Research Article |
Corresponding author: Leonardo Rodríguez-Sosa ( lrsosa@unam.mx ) Academic editor: Luis Ernesto Bezerra
© 2021 Gabina Calderón-Rosete, Juan Antonio González-Barrios, Celia Piña-Leyva, Hayde Nallely Moreno-Sandoval, Manuel Lara-Lozano, Leonardo Rodríguez-Sosa.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Calderón-Rosete G, González-Barrios JA, Piña-Leyva C, Moreno-Sandoval HN, Lara-Lozano M, Rodríguez-Sosa L (2021) Transcriptional identification of genes light-interacting in the extraretinal photoreceptors of the crayfish Procambarus clarkii. ZooKeys 1072: 107-127. https://doi.org/10.3897/zookeys.1072.73075
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Crayfish serve as a model for studying the effect of environmental lighting on locomotor activity and neuroendocrine functions. The effects of light on this organism are mediated differentially by retinal and extraretinal photoreceptors located in the cerebroid ganglion and the pleonal nerve cord. However, some molecular aspects of the phototransduction cascade in the pleonal extraretinal photoreceptors remain unknown. In this study, transcriptome data from the pleonal nerve cord of the crayfish Procambarus clarkii (Girard,1852) were analyzed to identify transcripts that potentially interact with phototransduction process. The Illumina MiSeq System and the pipeline Phylogenetically Informed Annotation (PIA) were employed, which places uncharacterized genes into pre-calculated phylogenies of gene families. Here, for the first time 62 transcripts identified from the pleonal nerve cord that are related to light-interacting pathways are reported; they can be classified into the following 11 sets: 1) retinoid pathway in vertebrates and invertebrates, 2) photoreceptor specification, 3) rhabdomeric phototransduction, 4) opsins 5) ciliary phototransduction, 6) melanin synthesis, 7) pterin synthesis, 8) ommochrome synthesis, 9) heme synthesis, 10) diurnal clock, and 11) crystallins. Moreover, this analysis comparing the sequences located on the pleonal nerve cord to eyestalk sequences reported in other studies reveals 94–100% similarity between the 55 common proteins identified. These results show that both retinal and pleonal non-visual photoreceptors in the crayfish equally expressed the transcripts involved in light detection. Moreover, they suggest that the genes related to ocular and extraocular light perception in the crayfish P. clarkii use biosynthesis pathways and phototransduction cascades commons.
Caudal photoreceptor, opsins, photoresponse, phototransduction, pleonal nerve cord
The freshwater crayfish is a model for studying locomotor behavioral and neurohormonal responses to light, which are mediated by retinal and extraretinal photoreceptors. The crayfish’s pleonal nerve cord (PNC), which consists of six ganglia, also responds to photostimulation previous studies have reported motor neuron activation (
In the invertebrate phototransduction mechanism, light initiates a signaling cascade that induces a depolarization of the cell membrane. One CPR is present in each half of the sixth pleonal ganglion (6th PG), with their axons coursing rostrally from the 6th PG to the brain. CPRs respond to a light stimulus with a high-frequency burst. In addition, these neurons respond trans-synaptically to mechanical stimuli. The CPR has been well-studied through electrophysiological recordings, along with analyses of the locomotor activity induced when sensing light (
The CPRs are “simple” photoreceptors due to their lack of specialized structures such as the microvilli or cilia that characterize the retinal photoreceptor (
The opsins identified include one that is sensitive to short-wavelength light (λmax = 440 nm, SWS, blue) and another sensitive to long-wavelength light (λmax = 530 nm, LWS, green). Other studies show that these simple photoreceptors have a spectral sensitivity peak at 500 nm, suggesting that they contain a rhodopsin-like photopigment (
Furthermore, a study seeking to identify the molecular mechanism of CPR transduction finds that the injection of inositol 1,4,5-trisphosphate (IP3), calcium, and guanosine nucleotide (GTP) mimics the light response (
In this study, we obtain and analyze the pleonal nerve cord transcriptome to identify potential light-interacting genes from the extraretinal photoreceptors of the freshwater crayfish P. clarkii. We also compare the encoded protein to the sequences of the eyestalk transcriptome reported in a study by
We used four adult crayfish (P. clarkii) two males and two females in their intermolt stage. The animals were acquired from a local provider in the autumn and kept in the laboratory in aerated water containers for two weeks before the experiments, with a program of 12:12 h light-dark cycles; they were fed with carrots and dried fish. The care and handling of the animals during the experimental procedures was carried out according to the policies established by the Ethics Commission. This study was approved by Research of the Faculty of Medicine, UNAM (code FM/DI/128/2019).
