
found to be downregulated, while 13 were upregulated, including 4 IRs or
iGluRs, 8 G-protein coupled receptors, 5 transient receptor potential
channels (TRP channels), 1 sodium-calcium exchanger, 1 olfactory
receptor, 1 isomerase, and 2 chemosensory proteins (CSPs). In addition,
the 9 sensory-related pathways and the number of DEGs are shown in Table
3.

Table 2 Olfactory and chemosensory regulation-related DEGs.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
NCBI reference sequence & Gene ID & Description & Categories & log2FC &
\emph{P}-value & Regulation\tabularnewline
\midrule
\endhead
AGJ51190.1 & TRINITY\_DN21918\_c0\_g1 & olfactory ionotropic receptor
IR93a & IR & -2.306 & 0.017 & down\tabularnewline
AAM47017.1 & TRINITY\_DN25415\_c0\_g2 & ionotropic glutamate receptor
subunit & IR & -3.724 & 4.187E-5 & down\tabularnewline
ROT79299.1 & TRINITY\_DN20582\_c0\_g1 & variant ionotropic glutamate
receptor & IR & -3.312 & 3.370E-5 & down\tabularnewline
XP\_023236077.1 & TRINITY\_DN21176\_c0\_g4 & peroxisomal acyl-coenzyme A
oxidase 1-like & GPCR & -6.572 & 0.021 & down\tabularnewline
AGM39710.1 & TRINITY\_DN521\_c1\_g2 & metal ion binding* & GPCR & -1.636
& 2.049E-4 & down\tabularnewline
XP\_027211194.1 & TRINITY\_DN1785\_c0\_g1 & threonine-protein kinase
26-like* & GPCR & -1.247 & 6.946E-4 & down\tabularnewline
AIT57587.1 & TRINITY\_DN3158\_c0\_g2 & crustacean cardioactive peptide
receptor, partial & GPCR & -2.250 & 1.191E-5 & down\tabularnewline
XP\_027208488.1 & TRINITY\_DN10908\_c0\_g3 & G-protein coupled receptor
GRL101-like & GPCR & -1.762 & 0.005 & down\tabularnewline
XP\_021365014.1 & TRINITY\_DN28245\_c0\_g1 & transient receptor
potential cation channel subfamily A member 1-like isoform X3 & TRP
channel & -4.519 & 0.034 & down\tabularnewline
XP\_027218241.1 & TRINITY\_DN518\_c0\_g1 & chemosensory protein & CSP &
8.280 & 6.233E-5 & up\tabularnewline
XP\_027236566.1 & TRINITY\_DN7269\_c0\_g2 & chemosensory protein* & CSP
& 7.496 & 1.077E-6 & up\tabularnewline
XP\_027224784.1 & TRINITY\_DN16621\_c0\_g1 & urotensin-2 receptor-like*
& GPCR & 1.092 & 0.033 & up\tabularnewline
XP\_018021398.1 & TRINITY\_DN22772\_c0\_g1 & G-protein coupled receptor
activity & GPCR & 1.039 & 0.010 & up\tabularnewline
PSN35490.1 & TRINITY\_DN22075\_c0\_g1 & G-protein coupled receptor
moody* & GPCR & 1.153 & 0.002 & up\tabularnewline
ROT70877.1 & TRINITY\_DN14467\_c0\_g4 & Transient receptor potential
channel pyrexia OS & TRP channel & 3.075 & 4.122E-4 & up\tabularnewline
ROT69786.1 & TRINITY\_DN24541\_c0\_g1 & putative transient receptor
potential cation channel subfamily A member 1 isoform X1 & TRP channel &
1.660 & 1.279E-4 & up\tabularnewline
XP\_027232413.1 & TRINITY\_DN4439\_c0\_g1 & ankyrin-1-like* & TRP
channel & 1.605 & 1.471E-5 & up\tabularnewline
XP\_027231576.1 & TRINITY\_DN2621\_c1\_g1 & transient receptor potential
channel pyrexia-like & TRP channel & 1.470 & 3.018E-4 &
up\tabularnewline
APG53786.1 & TRINITY\_DN5073\_c0\_g3 & transient receptor potential
cation channel pyrexia & TRP channel & 2.021 & 0.01 & up\tabularnewline
XP\_027234885.1 & TRINITY\_DN34955\_c0\_g1 & olfactory receptor 4C6-like
& OR & 1.333 & 0.006 & up\tabularnewline
AJO70196.1 & TRINITY\_DN32840\_c0\_g1 & sodium-calcium exchanger 1* &
sodium-calcium exchanger & 1.963 & 2.972E-4 & up\tabularnewline
ROT70691.1 & TRINITY\_DN4991\_c0\_g1 & putative E3 ubiquitin-protein
ligase TRIM32* & isomerase & 2.52 & 2.847E-6 & up\tabularnewline
\bottomrule
\end{longtable}

