RESEARCH ARTICLE

Neev, a novel long non-coding RNA, is expressed in chaetoblastsduring regeneration of Eisenia fetidaSurendra Singh Patel1,2, Sanyami Zunjarrao3 and Beena Pillai1,2,*

ABSTRACTEisenia fetida, the common vermicomposting earthworm, showsrobust regeneration of posterior segments removed by amputation.During the period of regeneration, the newly formed tissue initiallycontains only undifferentiated cells but subsequently differentiatesinto a variety of cell types including muscle, nerve and vasculature.Transcriptomics analysis, reported previously, provided a number ofcandidate non-coding RNAs that were induced during regeneration.We found that one such long non-coding RNA (lncRNA) is expressedin the skin, only at the base of newly formed chaetae. The spatialorganization and precise arrangement of the regenerating chaetaeand the cells expressing the lncRNA on the ventral side clearlysupport a model wherein the regenerating tissue contains a zone ofgrowth and cell division at the tip and a zone of differentiation at thesite of amputation. The temporal expression pattern of the lncRNA,named Neev, closely resembled the pattern of chitin synthase genes,implicated in chaetae formation. We found that the lncRNA has49 sites for binding a set of four microRNAs (miRNAs) while the chitinsynthase 8 mRNA has 478 sites. The over-representation of sharedmiRNA sites suggests that lncRNANeevmayact as amiRNA spongeto transiently de-repress chitin synthase 8 during formation of newchaetae in the regenerating segments of Eisenia fetida.

KEY WORDS: Earthworm, Chitin synthase, miRNA, lncRNA

INTRODUCTIONEarthworms are a large diverse group of segmented worms thatinhabit niches just under or deep within the soil. The ‘tube within atube’ body plan of the earthworm comprises a muscular outer wallenclosing a gut within. It also has a simple vascular system tocirculate blood and a nervous system comprising a nerve ganglion atthe anterior end and a long ventral nerve cord running the length ofthe body, with ring nerves in each segment.Earthworms vary widely in their ability to regenerate. Eisenia

fetida (commonly known as red wriggler worm) regenerates nearly2/3 of its posterior end (Xiao et al., 2011). The earthworm presentsan invertebrate model of epimorphosis, a type of regenerationinvolving the restoration of original anatomy and polarity followedby de-differentiation, proliferation and differentiation of cells (Bely,2014; Gazave et al., 2013; Planques et al., 2019; Xiao et al., 2011). Aseach segment consists of nerve, muscle, vasculature and additional

specialized structures, it provides a model for studying regenerationcoordinated across different tissue types. For instance, chaetae,specialized projections embedded in the skin used for gripping thesoil, are found embedded in the outer body wall in close proximityto muscle and peripheral nerves in each segment (Prosser, 1934).

We have previously characterized the genome and transcriptomeof the E. fetida (Bhambri et al., 2018). Injury and loss of theposterior 2/3rd of this regenerating worm was followed by apparentwound healing in 5–10 days. A stub of tissue largely consisting of amass of undifferentiated tissue was formed by 15 days anddifferentiated segments were formed by 20 days post-amputation.The period between 10 and 20 days after the injury presents a timewindow during which cell proliferation, growth and differentiationhappen simultaneously in a 4–5 mm long tissue amenable tomolecular and cellular visualization. Regenerating annelids areparticularly convenient for studying developmental gradients,because a single regenerating tail has many segments at varyingstages of development along the anterio-posterior axis.

The transcriptome of the regenerating worm revealed signatures ofrapid cell proliferation, reorganization of the extracellular matrix anddifferentiation of nerves. Besides these signatures, we also reported thedynamic expression of non-coding RNAs that potentially play roles inregulating the timing of expression, control and spatial organization ofthe transcriptome (Bhambri et al., 2018). We rationalized that novelnon-coding RNAs could play an important role in restoring anundifferentiated cellular state by interfering in the function of celltype-specific microRNAs (miRNAs) that are usually associated withdifferentiation. In mammalian systems, several long non-codingRNAs (lncRNAs) are known to carry tandem miRNA binding sites,allowing them to ‘sponge away’ miRNAs (Paraskevopoulou andHatzigeorgiou, 2016). Here, we report the expression pattern ofselected non-coding RNAs in the regenerating earthworm. Besidesvalidating our previous report, we focus on one lncRNA that showed aunique expression pattern at the base of the chaetae.

