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Published in final edited form as: Comp Biochem Physiol Part D Genomics Proteomics. 2021 May 12;39:100840. doi: 10.1016/j.cbd.2021.100840

RNA-Seq analysis of the blue light-emitting Orfelia fultoni (Diptera: Keroplatidae) suggest photoecological adaptations at the molecular level

Danilo T Amaral a, Carl H Johnson b, Vadim R Viviani a,c,*
PMCID: PMC8495875  NIHMSID: NIHMS1744411  PMID: 34022525

Abstract

Bioluminescence in Diptera is found in the Keroplatidae family, within Arachnocampininae and Keroplatinae subfamilies, with reported occurrences in Oceania, Eurasia, and Americas. Larvae of Orfelia fultoni, which inhabit stream banks in the Appalachian Mountains, emit the bluest bioluminescence among insects, using it for prey attraction, similarly to Arachnocampa spp. Although bioluminescence has a similar prey attraction function, the systems of Arachonocampininae and Keroplatinae subfamilies are morphologically/biochemically distinct, indicating different evolutionary origins. To identify the possible coding genes associated with physiological control, ecological adaptations, and origin/evolution of bioluminescence in the Keroplatinae subfamily, we performed the RNA-Seq analysis of O. fultoni larvae during day and night and compared it with the transcriptomes of Arachnocampa luminosa, and reanalyzed the previously published proteomic data of O. fultoni against the RNA-Seq dataset. The abundance of chaperones/heat-shock and hexamerin gene products at night and in luciferase enriched fractions supports their possible association and participation in bioluminescence. The low diversity of copies/families of opsins indicate a simpler visual system in O. fultoni. Noteworthy, gene products associated with silk protein biosynthesis in Orfelia were more similar to Lepidoptera than to the Arachnocampa, indicating that, similarly to the bioluminescent systems, at some point, the biochemical apparatus for web construction may have evolved independently in Orfelia and Arachnocampa.

Keywords: Arachnocampa, Bioluminescence, Chaperones, Diptera, Orfelia, RNA-Seq

1. Introduction

Bioluminescence, the light emission produced by living organisms, has been found in marine and terrestrial species, from bacteria to fishes. It is produced by the oxidation of substrates, called luciferins, by molecular oxygen in reactions catalyzed by enzymes known as luciferases, sometimes in the presence of cofactors. Biochemical and morphological data indicate that bioluminescence arose at least 40 times independently during the evolution of life on Earth. It is associated with vital functions for different organisms, such as defense, illumination, sexual communication, prey attraction, etc. (Hastings, 1983; Haddock et al., 2010).

In terrestrial organisms, bioluminescence is found mainly in insects, especially in Coleoptera and Diptera, however, the molecular origin and physiological control of bioluminescence in the latter group are still poorly investigated (Herring, 1978; Viviani, 2002; Viviani et al., 2002).

In Diptera, the bioluminescence is found only in four genera and two subfamilies of the Keroplatidae family: Arachnocampa in the Arachnocampininae subfamily, and Orfelia, Keroplatus, and Neoceroplatus in the Keroplatinae subfamily (Fulton, 1939; Harvey, 1952; Falaschi et al., 2019). Among them, the most well-studied genus with well-recognized distribution is Arachnocampa. This genus contains nine species, distributed in Australia, Tasmania, and New Zealand (Oceania), and all of them display bioluminescence (Viviani et al., 2002). The genus Orfelia has been described in several parts of the world, such as Europe and China (Caspers, 1991; Bechev, 2002; Cao et al., 2008), however, only Orfelia fultoni, which occurs in stream banks in the Appalachian Mountains in Eastern USA, is bioluminescent (Fulton, 1939; Viviani et al., 2020). The genus Keroplatus occurs in Eurasia with five luminescent species (Santini, 1982; Baccetti et al., 1987; Oba et al., 2011). Finally, a luminescent species of the genus Neoceroplatus was recently discovered in South-America (Falaschi et al., 2019).

Bioluminescence in Arachnocampa larvae is emitted by lanterns, which are located in a whitish area on the tip of the abdomen, consisting of the terminal ends of Malpighian tubules (Gatenby, 1959). The biochemistry of the bioluminescent system of this genus, which was originally studied by Lee (1976) and Viviani et al. (2002), was shown to involve luciferin, luciferase, and ATP. Transcriptional and proteomic analysis in Arachnocampa were done using the lantern and whole-body tissues showing the presence of several luciferase-like enzymes related to the Coleoptera luciferases (~30% identity), which were considered as the actual luciferases in this genus (Sharpe et al., 2015; Silva et al., 2015). Most studies agree that the Arachnocampa (AR) luciferin is different from other bioluminescent systems, except for a single study claiming that the luciferin of Arachnocampa is the same as fireflies (Trowell et al., 2016). Very recently, AR luciferin was shown to be a derivative of xanthurenic acid and tyrosine (Watkins et al., 2018).