* represents the selected DEGs for RT-qPCR.

Table 3 Number of olfactory- and chemosensory-related DEGs enriched in
different KEGG pathways.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Pathway ID & Description & Up number & Down number\tabularnewline
\midrule
\endhead
map04740 & Olfactory transduction & 1 & 0\tabularnewline
map04745 & Phototransduction - fly & 4 & 0\tabularnewline
map04750 & Inflammatory mediator regulation of TRP channels & 4 &
1\tabularnewline
map04024 & cAMP signaling pathway & 4 & 2\tabularnewline
map04020 & Calcium signaling pathway & 6 & 3\tabularnewline
map04010 & MAPK signaling pathway & 8 & 0\tabularnewline
map04072 & Phospholipase D signaling pathway & 3 & 2\tabularnewline
map04151 & PI3K-Akt signaling pathway & 7 & 4\tabularnewline
map04014 & Ras signaling pathway & 5 & 2\tabularnewline
\bottomrule
\end{longtable}

\textbf{Validation by RT-qPCR analysis}

To verify the accuracy of the RNA-Seq data, 8 olfactory- and
chemosensory-related genes were selected from among the DEGs and
validated by RT-qPCR analysis. All selected DEGs were significantly
expressed in the antennae. The results of RT-qPCR were consistent with
those of RNA-Seq, indicating the reliability of the RNA-Seq data (Figure
8 and Table S1).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

Figure 8 Relative levels of selected DEG expression by RT-qPCR and
RNA-Seq. Levels are measured using the 2\selectlanguage{greek}\textsuperscript{-ΔΔCT}\selectlanguage{english} method;
\selectlanguage{greek}β-\selectlanguage{english}actin is the internal normalization control. Data are shown as mean \selectlanguage{ngerman}±
SD; * \emph{P} \textless{} 0.05, ** \emph{P} \textless{} 0.01.

\textbf{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 \emph{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 \emph{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 \emph{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, \emph{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 \emph{Panulirus
argus}(1 GR) (Kozma et al., 2020b), \emph{Homarus americanus} (4 GRLs),
and\emph{Callinectes 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 \emph{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 \emph{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.

\textbf{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 \emph{P. clarkii} . The findings are expected to
clarify the olfactory and chemical communication mechanisms in \emph{P.
clarkii} and provide new targets for invasion management in the future.

\textbf{ACKNOWLEDGMENTS}

This work was supported by grants from the National Natural Science
Foundation of China (31960280), Science and Technology Base and Talent
Special Project of Guangxi Province (AD20159042), Guangxi Natural
Science Foundation of China (No. 2018GXNSFAA294024), Guangxi
Universities Young and Middle-aged Teachers Scientific Research Basic
Ability Improvement Project (2019KY0055), and Key Research Program of
Guangxi Normal University (2017ZD009).

\textbf{CONFLICT OF INTEREST}

The authors declare no competing interests.

\textbf{DATA ACCESSIBILITY}

The raw data files have been uploaded to the National Center for
Biotechnology Information (NCBI) sequence read archive (BioProject
accession number: PRJNA508983).

\textbf{REFERENCES}

Altner I., Hatt H., Altner H. (1983) Structural properties of bimodal
chemo- and mechanosensitive setae on the pereiopod chelae of the
crayfish, \emph{Austropotamobius torrentium} . Cell and tissue research
228:357-374.