The pleonal nerve cords were dissected and immediately placed in the Eppendorf tube with precooled TRIzol. The tissue was preserved at -80°C prior to extraction, the tissue was homogenized manually with a precooled mortar and pestle. Total RNA was extracted from the pleonal nerve cord using TRIzol reagent following the manufacturer’s protocol (Catalog number 15596018, Invitrogen Co., Carlsbad, CA, USA). TRIzol solubilizes the biological material after the addition of chloroform (Catalog number P3803, Sigma-Aldrich, St. Louis, MO, USA), producing three phases: the upper aqueous phase containing RNA, the interphase with DNA, and the organic phase containing proteins. The aqueous phase was transferred to a new tube; the RNA was precipitated with isopropanol (Catalog number I9516, Sigma-Aldrich, St. Louis, MO, USA) and collected via centrifugation; the pellet was then washed with 75% ethanol (Catalog number E7023, Sigma Aldrich Co., St. Louis, MO, USA). The ethanol was then removed, and the pellet was resuspended in RNase-free H2O and stored at -80°C. We used 5μg of total RNA to obtain the cDNA libraries, according to the manufacturer’s protocol for the Illumina TruSeq RNA Library Preparation Kit v2 (Catalog number RS-122-2001, Illumina, San Diego, CA, USA). We performed Illumina paired-end protocol 150 bp sequencing. The library obtained was sequenced using the MiSeq Reagent kit v3 system (Catalog number MS-102-3001) according to the manufacturer’s protocol, to obtain the PNC transcriptome.
The raw data from the Illumina system were uploaded to the Galaxy Web Portal to execute a de novo assembly process, using Trinity software (
The resulting sequences were processed via the “Get ORFs” program. Any sequences shorter than 100 amino acids were ignored, to produce the protein sequences to be analyzed (
The PIA pipeline uses tools to generate maximum-likelihood phylogenetic trees for 109 genes from a Light Interaction Toolkit (LIT), a gene collection regarding light-interacting structures and their functions and development in metazoans, including those in phototransduction, eye development, pigment synthesis, circadian cycles, and other light-interacting pathways; these genes are distributed across 13 functional gene sets. This bioinformatics program places uncharacterized genes into a gene family based in pre-calculated phylogeny in a secure and accessible web server. We used the e-value 1e-20 for a BLAST search of the cutoff.
The analysis with PIA generates two results files based on the functional set of genes that are selected for analyzing the amino acid sequences. One file contains the number and sequence with all the hit proteins retrieved by the initial BLAST search, while the other file contains all selected genes placed onto their corresponding gene trees. All PIA pipeline filtered transcripts were manually analyzed to determine which sequences correspond to the possible genes implicated within the photoreception process. This procedure facilitates the elimination of duplicates and fragments and the identification of overlapping sequence sections to integrate longer sequences. For protein sequence identification, we used the Prosite database to verify the preserved domain profiles; we correlated them with functions, using the Pfam or UniProt databases (https://pfam.xfam.org/search; https://www.uniprot.org). The amino acid sequences listed in the Suppl. material
This procedure facilitates the verification of sequence identities obtained via the PIA analysis; these sequence data have been submitted to the GenBank databases under the accession number indicated in the fourth column of Tables
The Illumina system displayed 40,867,860 raw data reads; with the Novo assembler Trinity software available on the Galaxy website, we obtained 53,967 assembled nucleotides sequences in FASTA files. The PIA phylogenetic analysis was carried out using 36,558 deduced amino acid sequences with open reading frames and a minimum length of 100 amino acids. The sequence translations were done automatically in the OSIRIS platform available on the Galaxy site. The PIA analysis generated 109 maximum-likelihood trees distributed across thirteen functional gene sets, using the metazoan Light Interaction Toolkit; with the software, we obtained results for all sets from the PNC transcriptome.
