Abstract
The red swamp crayfish, Procambarus clarkii, is globally an
invasive species and has caused huge damage to aquaculture,
biodiversity, and ecology worldwide. Antennae-expressed receptors are
important for P. clarkii to detect olfactory and chemosensory
cues for mate attraction. However, only a few olfactory and
chemosensory-related genes have been reported in P. clarkii to
date. In the present study, we performed RNA sequencing to investigate
the olfactory and chemosensory-related genes of the antenna of 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.
Keywords: Procambarus clarkii , antenna transcriptome,
RNA-Seq, olfactory, chemical communication
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, 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, 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.
MATERIALS AND METHODS
Animal collection and preparation
P. clarkii were purchased (300 in total) from Guilin city,
Guangxi province, China. The average weight of the mature male crayfish
was 16.22 ± 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 −80 °C until the extraction of total RNA.