Chaetae are stiff chitinous structures (appendages) that originatedeep within the muscular body wall, the outer ends of which areused to grip the surface and in locomotor activity (Hausen, 2005). Inanother annelid, Platynereis dumerilli, larval chaetogenesis startswith the appearance of chaetoblasts on the surface, which laterinvaginate to the base of the chaetae, forming chaetal sacs, while thesurrounding cells become follicle cells (Gazave et al., 2017). Wefound that this lncRNA, named Neev, is expressed only in the fewcells at the base of chaetae in newly regenerated segments close tothe site of injury. Its expression pattern closely resembles that ofchitin synthase and chitinases involved in the formation of chaetae.We therefore explored the relationship between Neev and chitinsynthase expression in regenerating segments of E. fetida.

MATERIALS AND METHODSExperimental conditionsEisenia fetida (Savigny 1826) earthworms were originally procuredfrom farmers engaged in vermicomposting and subsequentlyReceived 16 October 2019; Accepted 17 February 2020

1CSIR-Institute of Genomics and Integrative Biology, New Delhi, Delhi 110 025,India. 2Academy of Scientific & Innovative Research (AcSIR), Chennai, Tamil Nadu600113, India. 3Institute of Bioinformatics and Biotechnology, Savitribai Phule PuneUniversity, Pune, Maharashtra 411007, India.

*Author for correspondence (beena@igib.in)

S.S.P., 0000-0001-5827-9778; S.Z., 0000-0002-6441-7265; B.P., 0000-0002-9302-9878

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mailto:beena@igib.inhttp://orcid.org/0000-0001-5827-9778http://orcid.org/0000-0002-6441-7265http://orcid.org/0000-0002-9302-9878http://orcid.org/0000-0002-9302-9878

maintained in a plastic tray and fed with plant matter in thelaboratory at around 22°C for several years. No specific permissionswere required for procuring earthworms. They are not endangeredspecies and ethical approval is not required. Medium-sized wormswere collected before the experiment, rinsed in tap water to removeany soil sticking to the surface and amputated as described in ourprevious paper (Bhambri et al., 2018). The site of amputation was atabout 2/3rd of the body length from the anterior end, thus retainingabout 60 segments. After amputation, the worms were maintained ina separate container but under similar culture conditions.Regenerating tissue and about 2–3 mm of the adjacent tissue fromthe pre-amputated worms were collected for in situ hybridization.

Bioinformatic analysisThe sequence of Neev lncRNAwas used to search for contigs in thegenome assembly for E. fetida previously reported from our group(Bhambri et al., 2018). The longest contig within scaffoldSACV01159372.1 (Genome ID: SACV01) containing Neevsequence was 4884 nucleotides (nt) long. Further, this contig wasused to identify the similar regions from the genome of anyeukaryotes using a BLASTn search optimized for short stretches oflow similarity (Expect value threshold=10, Word size=11, Matchscore=2, Mismatch penalty=−3).

RNA isolation and qRT-PCRThe regenerated earthworm was rinsed thoroughly in running tapwater followed by autoclaved Milli-Q water; regenerated andadjacent control tissue from 20–30 earthworms was collected in1 ml Trizol kept on ice, at 15, 20 and 30 days post-amputation (dpa).A homogeneous cell suspension was made by grinding the tissueusing a homogenizer; 200 μl chloroform was then added, followedby vigorous shaking for 15 s. After incubation for 10 min at roomtemperature for phase separation, the mix was centrifuged at10,000 g, 15 min, 4°C. The upper aqueous layer was separated andan equal volume of isopropanol was added, incubated for 5 min, andcentrifuged at 10,000 g for 10 min. The resulting pellet was washedthrice with 70% ethanol at 10,000 g for 5 min each. The air-driedpellet was dissolved in 50 μl nuclease-free water. RNA (1 μg) wasused to make cDNA using a Transcriptor High Fidelity cDNASynthesis Kit (Roche, 5081955001) and primers (FP: ATATGG-TACCGTCTGCTCCCAGGGTTAG; RP: ATATGCGGCCGCC-TTGTGTCGAGTGTATTCAATTGC) designed to amplify full-length transcript. Gene-specific primers listed in Table S1 were usedin quantitative RT-PCR (qRT-PCR) reactions containing SYBRgreen master mix (Takara, RR820). The Ct values were used tocalculate fold-change against spike-in control lncRNA (see Resultsfor details) using the method described by Pfaffl (2001).