We combined the genes identified in the functional gene sets “Retinoid pathway vertebrate” and “Retinoid pathway invertebrate” into Set 1. Set 2 includes the functional gene set “Photoreceptor specification and retinal determination network”; thus, we present a total of 11 gene sets in 7 Tables. This filter identified 256 sequences with potential homology with some functional gene sets from the PIA pipeline. After the analysis for each sequence, we eliminated duplicate sequences; we obtained longer consensus sequences when the ends of shorter sequences overlapped correctly. Finally, we integrated a total of 62 different transcripts from the pre-calculated phylogenetic trees. The BLAST analyses for each of the amino acid sequences identified in P. clarkii show a high conservation grade (≥ 90 %) with some other crustacean species, especially the Pacific white shrimp Penaeus vannamei.
In addition, we compared the sequences that we identified in the transcriptome of the PNC to the sequences from the transcriptome of the eyestalk. As mentioned previously, the sequences used for this comparison were obtained directly from Table mmc3, included as Suppl. material
The Tables show the names of the sequences we identified in the PNC, the number of amino acids (as deduced from the nucleotides), and the accession number in GenBank, as well as a comparison with previously reported sequences in the eyestalk. The last column shows the identity percentage between both sequences. We identified 62 genes from the PNC 55 of these were also expressed in the eyestalk transcriptome, while 38 were 100% identical to their corresponding transcripts in the PNC; 19 sequences had 94–99% similarity, while two transcripts presented a similarity of 24–41% with the transcript of the same name from the eyestalk. Only five PNC identified genes were not found in the eyestalk transcriptome.
The first functional gene set in Table
Transcripts identified from PNC through PIA pipeline compared with crayfish eyestalk transcriptome data.
Pleonal nerve cord (Current study) |
Eyestalk ( |
|||||
Gene | Top BLAST hit-Protein | aa | Access number | Contig ID (Procl_ES) | aa | Homology percentage |
Set 1. Components of the retinoid pathway in vertebrates and invertebrates | ||||||
Ralbp | Retinal-binding protein | 159 | MN110026 | 5420_1 | 431 | 100 |
Rdh11 | Retinol dehydrogenase 11 | 346 | MT601680 | 12053_0 | 350 | 100 |
Rdh13 | Retinol dehydrogenase 13 | 149 | MT601681 | WCS | – | – |
Dhrs4 | Dehydrogenase/reductase SDR family member 4-like | 289 | MT601679 | 888_7 | 282 | 100 |
Sdr16c5 | Epidermal retinol dehydrogenase 2-like isoform X2 | 122 | MT601682 | 5911_0 | 309 | 98 |
Crabp1 | Cellular retinoic acid-binding protein 1-like | 115 | MT601683 | WCS | – | – |
ninaB | Carotenoid oxygenase (RPE65) | 108 | MT601684 | 4243_0 | 523 | 41 |
ninaD | Class B scavenger receptor | 111 | MT942649 | 2476_0 | 515 | 100 |
The eyestalk transcriptome contains two sequences denominated as retinol dehydrogenase 13 (Procl_ES_4929_1 and Procl_ES_29212_0), although they showed similarities of 46% and 44%, respectively, with the sequence that we identified in the PNC.
The sequence identified as Cellular retinoic acid binding protein 1 (CRABP) contains the domain that corresponds to the Lipocalin/cytosolic fatty-acid-binding protein family. Lipocalins are transporters for small hydrophobic molecules, such as lipids, steroid hormones, bilins, and retinoids. Cytosolic CRABPs may regulate the access of retinoic acid to the nuclear retinoic acid receptors (www.uniprot.org/uniprot/P40220).
Notably, in this set, we found a low identity grade (41%) between PNC and eyestalk sequences for the protein encoded by the ninaB gene, the carotenoid oxygenase. Carotenoid oxygenases are a family of enzymes involved in carotenoid cleavage to produce retinol, commonly known as vitamin A. There are five sequences reported in the eyestalk transcriptome (Procl_ES_659_0; 4243_0: 11203_0; 30934_0: 1244_0). All of them, including the PNC sequences, contain the RPE65 superfamily conserved domain. However, they have very low similarity among themselves (see https://doi.org/10.5061/dryad.pg4f4qrqp).
Set 2 in Table
Transcripts identified from the PNC through PIA pipeline compared with the crayfish eyestalk data.
