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.