Bao C., Yang Y., Zeng C., Huang H., Ye H. (2018) Identifying
neuropeptide GPCRs in the mud crab, \emph{Scylla paramamosain} , by
combinatorial bioinformatics analysis. General and Comparative
Endocrinology 269:122-130. DOI: 10.1016/j.ygcen.2018.09.002.

Benton R., Vannice K.S., Gomez-Diaz C., Vosshall L.B. (2009) Variant
Ionotropic Glutamate Receptors as Chemosensory Receptors in Drosophila.
Cell 136:149-162. DOI: 10.1016/j.cell.2008.12.001.

Bohbot J., Sobrio F., Lucas P., Nagnan-Le Meillour P. (1998) Functional
characterization of a new class of odorant-binding proteins in the
moth\emph{Mamestra brassicae} . Biochemical and biophysical research
communications 253:489-494. DOI: 10.1006/bbrc.1998.9806.

Breithaupt T. (2011) Chemical Communication in Crayfish. In T.
Breithaupt, M. Thiel (Eds.), Chemical communication in crustaceans (pp.
257--276). Springer, New York.

Chipman A.D., Ferrier D.E.K., Brena C., Qu J., Hughes D.S.T., Schroeder
R., \emph{et al.} (2014) The First Myriapod Genome Sequence Reveals
Conservative Arthropod Gene Content and Genome Organisation in the
Centipede \emph{Strigamia maritima} . Plos Biology 12. DOI:
10.1371/journal.pbio.1002005.

Conesa A., Gotz S., Garcia-Gomez J.M., Terol J., Talon M., Robles M.
(2005) Blast2GO: a universal tool for annotation, visualization and
analysis in functional genomics research. Bioinformatics (Oxford,
England) 21:3674-3676. DOI: 10.1093/bioinformatics/bti610.

Croset V., Cummins S.F., Benton R. (2010) Ancient Protostome Origin of
Chemosensory Ionotropic Glutamate Receptors and the Evolution of Insect
Taste and Olfaction. Journal of Neurogenetics 24:30-31. DOI:
10.1371/journal.pgen.1001064.

Derby C.D., Kozma M.T., Senatore A., Schmidt M. (2016) Molecular
Mechanisms of Reception and Perireception in Crustacean Chemoreception:
A Comparative Review. Chemical Senses 41:381-398. DOI:
10.1093/chemse/bjw057.

Derby C.D., Zimmer R.K. (2012) Neuroecology of predator-prey
interactions. In Brönmark,

C., Hansson, L.A. (Eds.), Chemical Ecology in Aquatic Systems (pp.
158-171). Oxford University Press, London.

Egly R.M., Annis G.M., Chadderton W.L., Peters J.A., Larson E.R. (2019)
Predicting the potential distribution of the non-native Red Swamp
Crayfish \emph{Procambarus clarkii} in the Laurentian Great Lakes.
Journal of Great Lakes Research 45:150-159. DOI:
10.1016/j.jglr.2018.11.007.

Enjin A., Zaharieva E.E., Frank D.D., Mansourian S., Suh G.S.B., Gallio
M., et al. (2016) Humidity Sensing in Drosophila. Current Biology
26:1352-1358. DOI: 10.1016/j.cub.2016.03.049.

Fowler M.A., Montell C. (2013) Drosophila TRP channels and animal
behavior. Life Sciences 92:394-403. DOI: 10.1016/j.lfs.2012.07.029.

Garm A., Derby C.D., Hoeg J.T. (2004) Mechanosensory neurons with bend-
and osmo-sensitivity in mouthpart setae from the spiny
lobster\emph{Panulirus argus} . The Biological bulletin 207:195-208.
DOI: 10.2307/1543208.

Garm A., Hallberg E., Hoeg J.T. (2003) Role of maxilla 2 and its setae
during feeding in the shrimp \emph{Palaemon adspersus} (Crustacea:
Decapoda). The Biological bulletin 204:126-137. DOI: 10.2307/1543548.