Pleonal nerve cord (Current study) | Eyestalk ( |
|||||
---|---|---|---|---|---|---|
Gene | Top BLAST hit-Protein | aa | Access number | Contig ID (Procl_ES) | aa | Homology percentage |
Set 2. Elements of photoreceptor specification and retinal determination network. | ||||||
Egfr | Tyrosine-protein kinase Fer | 873 | KY974273 | 3891_0 | 914 | 100 |
Pph | Putative retinal homeobox protein Rx2-like | 414 | MN110016 | 1058 | 422 | 100 |
Glass | Krueppel homolog 1-like | 608 | MN110021 | 652_0 | 608 | 100 |
En | Homeobox protein engrailed-1-like isoform X1 | 184 | MN110023 | WCS | – | – |
notch | Neurogenic locus Notch protein | 1210 | MN110012 | 9959 | 2464 | 100 |
Hh | Protein hedgehog-like | 190 | MN110017 | WCS | – | – |
dlx2b | Homeobox protein DLX2b-like | 299 | MT942642 | 33351_0 | 162 | 94 |
Dlx6 | Homeobox protein DLX-6-like | 305 | MT942643 | 18254_0 | 337 | 100 |
Zag-1 | Zinc finger E-box-binding homeobox protein zag-1-like | 204 | MT942647 | 12586_0 | 831 | 99 |
Zfhx3 | Zinc finger homeobox protein 3-like | 768 | MT942648 | 6525_0 | 2596 | 99 |
Set 3 of the genes, corresponding to the rhabdomeric phototransduction pathway associated with invertebrate eyes, had the highest number of PIA-identified genes, totaling 16 transcripts (Set 3, Table
Transcripts identified from PNC throught PIA pipeline compared with crayfish eyestalk transcriptome data.
Pleonal nerve cord (current study) |
Eyestalk ( |
|||||
Gene | Top BLAST hit-Protein | aa | Access number | Contig ID (Procl_ES) | aa | Homology (Percentage) |
Set 3. Elements of the rhabdomeric phototransduction pathway | ||||||
Rdgc | Serine/threonine protein phosphatase 1 | 329 | MN110024 | 983 | 329 | 100 |
Ppp2cb | Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform | 309 | MN110029 | 1697 | 309 | 100 |
G alpha | Guanine nucleotide-binding protein G(q) subunit alpha | 353 | MF279133 | 1935_0 | 353 | 94 |
Guanine nucleotide-binding protein G(s) subunit alpha | 379 | MN110031 | 1880_0 | 285 | 100 | |
Guanine nucleotide-binding protein G(i) subunit alpha | 355 | MN110025 | 6610_0 | 355 | 100 | |
Guanine nucleotide-binding protein G(o) subunit alpha | 262 | MN110018 | 2664_0 | 354 | 100 | |
G beta | Guanine nucleotide-binding protein subunit beta-5-like | 189 | MN110034 | 5560_0 | 354 | 98 |
Gnb1 | Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 | 340 | KY974308.1 | 1098_0 | 340 | 100 |
Ggamma1 | Guanine nucleotide-binding protein subunit gamma-1 | 100 | MT601685 | 3444_0 | 102 | 100 |
nonA | Protein no-on-transient A | 467 | MN110015 | – | – | – |
Dagk | Eye-specific diacylglycerol kinase isoform X3 | 902 | MF279134 | 1599_0 | 467 | 100 |
Plc | 1-Phosphatidylinositol 4,5-bisphosphate | 733 | MN110020 | 3323_0 | 1005 | 95 |
Phosphodiesterase delta-4-like | 2268_0 | 904 | 96 | |||
Pkc | cAMP-dependent protein kinase catalytic subunit 1 | 352 | MN110019 | 2373 | 507 | 96 |
Protein kinase C | 602 | MN110035 | 5727_0 | 747 | 100 | |
Arr | Beta-arrestin 1 | 263 | MN110013 | WCS | – | – |
rdgB | Phosphatidylinositol transfer protein beta isoform-like | 270 | MN110014 | 2227_0 | 270 | 100 |
Set 4. Opsins | ||||||
moody | Putative G-protein coupled receptor moody-like | 504 | MT601688 | 13547_0 | 739 | 100 |
moody | G-protein coupled receptor moody-like isoform X2 | 407 | MT601689 | 6096_0 | 411 | 99 |
Short wavelength-sensitive opsin | 391 | ALJ26468 | 11143_0 | 391 | 99 | |
Long wavelength-sensitive opsin | 377 | ALJ26467 | 23_0 | 377 | 100 |
In Set 4, the PIA pipeline identified two transcripts (Table
Set 5 includes genes identified by PIA analysis in the phylogenetic family of signaling cascades in ciliary photoreceptors (Table
Transcripts identified from PNC through PIA pipeline compared with crayfish eyestalk transcriptome data