PCR cloning and probe designPCR product from the RT reaction was cloned using TOPO™ TACloning™ Kit (Invitrogen, 450640) as per the manufacturer’sprotocol. Sanger sequencing was performed to confirm the sequenceof the clone. In vitro transcription to synthesize the probewas performed using a DIG RNA (SP6/T7) Labeling Kit (Sigma-Aldrich, 11175025910) with SP6 or T7 polymerase after linearizingthe plasmid using restriction enzymes. The probes were purifiedby using NucAway™ Spin Columns (Thermo Fisher Scientific,AM10070).

In situ hybridizationRegenerated earthworms were collected at 10, 15, 20 and 30 dpa andwashed thoroughly in running tap water followed by autoclaved

Milli-Q water. Earthworms were fixed overnight at 4°C in 4% (w/v)paraformaldehyde (PFA) prepared in 1× phosphate-buffered saline(PBS). After fixation, they were washed stringently in PBS withTween 20 (PBST; 0.1% Tween 20 in PBS) with subsequent storagein 100% methanol at 4°C. Prior to hybridization, the storedearthworms were rehydrated with a methanol gradient [90%, 75%,50%, 25% and 0% (v/v) in PBST] for 45–60 min each. Earthwormswere permeabilized by 20 µg ml−1 proteinase K for 45 min at 55°C,then fixed again in 4% PFA for 20 min and blocked usinghybridization buffer [50% formamide, 1.3× saline sodium citrate(SSC), 5 mmol l−1 EDTA, 5% dextran sulphate, 0.2% Tween 20,100 µg ml−1 heparin and 50 µg ml−1 yeast t-RNA in DEPC-treatedwater] for 60 min at 65°C. Hybridization was performed using senseand antisense probes, prepared in hybridization buffer, overnight at65°C in a water bath. Stringent washes were performed at 65°C withhybridization buffer thrice for 30 min each followed by washes inTris-buffered saline with Tween (TBST; 0.1% Tween 20 in TBS)for 15 min at room temperature. After incubation at roomtemperature for 4 h in 1:2000 dilution of anti-digoxigeninantibody (Roche, 11376623) prepared in TBST containing 10%FBS, 15 min washes in TBST at room temperature were performedthrice and subsequently the tissue was stained using Nitro BlueTetrazolium (NBT; working concentration 500 µg ml−1; Roche,11383213001) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP;working concentration 562.5 µg ml−1; Roche, 11383221001) indeveloping solution (0.1 mol l−1 NaCl, 0.1 mol l−1 Tris-HClpH 9.5, 0.05 mol l−1 MgCl2, 0.1% Tween 20 in DEPC-treatedwater). Images were captured by mounting earthworms in 2.5%methylcellulose at 3.2× and 5× magnification.

RESULTSOn the basis of transcriptomics analysis in the regeneratingearthworm in our previous report (Bhambri et al., 2018), weprioritized 16 potentially non-coding RNAs for further validation,as they were about 1 kb or larger and free of low complexity repeats.We designed qRT-PCR assays to detect four of the predictedlncRNA using the assembled transcript sequences (see Materialsand Methods; Fig. S1). Although GAPDH is widely used as acontrol in gene expression studies, it was not suitable fornormalization in our experiments because it is stronglyupregulated during regeneration. Instead, we used a spike-innormalization method, by adding an in vitro transcribed RNAfragment to the qRT-PCR reaction. The spike-in control lncRNAwas originally cloned from the zebrafish genome that has nosequence homology with the earthworm genome (Sarangdhar et al.,2017). We also verified that the primers for this fragment produce noproduct when provided with the earthworm cDNA as a template. Inclose agreement with the RNAseq data, all four lncRNAs werestrongly over-expressed in the regenerating tissue (Fig. 1). As shownin the figure, the newly regenerated segments expressed thelncRNAs at 4–8 times the levels in the adjacent control segments.