Bioluminescence in O. fultoni larvae (see Fig. Viviani et al., 2020) is produced mainly by translucent anterior and posterior regions, and at a lower level throughout the body of the larvae. Similar to Arachnocampa larvae, O. fultoni larvae are carnivorous, using their blue bioluminescence to attract and capture small invertebrates, which remain trapped in mucus sticky silk web constructed by the larvae (Fulton, 1941; Meyer-Rochow, 2007). Bassot (1978) showed that bioluminescence in O. fultoni is associated with rows of black bodies, which was recently corroborated by CCD imaging studies conducted by our group (Viviani et al., 2020). The biochemical system originally showed to involve a 140 kDa dimeric luciferase, however, recent proteomic and biochemical analyses suggested that the functional luciferase could be a 220 kDa trimeric enzyme; and a novel distinct luciferin which was named keroplatin, due to its widespread occurrence in the body of both luminescent and non-luminescent larvae of Keroplatinae subfamily (Viviani et al., 2020). The association of keroplatin with high molecular weight protein complexes and possibly mitochondria was originally called substrate-binding fraction (SBF). Keroplatin may display additional biological functions in non-luminescent Keroplatinae (Viviani et al., 2002, 2020).

Whereas biochemical, transcriptional, and morphological data were reported for Arachnocampa spp. (Sivinski, 1982; Pugsley, 1984; Merrit and Clarke, 2011), studies about the molecular origin and control of bioluminescence in O. fultoni and other members of the Keroplatinae subfamily are still scant.

To better understand the molecular origin and physiology of O. fultoni bioluminescent system and other Keroplatinae subfamily members, and its ecological adaptations, we performed three distinct analyses: (1) evaluated the transcriptional profile of O. fultoni during the day and night; (2) reanalyzed previous proteomic data generated to O. fultoni (Viviani et al., 2020) against the transcriptomic data to explore, with more details, possible protein candidates involved in the bioluminescence system in this species and (3) and test orthologous genes under positive selection in the Keroplatidae family. Based on previous proteomic and molecular studies in the family, which indicated the distinct bioluminescent systems origins in Arachnocampininae and Keroplatinae, here we also analyzed whether the ecological adaptations of vision and web construction may have evolved from a common ancestor or by convergence. The results obtained here provide the first transcriptional analysis of O. fultoni and of the Keroplatinae subfamily.

2. Material and methods

2.1. Sampling

O. fultoni larvae were manually collected during spring nights from 8 PM to 11 PM (May 3rd to 6th, 2016) in the Appalachian Mountains, Highlands, NC (USA). A total of six larvae were used to RNA-Seq. We froze at −80 °C the whole body of three individuals during the night (between 11:00 and 11:30 PM) and three individuals during the day (between 9:00 and 10:00 AM). To avoid as much as possible the stressful condition of the samples, although such samples may have undergone some physiological disturbance during the collecting, we kept them in a plastic bottle with soil and controlled humidity. During the sampling, we were not capable of distinguishing male and female larvae.

2.2. Total RNA extraction, cDNA libraries construction, and Illumina sequencing

We extracted the total RNA from the whole body of six large larvae; three for each tested condition (triplicates), during the night (high bioluminescent activity period) after the collection, and the others during the day (low bioluminescent activity period). Total RNA was extracted using TRIzol reagent (Life tech., USA), according to the instructions of the manufacturer with one additional chloroform step and two additional ethanol 70% wash step.

The RNA extraction quality was checked by spectrophotometry using a NanoDrop spectrophotometer (Thermo Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Tech., USA). The mRNA isolation and the cDNA library constructions were performed at the VANTAGE facility at Vanderbilt University (TN, USA), using the TruSeq RNA Sample Preparation Kit (Illumina, Inc., USA). The six tagged cDNA libraries were pooled in equal ratios and used for 2 × 150 bp paired-end sequencing on a single lane of the Illumina HiSeq3000, according to the manufacturer’s instructions (Illumina, Inc., USA). We deposited the raw reads files under project number PRJNA578979.

2.3. De novo transcriptome assembly, functional annotation, transcripts abundance, and differential expression gene analysis (DEG)

The raw reads were checked by FastQC 0.11.5 software (Andrews, 2010) and those one with low-quality were removed using FASTX TOOLKIT 0.0.13. The filtered reads were used to perform the de novo assembly in Trinity 2.2.0 (Grabherr et al., 2011). The data was in silico normalized and the default settings were used. The transcripts with more than 200 bp were translated to an amino acid by the TransDecoder tool (available at https://github.com/TransDecoder/TransDecoder/releases).

The transcripts were subjected to a similarity search against NCBI’s non-redundant (nr) and UNIPROT/SWISS-PROT databases using the BLASTp algorithm on Blast2GO 3.0 software (Conesa et al., 2005), with a cut-off e-value of ≤10–5. Following the steps of the Blast2GO mapping, we performed the gene ontology (GO) terms with e-value <10–6, annotation cut-off >55. We also determined the pathways annotations using the Kyoto Encyclopedia of Genes and Genomes (KEGG).

We compared the abundance of isoforms in each condition using the FPKM values, which were calculated by the align_and_estimate_abundance.pl script. We performed the differential expression analysis also comparing the normalized transcript abundance values between day-time and nighttime conditions. For that, we quantified the transcript and gene product abundance, qualified the samples and biological replicates (three in each condition), and performed the DE analysis methods using the DESeq2 package (Love et al., 2014).