Gherardi F., Aquiloni L., Dieguez-Uribeondo J., Tricarico E. (2011)
Managing invasive crayfish: is there a hope? Aquatic Sciences
73:185-200. DOI: 10.1007/s00027-011-0181-z.

Grabherr M.G., Haas B.J., Yassour M., Levin J.Z., Thompson D.A., Amit
I., et al. (2011) Full-length transcriptome assembly from RNA-Seq data
without a reference genome. Nature Biotechnology 29:644-U130. DOI:
10.1038/nbt.1883.

Groh K.C., Vogel H., Stensmyr M.C., Grosse-Wilde E., Hansson B.S. (2014)
The hermit crab's nose-antennal transcriptomics. Frontiers in
Neuroscience 7. DOI: 10.3389/fnins.2013.00266.

Harzsch S., Krieger J. (2018) Crustacean olfactory systems: A
comparative review and a crustacean perspective on olfaction in insects.
Progress in Neurobiology 161:23-60. DOI:
10.1016/j.pneurobio.2017.11.005.

Harzsch S., Rieger V., Krieger J., Seefluth F., Strausfeld N.J., Hansson
B.S. (2011) Transition from marine to terrestrial ecologies: Changes in
olfactory and tritocerebral neuropils in land-living isopods. Arthropod
Structure \& Development 40:244-257. DOI: 10.1016/j.asd.2011.03.002.

Hay M.E. (2011) Crustaceans as Powerful Models in Aquatic Chemical
Ecology. In: Breithaupt T, Thiel M (Eds), Chemical Communication in
Crustaceans (pp. 41--62). Springer Science \&

Business Media, New York.

Jacquin-Joly E., Vogt R.G., Francois M.C., Nagnan-Le Meillour P. (2001)
Functional and expression pattern analysis of chemosensory proteins
expressed in antennae and pheromonal gland of \emph{Mamestra brassicae}
. Chemical senses 26:833-844. DOI: 10.1093/chemse/26.7.833.

Johnson M.F., Rice S.P., Reid I. (2014) The activity of signal crayfish
(\emph{Pacifastacus leniusculus} ) in relation to thermal and hydraulic
dynamics of an alluvial stream, UK. Hydrobiologia 724:41-54. DOI:
10.1007/s10750-013-1708-1.

Johnson N.S., Tix J.A., Hlina B.L., Wagner C.M., Siefkes M.J., Wang H.,
et al. (2015) A Sea Lamprey (\emph{Petromyzon marinus} ) Sex Pheromone
Mixture Increases Trap Catch Relative to a Single Synthesized Component
in Specific Environments. Journal of Chemical Ecology 41:311-321. DOI:
10.1007/s10886-015-0561-2.

Kamio M., Derby C.D. (2017) Finding food: how marine invertebrates use
chemical cues to track and select food. Natural Product Reports
34:514-528. DOI: 10.1039/c6np00121a.

Keller-Costa T., Hubbard P.C., Paetz C., Nakamura Y., da Silva J.P.,
Rato A., et al. (2014) Identity of a Tilapia Pheromone Released by
Dominant Males that Primes Females for Reproduction. Current Biology
24:2130-2135. DOI: 10.1016/j.cub.2014.07.049.

Klopfenstein D.V., Zhang L., Pedersen B.S., Ramirez F., Vesztrocy A.W.,
Naldi A., et al. (2018) GOATOOLS: A Python library for Gene Ontology
analyses. Scientific Reports 8. DOI: 10.1038/s41598-018-28948-z.

Knecht Z.A., Silbering A.F., Cruz J., Yang L., Croset V., Benton R., et
al. (2017) lonotropic Receptor-dependent moist and dry cells control
hygrosensation in Drosophila. Elife 6. DOI: 10.7554/eLife.26654.

Kozma M.T., Hanh N.-V., Wong Y.Y., Shukla N.S., Pawar S.D., Senatore A.,
et al. (2020a) Comparison of transcriptomes from two chemosensory organs
in four decapod crustaceans reveals hundreds of candidate chemoreceptor
proteins. Plos One 15. DOI: 10.1371/journal.pone.0230266.

