Identification and Spatiotemporal Expression of a Putative New GABA Receptor Subunit in the Human Body Louse Pediculus humanus humanus

The human louse (Pediculus humanus) is an obligatory blood feeding ectoparasite with two ecotypes: the human body louse (Pediculus humanus humanus), a competent vector of several bacterial pathogens, and the human head louse (Pediculus humanus capitis), responsible for pediculosis and affecting millions of people around the globe. GABA (γ-aminobutyric acid) receptors, members of the cys-loop ligand gated ion channel superfamily, are among the main pharmacological targets for insecticides. In insects, there are four subunits of GABA receptors: resistant-to-dieldrin (RDL), glycin-like receptor of drosophila (GRD), ligand-gated chloride channel homologue3 (LCCH3), and 8916 are well described and form distinct phylogenetic clades revealing orthologous relationships. Our previous studies in the human body louse confirmed that subunits Phh-RDL, Phh-GRD, and Phh-LCCH3 are well clustered in their corresponding clades. In the present work, we cloned and characterized a putative new GABA receptor subunit in the human body louse that we named HoCas, for Homologous to Cys-loop α like subunit. Extending our analysis to arthropods, HoCas was found to be conserved and clustered in a new (fifth) phylogenetic clade. Interestingly, the gene encoding this subunit is ancestral and has been lost in some insect orders. Compared to the other studied GABA receptor subunits, HoCas exhibited a relatively higher expression level in all development stages and in different tissues of human body louse. These findings improved our understanding of the complex nature of GABA receptors in Pediculus humanus and more generally in arthropods.


Introduction
The human louse Pediculus humanus is an obligatory blood feeding ectoparasite.Two known ecotypes of human louse exist: the head louse Pediculus humanus capitis and the body louse Pediculus humanus humanus (Phh) [1,2].Currently, pediculosis due to head louse infestations remains one of the most prevalent parasitic infestation in humans, essentially due to the misuse of non-chemical products (silicon-based formulations) and development of resistance to commonly used pediculicides [3].The human body louse is a known vector of three bacterial pathogens: Rickettsia prowazekii, responsible for epidemic typhus [4,5]; Borrelia recurrentis, responsible for relapsing fever; and Bartonella quintana, which causes trench fever [6].In the past, massive epidemics of relapsing fever have affected Africa and Eurasia, and recently many cases of louse-borne relapsing fever have been diagnosed in Germany [7], the Netherlands [8], Switzerland [9], Finland [10], and Italy [11].
Genes 2024, 15, 844 2 of 14 GABA (γ-aminobutyric acid) receptors are members of the cys-loop ligand gated ion channels (cys-loop LGICs) superfamily [12,13].They share common molecular features: the cys-loop located in the extra cellular N-terminal domain, four transmembrane domains (TM1-TM4) architecting the body of the channel, and a large highly variable intracellular domain regulating the channel functions and containing most of the protein activation sites.
In insects, GABA receptors play an important role in the transmission of impulses in the central nervous system and neuromuscular junctions that makes them suitable targets for insecticides [14].Moreover, many physiological functions of GABA receptors like the regulation of central pattern generators and the modulation of information in higher brain processing centers have been described [15].In the honey bee Apis mellifera, picrotoxin and fipronil, known GABA receptor antagonists, impaired olfactory discrimination and sucrose perception, respectively [13].
Our previous exploration of GABA receptor subunits in the human body louse Pediculus humanus humanus revealed that the genome encodes for three GABA receptor subunits, Phh-RDL, Phh-GRD, and Phh-LCCH3, that are expressed throughout the developmental stages and in different tissues [30].Moreover, Phh-RDL was able to reconstitute anionselective functional homomeric receptor while Phh-GRD and Phh-LCCH3 combined to form cation-selective hetero-pentameric receptors [30,31].
Interestingly, besides the four well-described GABA receptor subunits, an additional gene closely related to GRD and 8916 has been described in the German cockroach Blattella germanica (Bg-8916_2) and the American cockroach Periplaneta americana (Pa-8916_2) [18] as well as in the Chelicerata species, the European sheep tick Ixodes ricinus (Ir-GABA3), the common house spider Parasteatoda tepidarorium (Pt-GABA3), and the western predatory mite Galendromus occidentalis (Go-GABA3) [32].
In the present study, our analysis in the genome of the human body louse allowed us to identify a homolog of these genes annotated as γ-aminobutyric-acid receptor α-2 subunit precursor (PHUM507160; XM_002430955.1).Based on a phylogenetic analysis among other GABA receptor subunits, this gene was found to form a new (fifth) clade.We renamed the newly identified gene hocas for Homologous to Cys-loop α-like subunit.