2.4. De novo transcriptome, functional annotation, transcripts abundance, and DE of Arachnocampa luminosa

We also performed the de novo transcriptome assembly to A. luminosa, using the Illumina data produced by Sharpe et al. (2015), from two different tissues: photogenic and non-photogenic. This transcriptome is useful as a comparison model to our O. fultoni transcriptome data. The A. luminosa read set was directly uploaded to the Galaxy server using the EBA tools (Project Number: PRJNA290397). In this server, we checked the quality of the samples, trimmed the reads, and de novo assembled using Trinity 2.2.0 software with default settings. The contigs were translated into an amino acid by TransDecoder.

All assembled proteins were subjected to a similarity search against the SwissProt database using BLASTp, as described above. We also compared the abundance of genes/isoforms in each tissue and performed the differential expression analysis also comparing the normalized transcript abundance values between the tissues.

2.5. Bioinformatic reanalyzes of mass spectrometry (MS) data of O. fultoni

Previously, we performed mass spectrometry analysis of partially purified luciferase fractions of O. fultoni larvae and cross-checked with the RNA-Seq data, to evaluate possible gene products associated with the bioluminescence (Viviani et al., 2020). Here, based on the transcriptomic approaches, including DEG analysis, we made further detailed analysis of the previous proteomic results using the transcriptome dataset. The MS/MS samples were analyzed using Sequest v.27 (Thermo Fisher Scientific, CA, USA), with a fragment ion mass tolerance of 0,00 Da and a parent ion tolerance of 2,5 Da, using the RNA-Seq obtained in this study as a database. We used Scaffold v.4.11.0 to validate MS/MS-based peptide and protein identifications.

2.6. Orthologous protein identification and test of positive selection

The orthologous protein groups were identified by OrthoFinder v.2.3.1 (Emms and Kelly, 2015) using the default settings. The single-copy orthologs identified were submitted to the test of positive selection. The nucleic acid sequences were codon aligned using the software PRANK v.150803 (Löytynoja and Goldman, 2005). We used the branch model (M = 2) of the codeml program in the PAML v4.8 (Yang, 2007) to calculate ω within each branch. We also applied the site models M7 and M8 to test whether positive selection had affected specific residues in each gene. We calculate the null model (M = 0) for the entire tree for the likelihood ratio test (LRT). The user tree was assumed to be ((O. fultoni, A. luminosa), (Bradysia odoriphaga)) for all genes. The Bayes empirical Bayes (BEB) was performed to calculate posterior probabilities for the site classes.

3. Results and discussion

3.1. Transcriptomes assembly

The high-throughput sequencing resulted in ca. 60 million reads/samples with a length of 150 bp. More than 94% of reads displayed Phred Quality Scores higher than 30. The total number of assembled bases obtained was 136,388,844 and the number of contigs was 186,422. After the protein translation, we obtained 45,536 putative gene products. The transcriptome assembly using all the six published libraries of A. luminosa (three libraries from photogenic tissues and three libraries from non-photogenic tissues) produced a total of 69,979 transcripts. After the protein translation, we obtained 28,981 possible gene products.

3.2. Functional annotation of O. fultoni transcriptome

The number of samples with blast hits was 40,242 (88.4%), while the number of annotated samples was 24,059 (52.8%). As expected, the first annotation hits in terms of species distributions were the closer mosquitoes Aedes sp. and Culex quinquefasciatus (Diptera: Culicidae). We plotted the annotation of the complete RNA-Seq from the whole body of Orfelia and Arachnocampa (Fig. 1). The KEGG pathway annotation, which performed a systematic analysis of inner-cell metabolic pathways and gene functions using Blast2GO software showed 134 pathways for the whole body of O. fultoni larvae (Table SM1).

Fig. 1.

Fig. 1.

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Annotation and enzyme statistics plot using WEGO: (A) Gene Ontology (GO) classification of O. fultoni and (B) Gene Ontology (GO) classification of A. luminosa. The y-axis indicates the percentage of total genes in each category.

The pathways recovered for O. fultoni were associated with thiamine metabolism, porphyrin metabolism, and arginine and proline metabolisms. All the pathways are strongly associated with general physiology, including energy metabolism, nervous system control, and nutrition, ‘cell development in flies (Bursell, 1981; Sannino et al., 2018; Orrego et al., 2019). We also identified a fifth pathway, the drug and xenobiotics metabolism-based on cytochrome P450. Recently, Silva et al. (2015) performed a first transcriptomic survey of A. luminosa lanterns, which also showed a high amount of cytochrome P450 and other gene products involved with detoxification. The CytP450 genes display important vital functions in the metabolism, providing hormone regulation and biosynthesis or inactivation of xenobiotic compounds (Martinez-Paz et al., 2012), displaying molecular function of monooxygenase and oxidoreductase activities, however, only a third of these genes are associated with xenobiotic metabolism (Giraudo et al., 2010).

Morphological and molecular studies with the dipteran Arachnocampa, already showed that the lanterns were derived from excretory Malpighian tubules (MT), therefore, the presence of these detoxification gene products is expected given (Gatenby, 1959; Sharpe et al., 2015; Silva et al., 2015). However, it is unclear why CytP450 is overexpressed in the whole body of O. fultoni.