Kozma M.T., Ngo-Vu H., Rump M.T., Bobkov Y.V., Ache B.W., Derby C.D.
(2020b) Single cell transcriptomes reveal expression patterns of
chemoreceptor genes in olfactory sensory neurons of the Caribbean spiny
lobster, \emph{Panulirus argus} . BMC Genomics 21. DOI:
10.1186/s12864-020-07034-7.

Kozma M.T., Schmidt M., Ngo-Vu H., Sparks S.D., Senatore A., Derby C.D.
(2018) Chemoreceptor proteins in the Caribbean spiny
lobster,\emph{Panulirus argus} : Expression of Ionotropic Receptors,
Gustatory Receptors, and TRP channels in two chemosensory organs and
brain. Plos One 13. DOI: 10.1371/journal.pone.0203935.

Krang A.-S., Knaden M., Steck K., Hansson B.S. (2012) Transition from
sea to land: olfactory function and constraints in the terrestrial
hermit crab \emph{Coenobita clypeatus} . Proceedings of the Royal
Society B-Biological Sciences 279:3510-3519. DOI:
10.1098/rspb.2012.0596.

Krieger J., Breer H. (1999) Olfactory reception in invertebrates.
Science (New York, N.Y.) 286:720-723. DOI: 10.1126/science.286.5440.720.

Kubec J., Kouba A., Buric M. (2019) Communication, behaviour, and
decision making in crayfish: A review. Zoologischer Anzeiger 278:28-37.
DOI: 10.1016/j.jcz.2018.10.009.

Lander E.S., Linton L.M., Birren B., Nusbaum C., Zody M.C., Baldwin J.,
et al\emph{.} (2001) Initial sequencing and analysis of the human
genome. Nature 409:860-921. DOI: 10.1038/35057062.

Langmead B., Trapnell C., Pop M., Salzberg S.L. (2009) Ultrafast and
memory-efficient alignment of short DNA sequences to the human genome.
Genome Biology 10. DOI: 10.1186/gb-2009-10-3-r25.

Larson E.R., Magoulick D.D. (2011) Life-History Notes on \emph{Cambarus
hubbsi} Creaser (Hubbs Crayfish) from the South Fork Spring River,
Arkansas. Southeastern Naturalist 10:121-132. DOI: 10.1656/058.010.0110.

Li B., Dewey C.N. (2011) RSEM: accurate transcript quantification from
RNA-Seq data with or without a reference genome. BMC Bioinformatics 12.
DOI: 10.1186/1471-2105-12-323.

Li H., Yang Y., Xu G., Wu D., Lv W., Jiang Q., et al. (2016) Cloning,
expression and localization of DacaCSP2 and DacaCSP3 during different
reproductive stages in \emph{Daphnia carinata} . Gene 582:59-68. DOI:
10.1016/j.gene.2016.01.048.

Li Y., Deng W., Yang K., Wang W. (2012) The expression of
prophenoloxidase mRNA in red swamp crayfish, \emph{Procambarus clarkii}
, when it was challenged. Genomics 99:355-360. DOI:
10.1016/j.ygeno.2012.04.001.

Liu Q.-N., Tang Y.-Y., Li Y.-T., Zha X.-H., Yang T.-T., Zhang D.-Z., et
al. (2021) Differentially expressed genes in hemocytes of red swamp
crayfish \emph{Procambarus clarkii} following lipopolysaccharide
challenge. Aquaculture 533. DOI: 10.1016/j.aquaculture.2020.735943.

Livak K.J., Schmittgen T.D. (2001) Analysis of relative gene expression
data using real-time quantitative PCR and the 2(-Delta Delta C(T))
Method. Methods (San Diego, Calif.) 25:402-408. DOI:
10.1006/meth.2001.1262.

Missbach C., Dweck H.K.M., Vogel H., Vilcinskas A., Stensmyr M.C.,
Hansson B.S., et al. (2014) Evolution of insect olfactory receptors.
Elife 3. DOI: 10.7554/eLife.02115.