Insects
Strain of the human body louse Pediculus humanus humanus was reared at BioMAP laboratory (University of Tours, France), maintained in standard conditions (temperature 30 • C, relative humidity 60-70%) and fed on rabbit blood four times per week.This strain of human louse was never exposed to chemical compounds.

RNA Extraction and cDNA Synthesis
Total RNA was extracted from 30 mg of young adult lice, nits, and larvae (L1, L2, L3), as well as the heads, thoraxes, and abdomens of adult lice using RNA plus extraction kit (Machery Nagel, Hoerdt, France) as per manufacturer's instructions.The cDNA was synthesized by reverse transcription from 1 mg of RNA using superscript III reverse transcriptase (Invitrogen TM , Thermo Fisher Scientific, Courtaboeuf, France) using 0.5 mM gene specific primer and 1.5 mM oligo dT primer according to manufacturer's instructions.
The phylogenetic trees were constructed in Molecular Evolutionary Genetics Analysis (MEGAXI) [36] using the neighbour-joining (Poisson model) or maximum likelihood methods based on the model of Whelan and Goldman [37], the best model being assessed with MEGAXI, and branch support being assessed with 1000 bootstrap replications.
The trees were constructed with the sequences of RDL, GRD, LCCH3, 8916, α-like, and GABA3 of the insects Collembola, Crustacea, and Chelicerata, obtained from the National Centre for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov, accessed on 20 September 2022); accession numbers of sequences were indicated in Figures 2 and 3.

RACE-PCR and Cloning of Full Length Transcripts
The 5 ends and 3 ends of γ-aminobutyric-acid receptor α-2 subunit precursor (PHUM507160; XM_002430955.1)were characterized by rapid amplification of cDNA ends (RACE-PCR) using a Gene Racer ® kit (Invitrogen, Thermo Fisher Scientific, Courtaboeuf, France) as previously described [30] using gene-specific and nested specific primers (Figure 1, Table 1).Cycles for the first and second nested PCR were 94 • C for 5 min and then 35 cycles of 94 • C for 30 s, 58 • C for 30 s, 72 • C for 1 min, and final extension 72 • C for 5 min.Fulllength γ-aminobutyric-acid receptor α-2 subunit precursor (PHUM507160; XM_002430955.1)was amplified using 1 µL of cDNA and 10 pmol of two gene specific primers (Table 1) using GoTaq DNA Polymerase (Promega, Charbonnières les Bains, France) according to the manufacturer's instructions.After migration on agarose gel, the PCR products were purified using Nucleo Spin Gel and a PCR clean-up kit (Macherey-Nagel, Hoerdt, France) according to the manufacturer's instructions.RACE-PCR and RT-PCR products were cloned in PGEM-T Easy vector (Promega) and sequenced by Eurofins genomics.