3.3. Differential analysis of circadian expressed genes in O. fultoni

Because O. fultoni exhibits circadian control of bioluminescence (Thérèse Wilson’s former observation), we performed the RNA-Seq of O. fultoni during day and night, to identify putative gene products associated with bioluminescence and nocturnal activity. The analysis identified 44 transcripts differentially expressed during the night and 56 differentially expressed during the day (Table SM2). Most of the expressed genes at daytime displayed oxidoreductase activity (14) or were involved with cellular nitrogen compound metabolism, while the great majority of night-time genes are involved with carbohydrate metabolism or displayed metal binding (8) and oxidoreductase activity (8). Some of these products appeared more than once (e.g. low-specificity L-threonine aldolase 2).

We plotted the GO terms to the differentially expressed genes during daytime and nighttime (Fig. 2) and observed distinct molecular activities, functions and localization among the conditions. The ligase activity and cellular processes, as well as several metabolic processes and biological processes associated with biological and biogenesis regulation, signaling, and stimulus response were common functions during daytime.

Fig. 2.

Fig. 2.

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GO enrichment analysis of differentially expressed gene products associated with the circadian rhythm in Orfelia fultoni. The red columns represent daytime conditions and the grey columns represent the nighttime conditions. The y-axis indicates the percentage of total genes in each category.

During the nighttime, catalytic and transferase activities were more represented among molecular functions, as well as development processes and localization in biological processes. The catalytic and transferase activities are evolutionary conditions associated with insect adaptation to the presence of natural toxins in the feeding process (Schuler and Berenbaum, 2013). Both CytP450 and UDP-glucuronosyl-transferase were differentially expressed during the nighttime and they may involve in the metabolism of toxic compounds and xenobiotics in Orfelia larvae. Studies demonstrated the importance of CytP450 and UDP in detoxification processes in insects and mammals (Heckel, 2018; Lu et al., 2021; Tian et al., 2019), such as the detoxification of phenolic compounds and organic acids molecules. These compounds are frequently found in nature and are also produced by the organism metabolism (Ashrap et al., 2017; Tian et al., 2019). In Orfelia larvae, the nighttime seems to be the most active period associated with the prey activity and associated processes, such as hunting, feeding, and locomotion, which may explain the increase of detoxification metabolism.

The KEGG pathway annotation showed a metabolic pathway associated with daytime conditions. The “Alanine, Aspartate, and Glutamate Metabolism” pathway is related to the biosynthesis of secondary metabolites, which seem to be represented during the diurnal activity (Fonken and Nelson, 2014). The asparagine synthase is responsible for the conversion of l-Aspartate to L-Asparagine, using ammonia or glutamine as a nitrogen sources. The absence or lower concentrations of these nitrogen sources, may delay the larval growth and survival (Dadd, 1978).

From the transcripts obtained at night, ca. 40% were unknown or uncharacterized. Most of the others with known functions were related to transcription, transport, organism development, tyrosine phosphorylation and degradation, exoskeleton formation, etc. Noteworthy, among all these gene products differentially expressed at night, again we observed several copies of cytochrome P450 (oxidoreductase activity and ion binding) (Table SM2).

Besides detoxification through hydroxylation of aromatic compounds, the group of Cyt P450 4d10 (ca. 30 kDa) has also the function associated with the reduction of oxidized flavoproteins in the presence of O2, into flavoprotein and oxygen radicals; and are directly involved in xenobiotic metabolism. The CytP450 was already found across multiple tissues and developmental stages in Diptera, including Plutella and Drosophila genera. Their higher abundance occurs in the head and midgut, near the Malpighian tubules, being associated with the detoxification of multiple xenobiotics (Yu et al., 2015; Oba et al., 2017). Although the overexpression of CytP450 in O. fultoni is still poorly understood, it could be involved with hydroxylation of metabolic compounds, including luciferin precursors, or detoxification processes in different tissues.

We also annotated a peroxisomal(S)-2-hydroxy-acid oxidase gene product (ca. 31 kDa), which is a FMN-dependent dehydrogenase with several FMN binding sites, responsible for oxidizing short-chain hydroxy acids (e.g. fusaric acid) (Esser et al., 2014). The presence of several gene products associated with the flavin metabolism, including the peroxisomal oxidase, could be an indication of their importance in a specific metabolic pathway during the night-time. However, additional molecular analyses are necessary to confirm it.