Missbach C., Vogel H., Hansson B.S., Grosse-Wilde E. (2015)
Identification of Odorant Binding Proteins and Chemosensory Proteins in
Antennal Transcriptomes of the Jumping Bristletail Lepismachilis
y-signata and the Firebrat Thermobia domestica: Evidence for an
Independent OBP-OR Origin. Chemical Senses 40:615-626. DOI:
10.1093/chemse/bjv050.

Monteclaro H.M., Anraku K., Matsuoka T. (2010) Response properties of
crayfish antennules to hydrodynamic stimuli: functional differences in
the lateral and medial flagella. Journal of Experimental Biology
213:3683-3691. DOI: 10.1242/jeb.046011.

Mortazavi A., Williams B.A., McCue K., Schaeffer L., Wold B. (2008)
Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature
methods 5:621-628. DOI: 10.1038/nmeth.1226.

Oyama T., Momohara Y., Yano H., Kamio M., Fujiyama N., Nagayama T.
(2020) Sex recognition and agonistic strategies of male and female
crayfish. Behaviour 157:575-596. DOI: 10.1163/1568539X-bja10014.

Peddio S., Sollai G., Podda C., Frau G., Palmas F., Sabatini A., et al.
(2019) The success in the short-distance communication for mating does
not depend on chemical signals in the crustacean
decapod\emph{Procambarus clarkii} (Girard, 1852). Advances in
Oceanography and Limnology 10:67-73. DOI: 10.4081/aiol.2019.8617.

Pelosi P., Iovinella I., Felicioli A., Dani F.R. (2014) Soluble proteins
of chemical communication: an overview across arthropods. Frontiers in
Physiology 5. DOI: 10.3389/fphys.2014.00320.

Pelosi P., Zhou J.J., Ban L.P., Calvello M. (2006) Soluble proteins in
insect chemical communication. Cellular and molecular life sciences :
CMLS 63:1658-1676. DOI: 10.1007/s00018-005-5607-0.

Regan M.C., Furukawa H. (2016) Deeper Insights into the Allosteric
Modulation of Ionotropic Glutamate Receptors. Neuron 91:1187-1189. DOI:
10.1016/j.neuron.2016.09.013.

Reiner A., Levitz J. (2018) Glutamatergic Signaling in the Central
Nervous System: Ionotropic and Metabotropic Receptors in Concert. Neuron
98:1080-1098. DOI: 10.1016/j.neuron.2018.05.018.

Rothemund S., Liou Y.C., Davies P.L., Krause E., Sonnichsen F.D. (1999)
A new class of hexahelical insect proteins revealed as putative carriers
of small hydrophobic ligands. Structure (London, England : 1993)
7:1325-1332. DOI: 10.1016/s0969-2126(00)80022-2.

Rump M.T., Kozma M.T., Pawar S.D., Derby C.D. (2021) G protein-coupled
receptors as candidates for modulation and activation of the chemical
senses in decapod crustaceans. Plos One 16. DOI:
10.1371/journal.pone.0252066.

Rytz R., Croset V., Benton R. (2013) Ionotropic Receptors (IRs):
Chemosensory ionotropic glutamate receptors in Drosophila and beyond.
Insect Biochemistry and Molecular Biology 43:888-897. DOI:
10.1016/j.ibmb.2013.02.007.

Sanchez-Gracia A., Vieira F.G., Rozas J. (2009) Molecular evolution of
the major chemosensory gene families in insects. Heredity 103:208-216.
DOI: 10.1038/hdy.2009.55.

Schmidt M., Gnatzy W. (1984) Are the funnel-canal organs the
'campaniform sensilla' of the shore crab, \emph{Carcinus
maenas}(Decapoda, Crustacea)? II. Ultrastructure. Cell and tissue
research 237:81-93.

Schmidt M., Mellon D., Jr. (2011) Neuronal Processing of Chemical
Information in Crustaceans.

Shen G., Zhang X., Gong J., Wang Y., Huang P., Shui Y., et al. (2020)
Transcriptomic analysis of \emph{Procambarus clarkii} affected by
''Black May'' disease. Scientific Reports 10. DOI:
10.1038/s41598-020-78191-8.