Next, we cloned each lncRNA gene into the TOPO TA cloningvector and generated digoxigenin-labelled probes by incorporatingdigoxigenin-linked rUTP in the in vitro transcription reaction. Theseprobes were used in in situ hybridizations with collected samplescontaining regenerating tissue closely juxtaposed with tissue fromthe worm before injury. Although there was a detectable signal inthe regenerating tissue, the expression of the lncRNAs did not, ingeneral, have a distinctive spatial pattern (Fig. S2). A notableexception was the 895 nt lncRNA, which showed a recurring patternof expression with four spots in each segment on the ventro-lateraland ventral side in the regenerating region (Fig. 2). From the Ct

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values, this lncRNA was estimated to be expressed at about one-tenth the abundance of GAPDH, making it quite abundant, aslncRNAs are usually expressed at low levels. Because this abundantlncRNA was restricted to tiny spots, the expression was strong andclearly visible.To understand the source of the signal (Fig. 3A), we made

longitudinal incisions on the dorsal region and spread out the innerbody wall. By gently teasing out the tissue around the signal, it wasclear that the spots were at the base of newly assembled chaetae(Fig. 3B,C). Notably, no such spots were seen at the base of chaetaefrom non-regenerating segments (Fig. 3A). Clearly, this lncRNAwas strongly but transiently induced in a very small group of cellsclosely associated with the chitinous setae. Because of thisinteresting expression pattern, we named the lncRNA Neev,which means base or foundation in Hindi.In transcriptomics experiments, fragments of protein-coding

mRNAs are sometimes erroneously annotated as non-codingtranscripts (Zhao et al., 2018). Some legitimate lncRNAs mayalso produce functional peptides from micro-open reading frames(microORFs) (Ruiz-Orera et al., 2014). To rule out spuriousannotation and detect conserved microORFs, we aligned thesequence of the Neev lncRNA to genome scaffolds assembledpreviously. Although there was an 82 amino acid open reading

frame, it did not show any similarity to known proteins (Fig. 4). Thescaffold from which the Neev gene was derived included aconserved region of 232 nt with strong similarity to manygenomes including vertebrate model systems like zebrafish. Thisregion was used to anchor genomic contigs from diverse species(Fig. 4C). However, the rest of the contig did not show anyconservation, suggesting that Neev is not present in otherorganisms. By aligning the transcript sequence to the genomicsequence, we found that the Neev gene comprises two exons and a191 nt long intron (Fig. 4B). Taken together, Neev codes for alncRNA that satisfies all the current criteria, i.e. multi-exon,polyadenylated transcript longer than 200 nt that is devoid of openreading frames (ORFs) larger than 300 nt (Clamp et al., 2007;Dinger et al., 2008; Frith et al., 2006; Niazi and Valadkhan, 2012).

Next, we tried to assign a potential function to the transcript. AslncRNAs often regulate overlapping genes or genes in closeproximity by RNA–DNA hybridization and recruitment ofchromatin modifiers, we first checked for relevant ORFs in the5 kb contig containing the Neev gene. Because there were no genesin this region, we speculated that the lncRNA might regulate theexpression of distant genes through RNA–RNA binding. Chaetae,i.e. chitinous setae, originate in bulbous cells called chaetoblasts,which put out microvilli that are subsequently coated with a large

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Fig. 1. Upregulation of novel long non-coding RNAs(lncRNAs) during regeneration of Eisenia fetida. Wormswere amputated at approximately the 60th segment from theanterior end and allowed to regenerate over a period of 30 days.At 15, 20 and 30 days post-amputation (dpa), the regeneratingtissue (15R, 20R, 30R) and adjacent control tissue (15C, 20C,30C) were collected. RNA isolated from these tissues was usedfor qRT-PCR (see Materials and Methods). The novel lncRNAswere named according to the size of the transcript assembledfrom RNAseq data: lncRNA895 is alternatively called Neevhere. (N=3 biological replicates; n=3 technical replicates; t-test,*P