Spatial and Temporal Expression
Quantitative PCR (qPCR) analysis was performed on 100 ng of RNA extracted from adult lice; nits; and L1, L2, and L3 larval stages, as well from heads, thoraxes, and abdomens of adult lice as described in section "RNA extraction, cDNA synthesis and RACE-PCR" using the primers listed (Table 2).A 100 ng of RNA was added to 10 µL of Sybergreen master mix (Thermo Fisher Scientific) and 10 pmol of gene-specific primers (Table 2) for a final volume of 20 µL.The qPCR was performed with primary denaturation at 95 • C for 10 min followed by 40 cycles of 95 • C for 15 s and 60 • C for 1 min using the StepOnePlus Real-time PCR System (Applied Biosystems, Thermo Fisher Scientific) following the conditions recommended by the manufacturer and by applying comparative cycle threshold experiment 2 −(∆Ct) with β-actin as an endogenous control.The data from resulting relative expressions were analyzed by ANOVA test followed by Tukey's multiple comparisons test and plotted as median using GraphPad Prism 7.
Moreover, arthropods 8916, GRD and the new (fifth) clade showed a close phylogenetic relationship, suggesting the possibility of having a common ancestor.Considering these results, we hypothesized the existence of a new GABA receptor subunit in arthropods that we named HoCas for Homologous to Cys-loop α-like subunit.At the sequence identity level, insect HoCas share identity from 37.2% to 62.7% whereas Chelicerata-HoCas share identity from 49.9% to 62.7% (Table 3).Inside insects, Phh-HoCas is more closely related to Bg-8916_2 with 39.5% sequence identities.Moreover, arthropods 8916, GRD and the new (fifth) clade showed a close phylogenetic relationship, suggesting the possibility of having a common ancestor.Considering these results, we hypothesized the existence of a new GABA receptor subunit in arthropods that we named HoCas for Homologous to Cys-loop α-like subunit.At the sequence identity level, insect HoCas share identity from 37.2% to 62.7% whereas Chelicerata- In order to confirm the existence of this new clade/subunit, using BLASTP, we compared the protein sequences of Phh-HoCas, Am-8916 and Phh-GRD as templates against different insect orders, namely Crustacea, Collembola, and Chelicerata.Firstly, our results confirmed the existence of the three separate clades (GRD, 8916, and HoCas) (Figure 3A).Secondly, 8916 is conserved among arthropods as sequences were present in Chelicerata, Collembola, Crustacea, and most of the tested insect orders (Blattodae, Orthoptera, Hemiptera, Coleoptera, Neuroptera, Hymenoptera, Lepidoptera, and Diptera (Figure 3B).A systematic search for the 8916-encoding gene in the genome of Pediculus humanus (Ph-thiraptera) ended up with no results, suggesting that the human body louse possesses the four GABA subunit genes: Phh-grd, Phh-rdl, Phh-lcch3, and Phh-hocas, but possibly misses Phh-8916 (Figure 3).Finally, the sequences of the newly described clade "HoCas" were found in Chelicerata, Crustacea, Collembola, and Hemimetabola, but not in Holometabola (Figure 3B).Inside each clade (GRD, 8916, HoCas), the sequences of Insecta, Collembola, Crustacea, and Chelicerata are well segregated (Figure 3A).Interestingly, Bombyx mori Bm-GRD clustered with other insect 8916 genes (Figure 3A), confirming that, as previously described, the Bm-GRD sequence deposited in NCBI (Accession numbers: GQ890659.1 or NP_001182633.1)may be the Bm-8916 gene [38].

Cloning and Sequence Analysis of Phh-Hocas
We characterized the Phh-hocas mRNA ends by 5 and 3 RACE-PCR on total RNA extracted from nits by using set of primers binding in exon 2 and in exon 6, respectively (Table 1; Figure 1A).For 5 RACE PCR, we identified one TSS 38 nt upstream from the starting codon, and for 3 RACE two 3 end sites located, respectively, 146 nt and 68 nt downstream from an ATTAAA/AT polyA signal (Figure 1A).Reverse transcription PCR (RT-PCR) on total RNA extracted from lice with the pair of primers F3-R3 and F3-R4 allowed us to amplify 3 transcripts (Figure 1B).The sequences of Phh-hocas transcripts were deposited in GenBank with the corresponding accession numbers: the transcript Phhhocas-1 (OQ831857) confirmed the in silico annotation, the second transcript Phh-hocas-2 (OQ851502) resulted from a partial retention of intron 5, and the third transcript Phh-hocas-3 (OQ851503) resulted from alternative splicing of exon 5 and ended early at the beginning of E7.Translation and analysis of the encoded proteins revealed that both Phh-hocas-2 (with a premature stop codon in the retained intron) and Phh-hocas-3 code for truncated proteins missing TM4.Finally, the full-length cDNA of Phh-hocas-1 is 1819 bp, with an ORF of 1713 bp encoding a protein of 570 amino acids.The multiple sequence alignment of HoCas in Insecta and Chelicerata revealed that they possess all the typical features of cys-loop LGICs: the two cysteines, Phh-HoCas-C 161 and Phh-HoCas-C 175 , characteristic of the cys loop motif; 4 TM domains, TM1 (Phh-HoCas-Y 242 -S 261 ), TM2 (Phh-HoCas-R 272 -S 292 ), TM3 (Phh-HoCas-V 307 -L 334 ), and TM4 (Phh-HoCas-S 533 -F 552 ); the six loops (A-F) involved in binding to the natural ligand; and a signal peptide at position 1 to 23 (Figure 4).
hocas-1 (OQ831857) confirmed the in silico annotation, the second transcript Phh-hocas-2 (OQ851502) resulted from a partial retention of intron 5, and the third transcript Phh-hocas-3 (OQ851503) resulted from alternative splicing of exon 5 and ended early at the beginning of E7.Translation and analysis of the encoded proteins revealed that both Phh-hocas-2 (with a premature stop codon in the retained intron) and Phh-hocas-3 code for truncated proteins missing TM4.Finally, the full-length cDNA of Phh-hocas-1 is 1819 bp, with an ORF of 1713 bp encoding a protein of 570 amino acids.