In the day-time, we observed an expressed isoform with high similarity to the adenylate forming enzymes class I superfamily, being 10 times more expressed than during the night. The similarity of this gene product to other AFDs included: 4-luciferin monooxygenase or luciferases (~62%), 4-coumarate-CoA ligase genes (~55%), AMP-dependent CoA ligases (~40%), and the putative Arachnocampa luciferase (~25%). The molecular weight of this enzyme was ~60 kDa, with 552 amino acid residues, therefore quite similar to the luciferases from Coleoptera (Elateroidea). Silva et al. (2015) and Sharpe et al. (2015) studying A. luminosa, and more recently in Trowell et al. (2016) studying the Arachnocampa richardase, identified a luciferase-like enzyme that displayed some similarity to current beetle luciferases (31 to 37%) and suggested that this enzyme could be the putative luciferase enzyme of Arachnocampa. However, such luciferase-like enzymes have no apparent relationship with Orfelia luciferase, which is a distinct oligomeric protein with distinct substrate requirements (Viviani et al., 2002, 2020). The presence of this AFD isoform of high expression during the day-time also indicates the absence of a relationship between Orfelia and fireflies/Arachnocampa bioluminescent system, suggesting that the ligase could be associated with a fatty acids or other carboxylic acids metabolism in Orfelia.

3.4. Gene products putatively associated with bioluminescence in Orfelia

Recently, our proteomic results suggested that the functional Orfelia luciferase is a trimeric protein with 220 kDa constituted by ~70 kDa monomers (Viviani et al., 2020). Based on this result, we conducted a data mining approach to select within the Orfelia transcriptome dataset the transcripts that displayed polypeptide length between 630 aa and 760 aa (~70 to ~90 kDa), similar to the MW suggested by proteomic analyses, which resulted in 1630 transcripts (Viviani et al., 2020). Comparison among MS/MS samples and the 1630 transcripts indicates two distinct isoforms of hexamerin as possible enzymes associated with the Orfelia luciferase (Viviani et al., 2020).

Here, we further analyzed the proteomic information against the downstream analyses of DEG. The two distinct isoforms of hexamerin, type 1 and type 2, with different polypeptide lengths, 750 aa (~83 kDa) and 715 aa (~79 kDa), were not differentially expressed during daytime and nighttime conditions. However, they were highly abundant (~3 fold) during nighttime (FPKM, night: 14,265.83 (type 1)/11,597.05 (type 2); day: 6394.31 (type 1)/2475.444 (type 2). Both proteins displayed three conserved domains, Hemocyanin-N (all-alpha domain), Hemocyanin-M (copper-containing domain), and Hemocyanin-C (Ig-like domain). The identity between these sequences was ~50% (Viviani et al., 2020).

We also reevaluated the MS results of 6 protein samples (bands) generated in Viviani et al. (2020), blasting these results against the transcriptome data, displaying a total of 221 gene products associated with these samples. From them, sample 3 displayed possible similarity to partial amino acid sequences of 192 gene products, while the other samples displayed a range from 10 to 25 gene products. This information indicate that a pool of proteins was isolated in one band and sequenced ducts, such as myosin heavy chain and muscle isoform X10. The total spectrum count, a semiquantitative relative measure of protein abundance in proteomic studies, was used to identify the most common gene products associated among all samples. Based on that, we observed the 6 most abundant proteins among the 6 bands (Table 1). Four hexamerins (two of them just a partial sequence), two heat-shock (chaperones), and a myosin heavy chain (only in sample 3). The chaperones were also observed in samples 4 to 6, associated with SBF purified fraction. For the downstream analysis, we excluded the two partial sequences of hexamerin. We aligned both groups of enzymes, hexamerin, and chaperone, to identify the possible common spectra among them.

Table 1.

Common transcripts and total spectrum count among putative luciferase and SBF fraction of O. fultoni.

Band/samples Transcripts identified Number of proteins with identification probability > 90% Number of proteins with coverage > 10% Associated gene products Transcript - total spectrum count > 20
1 15 10 2 larval serum protein 2-like [Anopheles stephensi]/hexamerin-1.1 [Aedes aegypti] c40889_g2_i1 c47146_g1_i1
2 16 9 3 larval serum protein 2-like [Anopheles stephensi]/hexamerin-1.1 [Aedes aegypti] c47146_g1_i1 c40889_g2_i1
3 192 187 139 myosin heavy chain, muscle isoform X10 [Contarinia nasturtii]/larval serum protein 2-like [Anopheles stephensi] c40889_g2_i1
4 18 16 7 larval serum protein 2-like [Anopheles stephensi]/hexamerin-1.1 [Aedes aegypti]/heat shock cognate 70 [Rhynchosciara americana] c40889_g2_i1 c37503_g1_i2 c46738_g1_i1
5 18 15 3 larval serum protein 2-like [Anopheles stephensi]/hexamerin-1.1 [Aedes aegypti]/endoplasmic reticulum chaperone BiP [Aphantopus hyperantus] c37503_g1_i2 c40889_g2_i1 c43509_g1_i2
6 28 24 2 larval serum protein 2-like [Anopheles stephensi]/hexamerin-1.1 [Aedes aegypti]/heat shock cognate 70 [Rhynchosciara americana] c40889_g2_i1 c46738_g1_i1
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The number of spectra for all samples shows higher coverage to the isoform c40889_g2_i1 (hexamerin), which indicates this isoform is the main target in our samples. For the chaperones/heat-shock proteins, we observed a high similarity among them (~63% of identity), which was also observed in the number of spectra among them. However, the isoform c46738_g1_i1 was ~2× more abundant than the isoform c43509_g1_i2 (FPKM:1249.01 and 659.48, respectively). This MS reanalysis shows, for the first time, the presence of chaperones associated with the hexamerin-like enzyme in the bioluminescence process in Orfelia. However, additional biochemical analyses are necessary to provide direct evidence of the interaction between these proteins. Based on the gene function, the chaperone could be involved in the hexamerin folding (Porter et al., 2020).