amount of chitin, presumably produced within these cells(Schweigkofler et al., 1998). We reasoned that the chaetoblastswould also need to express chitin synthase genes transiently and in ahighly regulated manner. We checked our transcriptomics data forthe expression pattern of chitin synthase genes and chitinases.Amongst 29 genes with the word chitin in their name, 10 changedexpression during regeneration. We retrieved the basal expressionlevel of these genes and in agreement with our prediction, one of thechitin synthases, chitin synthase 8 (Chs8; Q4P9K9), showed astrong induction of 11- to 23-fold in the regenerating tissue,compared with the adjacent control tissue (Fig. 5).We checked the spatial expression pattern on Chs8 by in situ

hybridization in the regenerating earthworm at 10 and 15 dpa. Theexpression signal was strikingly similar to that of Neev, although ofmuch lesser intensity. This was in agreement with the relativeexpression (as interpreted from baseMean values) in thetranscriptomics data. At higher magnification, expression was alsorevealed at the neck of the chaetae closer to the surface, while Neevwas expressed at the base (Fig. 6).Next, we looked for potential RNA–RNA interactions that

implicate the lncRNA in chitin synthase regulation. We aligned thelncRNA sequence with those of differentially expressed chitinsynthases. Similarity in the anti-sense orientation would indicate thatlncRNA–mRNA duplexes could potentially form, which are usuallytargeted for degradation. More complex regulatory mechanisms likeguidance of splicing or RNA–RNA scaffold formation are also

possible. In the anti-sense orientation, the mRNA of Chs8 couldpotentially bind to the lncRNA only at four stretches of 7 nt each.

We also looked for potential miRNA sponge-like activity in thelncRNA sequence, because sequestration of miRNAs maytransiently de-repress chitin synthesis genes to facilitateregeneration of chaetae. As miRNAs tend to be highly conserved,we used the list of mouse miRNAs (Griffiths-Jones, 2004; Griffiths-Jones et al., 2006, 2008; Kozomara and Griffiths-Jones, 2011,2014) to predict targets and verified that the earthworm genomecontained sequences corresponding to the miRNAs of interest(Bhambri et al., 2018). We used the well-accepted miRNA targetprediction tool miRanda (Betel et al., 2010) to identify the mostfrequently occurring miRNA targets in Neev (Table S2). TwomiRNAs were discarded from further analysis because we could notfind the corresponding region in the earthworm genome. FourmiRNAs, each with more than five target sites in the Neev lncRNA(see Fig. S2; Fig. 4A) collectively had 49 sites of ΔG100 sites on average andcollectively had 478 potential binding sites (ΔG

in close proximity within the 5 kb contig containing the gene forNeev. Translating the 895 nt transcript in all potential readingframes did not reveal any peptide with even minimal homology to aknown gene. Unlike the typical lncRNA, Neev is expressed at highlevels, to about one-tenth of the abundant GAPDH mRNA.Comparing the expression levels from the RNAseq data, it

appears that Neev is more abundant than the Chs8 gene. Takentogether, the reliable expression, poor conservation and presence ofmultiple exons agrees with it being a functional non-coding RNA.

Non-coding RNAs are found in large numbers in every genome,frequently outnumbering their protein-coding counterparts (Derrienet al., 2012). The transient and localized expression of Neev is in

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Fig. 4. Alignment of Neev with E. fetida genomic scaffold SACV01159372.1. (A) Predicted open reading frames (ORFs) in the genomic contig showed38 small ORFs with no functional annotation. (B) Neev contains two exons and a 191 nt intron. (C) Only one ORF of ∼900 nt in the 3′-direction wasconserved in other organisms.

sp|Q91XA9|CHIA_MOUSE

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Fig. 5. Neev expression coincides with expression ofseveral chitin metabolism genes. The pre-amputatedzone at 15, 20 and 30 dpa (15C, 20C and 30C) does notshow any expression of these genes while four chitinmetabolism genes and Neev are strongly induced in theregenerating region at corresponding time points (15R,20R and 30R). The numbers depict log2 fold-change withrespect to a similar region of the pre-amputated worm.