Shen H., Hu Y., Ma Y., Zhou X., Xu Z., Shui Y., et al. (2014) In-Depth
Transcriptome Analysis of the Red Swamp Crayfish \emph{Procambarus
clarkii} . Plos One 9. DOI: 10.1371/journal.pone.0110548.

Solari P., Sollai G., Masala C., Loy F., Palmas F., Sabatini A., et al.
(2017) Antennular Morphology and Contribution of Aesthetascs in the
Detection of Food-related Compounds in the Shrimp \emph{Palaemon
adspersus} Rathke, 1837 (Decapoda: Palaemonidae). Biological Bulletin
232:110-122. DOI: 10.1086/692696.

Tu S., Xu R., Wang M., Xie X., Bao C., Zhu D. (2021) Identification and
characterization of expression profiles of neuropeptides and their GPCRs
in the swimming crab, \emph{Portunus trituberculatus} . Peerj 9. DOI:
10.7717/peerj.12179.

Urbschat N., Scholtz G. (2019) Comparative analysis of the antennae of
three amphipod species with different lifestyles. Arthropod Structure \&
Development 53. DOI: 10.1016/j.asd.2019.100886.

Van den Eynde C., Vriens J., De Clercq K. (2021) Transient receptor
potential channel regulation by growth factors. Biochimica et biophysica
acta. Molecular cell research 1868:118950-118950. DOI:
10.1016/j.bbamcr.2021.118950.

Varet H., Brillet-Gueguen L., Coppee J.-Y., Dillies M.-A. (2016)
SARTools: A DESeq2-and EdgeR-Based R Pipeline for Comprehensive
Differential Analysis of RNA-Seq Data. Plos One 11. DOI:
10.1371/journal.pone.0157022.

Venkatachalam K., Luo J., Montell C. (2014) Evolutionarily Conserved,
Multitasking TRP Channels: Lessons from Worms and Flies, in: B. Nilius
and V. Flockerzi (Eds.), Mammalian Transient Receptor Potential. pp.
937-962.

Venkatachalam K., Montell C. (2007) TRP channels. Annual review of
biochemistry 76:387-417. DOI: 10.1146/annurev.biochem.75.103004.142819.

Waldrop L.D. (2013) Ontogenetic Scaling of the Olfactory Antennae and
Flicking Behavior of the Shore Crab, \emph{Hemigrapsus oregonensis} .
Chemical Senses 38:541-550. DOI: 10.1093/chemse/bjt024.

Wanner K.W., Willis L.G., Theilmann D.A., Isman M.B., Feng Q., Plettner
E. (2004) Analysis of the insect os-d-like gene family. Journal of
chemical ecology 30:889-911. DOI: 10.1023/B:JOEC.0000028457.51147.d4.

Wittwer C., Stoll S., Strand D., Vradalstad T., Nowak C., Thines M.
(2018) eDNA-based crayfish plague monitoring is superior to conventional
trap-based assessments in year-round detection probability.
Hydrobiologia 807:87-97. DOI: 10.1007/s10750-017-3408-8.

Yoder J.A., Stoeckel J.A., Helms B.S., Lorenz A.L., Jajack A.J. (2016)
Hygric stresses and strategies in maintaining the association between
crayfish and ectosymbiotic worms across vastly different environments.
Symbiosis 69:141-150. DOI: 10.1007/s13199-016-0394-y.

Zhang D., Lin J., Huang C. (2009) Relationship between olfactory sensor
number and mating in a marine shrimp, \emph{Lysmata wurdemanni} . Marine
and Freshwater Behaviour and Physiology 42:265-273. DOI:
10.1080/10236240903174750.

Zhou J.-J., Kan Y., Antoniw J., Pickett J.A., Field L.M. (2006) Genome
and EST analyses and expression of a gene family with putative functions
in insect chemoreception. Chemical senses 31:453-465. DOI:
10.1093/chemse/bjj050.

Zhu F., Du H., Miao Z.-G., Quan H.-Z., Xu Z.-R. (2009) Protection
of\emph{Procambarus clarkii} against white spot syndrome virus using
inactivated WSSV. Fish \& Shellfish Immunology 26:685-690. DOI:
10.1016/j.fsi.2009.02.022.

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