3.5. Comparison of Orfelia fultoni and Arachnocampa luminosa orthologs

The keroplatids O. fultoni and A. luminosa display several ecological and behavioral traits in common, such as the use of bioluminescence and web construction for prey attraction and capture. We compared the orthologue gene products present in both species to find similarities and associations with the phenotype.

The orthology search identified in our dataset a total of 2151 single-copy orthologous gene products (Table SM3; Supplementary material). Phosphatase enzymes were observed in both species distributed in photogenic and non-photogenic tissues and all day. However, in the non-luminescent Diptera species Aedes and Drosophila, phosphatases is most commonly observed in Malpighian tubules (Yang et al., 2000; Cabrero et al., 2004; Jagge and Pietrantonio, 2008). The catabolism of purine is vital for insect physiology, producing, as the final product, uric acid, which will be excreted by the organism. The number of distinct phosphatase isoforms in Orfelia and Arachnocampa transcriptomes indicates that the Malpighian tubules are highly active during larval stages.

In the drug metabolism pathways, we observed esterases, N-acetyltransferase, dehydrogenase, and lactoperoxidase. These enzymes are involved in different pathways related to conversion, degradation, and detoxification of xenobiotic compounds, such as imidazolamine, environmental toxicants, among others (Hogg and Jago, 1970; Fermino et al., 2010; Hiragaki et al., 2015; Li et al., 2017). Two highly expressed isoforms of CytP450 (6a1 and 9e2 isoforms) were found in the photogenic tissues of A. luminosa and an isoform (CytP450 4d10) in O. fultoni. They are associated with insecticide detoxification (6a1), xenobiotic metabolism (4d10), and resistance against fungi (9e2) (Xing et al., 2017).

3.6. Evidence for positive selection and functional categories

Here, we also investigated gene products that undergo positive selection in the family Keroplatidae, to evaluate their importance in the adaptation of the group to similar habitats, such as crevices on damp stream banks (Viviani, 2002).

The positive selection analysis using the branch model in the Keroplatidae family allowed us to detect selection acting upon some gene products. The LRTs and the multiple tests using an FDR of 5% showed 148 orthologs under positive selection, which represents about 9% of all orthologous genes (Table SM4; Supplementary Material).

We determined their gene ontology and assigned the molecular function. They were associated with transmembrane transporter activity, mainly carboxylic acid and carbohydrate transmembrane transporter activity, being involved in the cellular response to fluoride, to acids, and cellular response to a chemical stimulus.

The KEGG database identified several pathways that are involved in the transferase activity, specifically the Glutathione-S-transferase, which is involved in GSH and redox homeostasis, and which is also involved in drug and xenobiotic metabolism with cytochrome P450. We also identified a fatty acid synthetase gene product associated with carboxylic acid biosynthesis, including ethanedioic acid, tetradecanoic, hexadecanoic, and octadecanoic acid.

3.7. Gene products associated with other ecological adaptations in Orfelia

Larvae of O. fultoni and Arachnocampa spp. display common ecological adaptation to maximize the effect of prey attraction using bioluminescence, its visual detection, and the construction of sticky webs (Fulton, 1939, 1941; Meyer-Rochow, 2007). We analyzed opsin gene products, responsible for color discrimination, as well as, a gene product related to web silk formation.

3.7.1. Opsin genes and vision

The visual and bioluminescent systems display vital functions for bioluminescent organisms, such as defense, prey attraction, illumination, communication in fireflies, among others. In bioluminescent beetle species, for example, the detection of a specific light color and flash patterns are important for intraspecific sexual communication (McDermott, 1964; Lloyd, 1978; Lall et al., 2000, 2010; Branham and Wenzel, 2003).

Larvae of O. fultoni and A. luminosa display blue and blue-green bioluminescence, with peaks at 460 nm and 485 nm, respectively. These blue-shifted emissions seem to be an ecological adaptation to maximize prey attraction (dark places) of flying insects, which usually display higher visual spectral sensitivity in the blue region. Similarly, the larvae of the Pyrearinus pumilus click beetle group (Elateroidea, Coleoptera), which colonize termite mounds in savannas and clayish caves in the Amazon forest in Brazil, also use their greenish-blue bioluminescence for prey attraction (Viviani et al., 1999; Martin et al., 2015; Sander and Hall, 2015; Viviani and Amaral, 2016). Blue bioluminescence could also be involved in the illumination of the silk web during prey attraction at night.