Pleonal nerve cord (current study) |
Eyestalk ( |
|||||
Gene | Top BLAST hit-Protein | aa | Access number | Contig ID (Procl_ES) | aa | Homology percentage |
Set 5. Components of ciliary phototransduction | ||||||
Rcvrn | Neurocalcin homolog isoform X2 | 192 | MN110027 | 2966_0 | 192 | 99 |
ncs-2 | Neuronal calcium sensor 2-like | 188 | MN110022 | 3948_0 | 188 | 100 |
Rgs9 | Regulator of G-protein signaling 9-like | 170 | MN110033 | 5623_0 | 962 | 100 |
Regulator of G-protein signaling 7-like | 125 | MN110036 | 3602_1 | 486 | 99 | |
Putative regulator of G protein signaling | 255 | MN110028 | 4872_0 | 1534 | 100 |
Sets 6-9 contain enzymes in several pigment biosynthesis pathways (Table
Transcripts identified from PNC through PIA pipeline compared with crayfish eyestalk transcriptome data.
Pleonal Nerve Cord (Current study) | Eyestalk ( |
|||||
---|---|---|---|---|---|---|
Gene | Top BLAST hit-Protein | aa | Access Number | Contig ID (Prcl_ES) | aa | Homology (Percentage) |
Set 6. Elements of melanin synthesis pathway | ||||||
Csad | Cysteine sulfinic Acid Decarboxylase | 417 | MN110038 | 4782_0 | 603 | 100 |
Ppo | Prophenoloxidase | 441 | MH156427 | 2348_0 | 495 | 99 |
Set 7. Elements of pterin synthesis pathway | ||||||
Xdh | Aldehyde oxidase | 435 | MN110003 | 7559_0 | 1314 | 99 |
Indole-3-acetaldehyde oxidase-like | 536 | MN110004 | 8366_0 | 1340 | 99 | |
Sepia | Pyrimidodiazepine synthase | 241 | MN110006 | 5690_1 | 102 | 100 |
Dhpr | Dihydropteridine reductase-like | 235 | MN110005 | 1504_0 | 235 | 100 |
Pcd | Pterin-4-alpha-carbinolamine dehydratase-like | 101 | MN110007 | 2287_0 | 157 | 100 |
Spr | Sepiapterin reductase-like | 185 | MN110009 | 12527_0 | 274 | 100 |
Set 8. Elements of ommochrome synthesis pathway | ||||||
Abcg1 | ATP-binding cassette sub-family G member 1-like | 156 | MN110008 | 6760_0 | 700 | 100 |
ABC transporter, subfamily ABCB/MDR | 270 | MT942646 | 8046_0 | 1341 | 100 | |
Alad | Delta-aminolevulinic acid dehydratase | 280 | MN110039 | 3984_0 | 338 | 100 |
Alas2 | 5-aminolevulinate synthase, Erythroid-specific, Mitochondrial-like isoform X5 | 215 | MT942644 | 2230_0 | 534 | 99 |
Uros | Uroporphyrinogen-III synthase | 252 | MH156441 | 5238_0 | 345 | 99 |
Urod | Uroporphyrinogen decarboxylase | 107 | MN110037 | 4848_0 | 359 | 100 |
In the Ommochrome synthesis set, we recognized the scarlet-brown gene that encodes an ATP-binding domain of the ABC transporters family. This is a water-soluble domain of transmembrane ABC transporters; it uses the hydrolysis of ATP to translocate a variety of compounds across biological membranes and is also responsible for the transportation of guanine, tryptophan, and histamine precursors of eye pigments in planthopper (
Set 9 in Table
Because light is the primary synchronizer in the regulation of circadian rhythms, the PIA pipeline facilitates identification of some transcripts related to the molecular pathway of the circadian clock. In Set 10 of Table
Transcripts identified from PNC though PIA pipeline compared with crayfish eyestalk transcriptome data
Pleonal Nerve cord (current study) | Eyestalk (Manfrin et el. 2015) | |||||
---|---|---|---|---|---|---|
Gene | Top BLAST hit-Protein | aa | Access Number | Contig ID (Procl_ES) | aa | Homolog Percentage |
Set 10. Elements identified in the set of circadian clock | ||||||
Slo | Calcium-activated potassium channel variant 4 | 263 | QIA97593 | 4724_0 | 1172 | 100 |
Lark | RNA-binding protein lark | 308 | QIA97594 | 2543_0 | 308 | 100 |
The identified gene lark in PNC has 100% identity to the corresponding sequence identified in the eyestalk (Procl_ES_2543_0) of the crayfish; it is 52% similar to that found in Drosophila melanogaster (GenBank: Q94901.1) (see https://doi.org/10.5061/dryad.pg4f4qrqp).