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agreement with the view that lncRNAs may be involved inestablishing the fine spatio-temporal regulation of genes in theregenerating tissue. lncRNAs are known to regulate gene expressionat various levels: by recruiting chromatin modifiers (Campos andReinberg, 2009; Kanhere et al., 2010), directing splicing (Bernardet al., 2010; Tripathi et al., 2010), modifying stability of mRNAs by

masking motifs within the mRNAs (Kung et al., 2013; Matsui et al.,2008) or sequestering miRNAs (Faghihi et al., 2010; Franco-Zorrilla et al., 2007). Some of these mechanisms inherently involveRNA–protein complexes, which cannot be predicted on the basis ofRNA sequence. However, some of the mechanisms can be predictedfrom the sequence of mRNA and lncRNA. The direct binding of

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Fig. 6. Chitin synthase 8 (Chs8) expression pattern in regeneratingE. fetida tissue.Earthworms were allowed to regenerate for 10 and 15 dpa.Whole-mountin situ hybridization of the regenerating tissue was performed using probe RNA synthesized by in vitro transcription. The antisense probe (right) showeda distinct expression signal on the ventral side, while the sense probe (left) served as a negative control (n=5; typical results are shown here). Scale bars: 100 µm,unless indicated otherwise. The tissue was dissected from the dorsal side to expose the chaetae, and to visualize Chs8 expression (arrows) at highermagnification (lower panels).

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Fig. 7. Schematic depiction of generegulation in the chaetoblast duringregeneration. In the pre-amputationzone, Chs8 is repressed by microRNAs(miRNAs), while in the differentiationzone, expression of Neev leads tosequestration of miRNAs and de-repression of Chs8. The posterior growthzone at the tip of the regeneratingearthworm comprises undifferentiatedcells.

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lncRNA and mRNA can be deciphered from stretches of apparentcomplementarity while similarity at miRNA binding sites indicatesthe possibility of a miRNA sponge mechanism. On the basis of thehigh frequency of binding sites for miR-667-5p and miR-7658-5p inthe mRNA of Chs8 mRNA and Neev lncRNA, we speculate that thetransient induction of the lncRNA in a few cells at the location of thechaetae temporarily releases chitin synthase mRNA from repression,and facilitates the development of chaetae in the regeneratingsegments (Fig. 7). Further experiments are needed to test thepredicted miRNA sponge-like activity of the lncRNA in vivo.Drawing upon other studies and our own results, we propose the

following hypothetical scenario in the regenerating tissue ofearthworm. Immediately following injury, a mass ofundifferentiated tissue, called the blastema, caps the site of injury.Within days, the mass of cells starts differentiating to structuresfound within each segment even as rapidly dividing cells increasethe volume of tissue. It was not clear whether the temporal period ofgrowth and cell division is completed before differentiation ensues;our data imply that differentiation and cell proliferation happensimultaneously. The rapidly dividing cells are concentrated at theposterior tip while the differentiation zone abuts the site of injury.This is broadly in agreement with a recent report of zones within theposterior regenerating region of another annelid, Platynereisdumerilii (Gazave et al., 2013). Cells that exhaust their divisionpotential are perhaps pushed down into the zone of differentiationwhere, presumably, local signals precisely position the chaetoblasts,which then adopt a transcriptional programme distinctive from thatof the neighbouring cells. At the site of injury, the newly formedsegment has chaetoblasts expressing the Neev lncRNA, even as thepre-existing chaetoblasts (in the adjacent segment on the other sideof the site of injury) remain immune to these signals. Thus, theposition of the regenerating chaetae and the pattern of Neevexpression can help in inferring an invisible developmentalboundary within the regenerating tissue.

AcknowledgementsThe authors acknowledge Aksheev Bhambri for support with genome analysis. Ananonymous reviewer is acknowledged for suggestions that improved the manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: S.S.P., B.P.; Methodology: S.S.P., S.Z.; Validation: S.S.P., S.Z.;Formal analysis: S.S.P.; Investigation: S.S.P.; Resources: B.P.; Data curation:S.S.P., B.P.; Writing - original draft: B.P.; Writing - review & editing: S.S.P., B.P.;Visualization: B.P.; Supervision: B.P.; Project administration: B.P.; Fundingacquisition: B.P.

FundingS.S.P. is supported through a Senior Research Fellowship from the UniversityGrants Commission.

Supplementary informationSupplementary information available online athttp://jeb.biologists.org/lookup/doi/10.1242/jeb.216754.supplemental

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