Opsin gene products for all the three main opsin classes, UV (ultra-violet), SW (short-wavelength), and LW (long-wavelength) were found in both O. fultoni and A. luminosa species. In O. fultoni, two copies of UV (Orf_UVS1, daytime: 0.29; nighttime: 0.37; Orf_UVS2, daytime: 18.3; nighttime: 16.68), a single copy of SW (a partial sequence; Orf_SWS_partial, daytime: 0.568; nighttime: 0.733), and a single copy of LW (Orf_LWS, daytime: 6.65; nighttime: 4.67) were identified (Fig. S1). We also identified a pteropsin (Orf_pteropsin, daytime: 0.7; nighttime: 1.28), which is classified as an ancestral class of opsins. In A. luminosa, one copy of SW (Arach_SWS, photogenic tissue: 0.0; non-photogenic tissue: 1.77), one copy of UV (Arach_UVS, photogenic tissue: 0.4; non-photogenic tissue: 1.5), and two copies of LW were identified (Arach_LWS1, photogenic tissue: 3.7; non-photogenic tissue: 5.1; Arach_LWS2, photogenic tissue: 0.0; non-photogenic tissue: 4.2). These results indicate that Orfelia fultoni has a simple visual system with fewer events of gene duplication.

These results support that O. fultoni and Arachnocampa display a complex visual system, with the presence of gene duplication events in some opsins classes. In O. fultoni, similarly to Keroplatus spp., the larva reacts to the environmental light, indicating that the eyes have the main function to detect it (Meyer-Rochow and Eguchi, 1984; Meyer-Rochow and Yamaham, 2017). In Arachnocampa, the presence of duplicate LW genes, the developed lens, and the response to monochromatic isophotonic light with distinct peaks (Meyer-Rochow and Eguchi, 1984; Meyer-Rochow and Yamaham, 2017), suggest that the bioluminescent system is important not only for prey attraction but also for intra-specific communication in Arachnocampa (eg. for a synchronism event) (Berry et al., 2017). Besides displaying bioluminescence spectrum in the blue region, the isolated gene duplication events among distinct opsin classes in O. fultoni and Arachnocampa also suggest slightly distinct evolution patterns in these species (Fig. 3), which could be associated with the adaptation to slightly distinct habitats (eg., streams banks versus caves) and to their respective bioluminescent spectra.

Fig. 3.

Fig. 3.

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Cladogram summarizing the bioluminescent, visual, and web construction systems evolution within Keroplatidae. The results reinforce the distinct evolution of these systems in both species associated with prey attraction.

3.7.2. Silk formation

The web construction by silk secretion represents another important adaptation for prey capture in Keroplatidae larvae. The organs responsible to secrete the silk are the same in Arachnocampa and Orfelia, the labial glands, or salivary glands (Fulton, 1939; von Byern et al., 2016).

We identified a gene product related to silk formation in O. fultoni, the fibroin heavy chain (daytime: 25.43; nighttime: 32.42), with high identity to Bombyx mori fibroin (65%). This gene is related to silk formation in silkworms, spiders, and it is also observed in other insects, such as Antheraea and Samia (Lepidoptera) (Hakimi et al., 2007). Usually, the silk is formed by two associated proteins in Lepidoptera and Trichoptera, the fibroin, which is responsible for 70–80% of the silk, and sericin, which is a glue-type protein, important for cocoon formation, which consists of 20–30% of the silk (Hakimi et al., 2007; Shimizu et al., 2007; Sehnal and Sutherland, 2008).

We did not observe any copy of sericin in O. fultoni and A. luminosa, however, we identified mucin (Orfelia, daytime: 51.73; nighttime: 75.44; Arachnocampa, photogenic tissue: 0.0; non-photogenic tissue: 0.86) and sialomucin (Orfelia, daytime: 54.60; nighttime: 42.45; photogenic tissue: 3.886; non-photogenic tissue: 2.614) proteins (produces a sticky mucus) and a venom peptide HsVx1 protein (Orfelia, daytime: 36,043.9; night-time: 32,347.98; it was not observed in Arachnocampa,), which was the second most expressed gene product during night time in Orfelia (Table 2) and it is a precursor of some types of insect toxins, such as La1 and PC present in scorpions (Palma, 2013). Both mucus (Richards, 1960; Meyer-Rochow, 2007) and a toxic compound, previously identified as oxalic acid (Fulton, 1939), seem to be important to capture the prey, which is corroborated by our results.

Table 2.

Top 30 most abundant transcripts during the nighttime condition.