Spatial and Temporal Expression of GABA Receptor Subunits
In order to verify the expression of Phh-hocas in the human louse, we performed qPCR experiments with RNA extracted throughout the development stages (nits; L1, L2, L3 larval stages; and adults) and from different parts (head, thorax, and abdomen) (Figure 5).Phh-hocas was found to be expressed in all parts of adult body louse (Figure 5 bottom) and throughout all development stages (Figure 5 top and center).Relative to actin, the expression of Phh-hocas is highest in nits (about 100-fold) (Figure 5 top) followed by larval stages (20-fold) (Figure 5).In adults, relative to actin, the highest expression of Phh-hocas was in the head (40-fold) then in the thorax and abdomen (about 10-fold) (Figure 5 bottom).Furthermore, Phh-hocas was always much more highly expressed than the other subunits.and throughout all development stages (Figure 5 top and center).Relative to actin, the expression of Phh-hocas is highest in nits (about 100-fold) (Figure 5 top) followed by larval stages (20-fold) (Figure 5).In adults, relative to actin, the highest expression of Phh-hocas was in the head (40-fold) then in the thorax and abdomen (about 10-fold) (Figure 5 bottom).Furthermore, Phh-hocas was always much more highly expressed than the other subunits. .

Discussion
GABA is the main inhibitory neurotransmitter in insects targeted by insecticides.In insects, four GABA subunits segregated in 4 separate clades are well described: RDL, GRD, LCCH3, and 8916.Depending on their association to formulate functional receptors, they could have distinct sensitivities to the natural ligand and different pharmacological profiles [13,17,20,22,23,27,28].
In the present work, in addition to our previous studies describing three GABA subunits in the human body louse, Phh-rdl [31], Phh-grd, and Phh-lcch3 [30], we have now identified another gene encoding for a putative GABA receptor subunit that we named Phh-hocas for Homologous to Cys-loop α like subunit.Phh-HoCas formed a separate clade distinct from the well-known GRD, LCCH3, RDL, and 8916 subunits.In this clade, Phh-HoCas clusters with the non-classified Bg-8916_2, Pa-8916_2, Ir-GABA-3, Go-GABA3, Pt-GABA3, and Ls-α-like sequences, suggesting the existence of a new (fifth) GABA receptor clade in arthropods.
Our systematic search revealed that HoCas and 8916 subunits are present in Insecta, Collembola, Crustacean, and Chelicerata and share a common ancestor with GRD subunits who lived about 600 million years ago [39,40].It has been shown that, in the genome of A. mellifera, D. melanogaster, and T. castaneum, the 8916 gene is located close to lcch3, while in the genome of Pediculus humanus, Phh-hocas is close to Phh-lcch3.Interestingly, our search revealed that in the genomes of Hemiptera, Chelicerata, and Blattodae 8916, hocas and lcch3 genes are located close to each other, suggesting that these genes originated from three duplication events: the first duplication event from the ancestral gene A1 leading to LCCH3 and the ancestral gene A2, A2 was further duplicated in HoCas and the ancestral gene A3, which finally duplicated to give 8916 and GRD (Figure 2).However, the absence of hocas in all Holometabola (Figure 3B) is intriguing.If a common ancestor of hocas existed as suggested previously, one hypothesis would be the loss of this gene during speciation event of Holometabola sub-order [40].
In the genome of P. humanus humanus, our analysis failed to identify the gene encoding for 8916.Assembly of insect genomes is challenging due to the large stretch of AT; we cannot exclude defaults in assembly of the human louse genome, as revealed by the missed sequences found in the Phh-GRD genomic annotation and the strikingly very long Phh-GRD gene (>7 kb) encompassing 3 contigs [30].Confirmation of the genomic annotation by comparing with the transcriptomics data is a good method to have a complete view of cys-loop LGICs as observed in I. ricinus and I. scapularis [32].In the human body louse, transcriptomics analysis [41] validated the genomic annotation [36] confirming the absence of Phh-8916.The obligatory parasitism status of P. humanus could explain the loss of some genes like 8916 in favor of others, similar to what described in Acyrtosyphon pisum lacking grd and lcch3 but having a second GluCl subunit [42].
In this study, we cloned the complete cDNA sequence of Phh-hocas of Pediculus humanus humanus (Phthiraptera) using qPCR and RACE-PCR techniques.Moreover, besides the full-length transcript, we identified two transcript variants of Phh-hocas resulting from a partial intron 5 retention/alternative splicing and premature polyA signal, both encoding proteins missing TM4.The possible role of these truncated variants is not clear and requires more investigation.Alternative splicing and retention of introns in the genes encoding cys-loop LGICs have been described in many insects and result in proteins with variable functionalities and sensitivities to insecticides.For example, retentions located at the intracellular domain between TM3 and TM4 have been observed in varroa destructor (Vd-GRD), leading to marked changes in GABA sensitivities compared to variant without retentions [27].
Results of the relative expressions revealed a remarkable higher expression of Phh-HoCas compared to other subunits in all tested parts of human body louse and throughout the development stage, while the expression of Phh-grd, Phh-lcch3 and Phh-rdl is almost the same, raising a question about the possible physiological role of Phh-hocas.It would be interesting to see if similar expression profiles can be obtained in other species and to test the effect of inhibition of gene expression in the louse physiology using RNAi.Since Phh-hocas was found to be expressed at higher levels compared to other GABA subunits of human louse especially in nits, it remains to determine whether Phh-HoCas is able to reconstitute homo or hetero pentameric functional GABA receptor.If so, and hence most of the commonly used pediculicides are not active against nits, Phh-HoCas could be a potential target to design a novel pediculicides against adult louse and nits.