Set 11 contains transcripts related to soluble proteins called crystallins (Table
. Transcripts identified from PNC through PIA pipeline compared with crayfish eyestalk transcriptome data
Pleonal Nerve Cord (Current study) |
Eyestalk ( |
|||||
Gene | Top BLAST hit-Protein | aa | Access Number | Contig ID (Procl_ES) | aa | Homology Percentage |
Set 11. Elements associated with crystalline proteins | ||||||
GstS1 | Glutathione S-transferase theta | 221 | MH156430.1 | 5690_1 | 241 | 100 |
Aldh | Aldehyde dehydrogenase (omega-crystallin) | 523 | MN110030 | 2528_0 | 523 | 100 |
Cryaa | Alpha-crystallin A chain | 139 | MT601686 | 721_0 | 163 | 24 |
ibpB | Small heat shock protein, | 184 | MG910470 | 554_0 | 184 | 100 |
hif1an | Hypoxia inducible factor 1 alpha | 1054 | MW981273 | 2830_0 | 523 | 96 |
All genes identified here from the PNC were edited for annotation and submitted to the GenBank database of the National Center for Biotechnology Information (NCBI); the assigned accession number appears in the fourth column of each table. We have included Suppl. material
Invertebrates preserve various organs to sense light. In addition to retinal photoreceptors, crayfish possess extraretinal photoreceptors in the cerebroid ganglion and the abdominal nerve cord. These photoreceptor groups contribute differentially to phototactic motor behaviors and the synchronization of circadian rhythms (
We present in this study the putative molecular components of the extraocular phototransduction system identified from the transcriptome of the PNC of the crayfish P. clarkii. We identify 62 transcripts that encode proteins potentially involved in the development processes of photoreceptor structures, phototransduction cascades, pigment biosynthesis, crystalline structures, and circadian rhythms. This constitutes the first report on the comprehensive identification of genes with a putative functional identification in extraretinal phototransduction from the PNC of the crayfish P. clarkii.
The genetic information on the PNC in this study allows us to make comparisons to the eyestalk transcriptome of the same species, as reported by
However, we also found five transcripts in the PNC that we could not identify in the eyestalk transcriptome. These differences were in Sets 1, 2, and 3, suggesting some functional peculiarities between retinal and extraretinal photoreceptors. Set 1 (corresponding to the phylogenetic family of the retinoid pathway) contains the first 2 differences. One of these genes is Rdh13, which encodes retinol dehydrogenase 13; in humans, this enzyme participates in retinoid metabolism and oxidizes all-trans-retinol, although it seems to reduce all-trans-retinal with much greater efficiency (
The second group of genes listed in Table
Interestingly, in the same set, we identified the expression of the Pph gene in the PNC. The protein encoded by this gene is the putative retinal homeobox protein Rx2, which plays a critical role in eye formation by regulating the initial specification of retinal cells. This transcription factor is necessary for mushroom body development in the Drosophila brain and is conserved between vertebrates and flies (
Generally, the rhabdomeric photoreceptors are associated with invertebrate eyes; functional Set 3 corresponds to elements of rhabdomeric phototransduction. From the identified transcripts, we can identify the 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-4-like protein (plcd4), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two second messenger molecules: DAG and inositol 1,4,5-trisphosphate (IP3). This confirms a previous study by
In this set, we have also identified four different transcripts that encode the α subunits of the heterotrimeric G proteins. These proteins can be identified by their α subunits, and they are grouped into four families based on their sequences and functionality. The four G-protein families are Gαs, Gαi, Gαq, and Gα12 (
Notably, we did not identify any of the two opsins previously reported in both the eyestalk and the PNC of this species (
Comparative alignments of opsins reported in the crayfish Procambarus clarkii A long-wavelength-sensitive opsin (UniProtKB/Swiss-Prot: P35356.1;
While the eyestalk transcriptome contains 19 sequences identified or related to the protein beta-arrestin, none were like the beta-arrestin-1 identified in the PNC. This protein participates in the deactivation of the ciliary and rhabdomeric cascades and is regenerated by retinal binding proteins (Peterson et al. 2017). This particularity merits further exploration in future studies, since beta-arrestin-1 may be a determining element in the characteristics of retinal and extraretinal photoresponsiveness in this crustacean.