Contig number Putative functional annotation FPKM
c33076_g1_i1 —NA— 35,446.57
c47750_g2_i2 venom peptide HsVx1 32,347.98
c43897_g5_i1 AAEL013757-PA [Aedes aegypti] (Hexamerin) 19,521.52
c47127_g1_i3 —NA— 18,827.67
c40889_g2_i1 hexamerin 2 beta 14,265.83
c43818_g4_i1 —NA— 11,694.36
c37503_g1_i2 larval serum 2 [Culex quinquefasciatus] 11,597.05
c15034_g1_i1 chitin binding [Blastocystis ST4] 9988.51
c73097_g2_i1 —NA— 8934.78
c40586_g2_i1 —NA— 8456.04
c1847_g1_i1 —NA— 8093.49
c36232_g1_i3 hypothetical protein GALMADRAFT_224445 7307.83
c44391_g2_i2 AAEL004631-PA [Aedes aegypti] 6348.41
c34319_g1_i1 —NA— 5881.16
c42697_g1_i3 —NA— 5828.43
c4084_g1_i1 NPC2 homolog 5540.99
c33561_g2_i1 hypothetical protein GALMADRAFT_224445 5074.56
c44431_g3_i1 serine protease 1 4829.19
c42001_g3_i1 PREDICTED: uncharacterized protein LOC105211473 4674.76
c18267_g1_i1 myosin light chain 2 [Culex quinquefasciatus] 4665.02
c47146_g1_i1 AGAP001659-PA [Anopheles gambiae PEST] 4255.81
c45803_g5_i1 GL15515 [Drosophila persimilis] 3863.74
c29051_g1_i1 class III chitinase 1 [Cordyceps militaris CM01] 3645.35
c7995_g1_i1 larvae cuticle 3553.67
c47127_g1_i2 —NA— 3398.42
c47556_g1_i2 60S ribosomal L27a 2926.79
c44749_g2_i1 AAEL001061-PB [Aedes aegypti] 2847.79
c40446_g2_i1 uncharacterized protein Dwil_GK22048 [Drosophila willistoni] 28,19.56
c40114_g1_i1 —NA— 2809.88
c44723_g1_i2 alaserpin-like isoform X6 2720.65
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—NA—: no similarity observed.

When compared to spiders and silkworms, the O. fultoni web looks more rudimentary, less structured, and displays small capsules with toxic compounds, corroborating the previous descriptions made by Fulton (1939). These results suggest that O. fultoni larvae have a simpler system of web construction, however with an efficient adaptation for capturing prey.

However, we did not observe these gene products (fibroin and sericin) in A. luminosa transcriptional profile. Rather, we identified a fibronectin type III (Arachnocampa. photogenic tissue: 0.0; non-photogenic tissue: 112.9; Orfelia: daytime: 12.4; nighttime: 15.9) protein and collagen alpha I (Arachnocampa. photogenic tissue: 0.0; non-photogenic tissue: 218.4; Orfelia: daytime: 0.0; nighttime: 24.2). The fibronectin type III protein is a component of the cellular matrix that is responsible of collagen polymerization, improving cell adhesion, its viability, and fibrous strength (Velling et al., 2002; Zhao et al., 2017). In Hymenoptera, the collagen-type genes and other compounds are responsible to form a ß-sheet structure of the web (Sehnal and Sutherland, 2008). Recent comparison of X-ray scattering, infrared spectroscopy, and amino acid analysis data also suggested that in Arachnocampa spp., the web consists of a cross-β-sheet structure (Walker et al., 2015). Regarding the mucus, Arachnocampa spp. also expressed the mucin gene product, however, the most expressed mucus gene product is the sialomucin, a specific type of mucin.

Altogether, these results may indicate two possible adaptations for the silk web synthesis apparatus in the Keroplatidae family, one for O. fultoni (Keroplatinae) which is more related to that of the Lepidoptera, and another for Arachnocampa (Arachnocampininae), which is more related to that of the Hymenoptera. Web construction and silk biosynthesis, similarly to bioluminescence, could be another phenotypic trait that took slightly different evolutive pathways in insects (Fig. 3) (Betz, 2010; Walker et al., 2015).

4. Concluding remarks

We reported the first transcriptional profile of bioluminescent O. fultoni (Keroplatidae: Keroplatinae) during day and night and compared it to the RNA-Seq dataset of A. luminosa (Keroplatidae: Arachnocampininae). The high representation of chaperones and hexamerins isoforms in the transcriptomes and MS data analysis of partially purified fractions of luciferase and SBF, especially at night, suggest for the first time a possible association of these proteins in the bioluminescence of O. fultoni and other nocturnal activities.

Larvae of O. fultoni also display visual system that cover a specific (and narrow) spectral range, with opsins isoforms displaying blue light sensitivity only in the wavelength range from 400 nm to 500 nm, while the Arachnocampa displays two LW copies (480 nm to 600 nm), adapted to blue-green light detection, agreeing with the adaptation of the visual systems to their respective bioluminescence spectra. Despite using the same strategies for prey attraction, using bioluminescence and web construction, the distinct gene product patterns associated with silk, mucus, and toxin biosynthesis in O. fultoni and A. luminosa transcriptomes suggest that silk production, similarly to bioluminescence, may have evolved independently in these subfamilies.

Supplementary Material

Supplement

Acknowledgments

We are deeply thankful to Prof. Anthony Rokas (Vanderbilt University, TN, USA) for the discussions about the NGS sequencing methodology, Dr. Hayes McDonald for access and discussion of proteomic results (Vanderbilt University, TN, USA), and to Prof. James Costa and Highlands Biological Station (Highlands, NC, USA) for allowing collecting and making observations of Orfelia fultoni in this station. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2010/05426-8; FAPESP/NSF (UFSCar/Vanderbilt) 2014/50583-5; FAPESP 2015/25051-2; FAPESP 2014/20176-9); Conselho Nacional de Pesquisa (CNPq 401867/2016-1) from Brazil; Amazon Web Service (AWS) (PS_R_OTH_FY2017_Q3_FU_Sao_Carlos_Amaral); and R21 MH116150 from the USA National Institutes of Health to CHJ.

Footnotes

Declaration of competing interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cbd.2021.100840.

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