Figure 1 .
Figure 1.Genomic and transcript organizations of Phh-hocas.Exons are represented by black boxes in expanded views of the contigs DS235842.1 with the annotated coding sequence ((A), top drawing) and transcripts (B).Positions of the primers are indicated by triangles; 5′ and 3′ extremities are indicated by black arrows.The bottom drawings (B) show the organization of the 3 cloned Phh-HoCas transcripts.* premature stop codon.

Figure 1 .
Figure 1.Genomic and transcript organizations of Phh-hocas.Exons are represented by black boxes in expanded views of the contigs DS235842.1 with the annotated coding sequence ((A), top drawing) and transcripts (B).Positions of the primers are indicated by triangles; 5 and 3 extremities are indicated by black arrows.The bottom drawings (B) show the organization of the 3 cloned Phh-HoCas transcripts.* premature stop codon.

Figure 2 .
Figure 2. Phylogenic tree of Phh-GABA receptor subunits compared with those of other arthropod species.The phylogenic tree was constructed by maximum likelihood methods based on Whelan and Goldman, the best model being assessed with MEGAXI, and branch support being assessed with 1000 bootstrap replications.P. humanus sequences are labeled with red triangles.A1: common ancestor of LCCH3, HoCas, 8916 and GRD.A2: common ancestor of HoCas, 8916 and GRD.A3: common ancestor of 8916 and GRD.Am for Apis mellifera; Bg for Blatella germanica; Dm for Drosophila melanogaster; Cs for Chilo suppressalis; Go for Galendromus occidentalis; Ir for Ixodes ricinus; Ls for laodelphax striatellus; Nv for Nasonia-vitripenis; Pa for Periplaneta americana; Phh for Pediculus humanus humanus; Pt for Parasteatoda tepidarorium; Sl for Spodoptera litura; Tc for Tribolium castaneum.

Figure 5 .
Figure 5. Relative expression of Phh-hocas, Phh-grd, Phh-rdl, and Phh-lcch3 throughout the development stages and in different parts of the human body louse.qPCR applying comparative cycle threshold experiment (2^− ΔCt ) was performed.Data presented as median and normalized to the

Figure 5 .
Figure 5. Relative expression of Phh-hocas, Phh-grd, Phh-rdl, and Phh-lcch3 throughout the development stages and in different parts of the human body louse.qPCR applying comparative cycle threshold experiment (2ˆ− ∆Ct ) was performed.Data presented as median and normalized to the expression of actin as endogenous control.Statistical difference (p value ≥ 0.05) was calculated using ANOVA followed by Tukey's multiple comparisons test.The results of Phh-grd, Phh-rdl, and Phh-lcch3 have been already published without the results of Phh-hocas, analyzed at the same time[30].

Table 1 .
Primers used to characterize the 5 and 3 ends of Phh-hocas by RACE-PCR and to amplify full-length cDNA.

Table 3 .
Percentage identity among the amino acid sequences of Phh-HoCas, GABA-3, and α-like GABA receptor subunits from insects (Pa for P. americana, Bg for B. germanica, Ls for Laodelphax striatellus, and Phh for Pediculus humanus humanus) and arachnids (Pt for P. tepidariorum, Ir for Ixodes ricinus, and Go for Galendromus occidentalis).Phylogenic tree of Phh-GABA receptor subunits compared with those of other arthropod species.The phylogenic tree was constructed by maximum likelihood methods based on Whelan and Goldman, the best model being assessed with MEGAXI, and branch support being assessed with 1000 bootstrap replications.P. humanus sequences are labeled with red triangles.A1: common ancestor of LCCH3, HoCas, 8916 and GRD.A2: common ancestor of HoCas, 8916 and GRD.