Because ciliary photoreceptors are generally associated with vertebrate eyes, we did not expect to identify genes of both phototransduction cascades in this structure with simple photoreceptors. This finding suggests that these light-mediated biochemical processes are highly conserved and coexist in various invertebrate species, as previous studies have shown (
The physical appearance of the nervous tissue in the crayfish is of a whitish color; the presence of numerous enzymes that participate in the synthesis pathways of various pigments is remarkable. The pigment expression in this structure suggests that the pigments are associated with various functions. For example, one of the functional gene sets is related to the melanin synthesis pathway; melanin is a unique pigment with several functions and is found in all biological kingdoms (
Similarly, pterin is a member of the group of compounds called pteridines. Some microorganisms utilize cyanide and heavy metals for the efficient production of pterin compounds, and the antimicrobial activity of pterin has been studied and substantiated by antagonistic activity against Escherichia coli and Pseudomonas aeruginosa. Furthermore, the pterin compound has been proven to inhibit the formation of biofilm. The extracted pterin compounds may function as antioxidants or antimicrobials (
We also identify four enzymes that participate in the biosynthesis of the heme group, a cofactor involved in multiple cellular processes. One of the best known of these is the binding of oxygen to hemoglobin and myoglobin, although it has also been established that heme can interact with transcription factors that regulate genes participating in the maintenance of circadian rhythms (
In the PNC transcriptome, we have identified two transcripts that encode proteins involved in diurnal rhythms (Table
Finally, crystallins are proteins that contribute to the transparency and refractive index of the lens in vertebrates. However, their expression in the PNC is probably associated with other functions that have been described for crystallins outside of the lens; primarily, they have been linked to protective functions against some stressors and the maintenance of cytoplasmic order (
Although retinal and extraretinal photoreceptors in crayfish show significant morphological differences regarding structure, the phototransduction pathways at the molecular level have common pathways, as we show in this study. Interestingly, these very different cell types share molecular components of photoreception and other associated metabolic pathways.
We believe that the knowledge of the molecular components involved in the phototransduction of the caudal photoreceptors and other associated metabolic pathways which we present in this study can serve as an essential primary resource for future research while also facilitating the comparative analysis of photoreception processes with other species of decapod crustaceans.
Unlike the image-forming function in the eyes, extraretinal photoreception has not been deeply studied, particularly at the molecular level. In this study, we have described 62 transcripts from the PNC of the crayfish Procambarus clarkii, using a bioinformatics tool that identifies phylogenetic families of light-interacting transcripts. We compared these results to the crayfish eyestalk transcriptome described by other researchers (
The molecular components described here potentially underlie photoreceptor development, pigment synthesis, phototransduction, and the regulation of circadian rhythm from the pleonal nerve cord of this species. We identify 5 transcripts that are expressed only in the transcriptome of the PNC. Furthermore, phototransduction in the extraretinal photoreceptors presents differences that merit further elucidation in future studies.
All these sequences are available in the GenBank database. We hope that the availability of these sequences will facilitate access for other researchers performing molecular-level studies and comparative analyses on these processes in future studies on decapod crustaceans.
This research was funded by Facultad de Medicina, UNAM, Grant FM/DI/128/2019 to LRS.
Appendix S1, S2
Data type: Text
Explanation note: We have additional supporting information available online in the supporting tab for this article. Appendix S1. Some sequence alignments allow us to appreciate the degree of similarity among Drosophila melanogaster, the crayfish (P. clarkii) pleonal nerve cord, and the eyestalk. Appendix S2. Nucleotide sequence list referred from Tables