Diversity of adipokinetic hormones in termites ( Isoptera )

In this study we report on the structural diversity of adipokinetic hormones (AKHs) in the evolutionarily oldest group of social insects, the termites (Isoptera). Using molecular methods combined with in silico bioinformatic analysis, we studied and compared the sequences coding for these neuropeptides in thirteen species of fi ve families of Isoptera. There are three types of AKH octapeptides in termites: Empusa pennata adipokinetic hormone (Emppe-AKH; pQVNFTPNWamide), Microhodotermes viator corpus cardiacum peptide (Micvi-CC; pQINFTPNWamide) and Periplaneta americana cardioaccelerating hormone (PeramCAH-I; pQVNFSPNWamide). Of these the Peram-CAH-I was the most frequently bioactive form detected in representatives of four out of the six families studied. The complete AKH preprohormones in the termites studied shared at least an 84% amino acid similarity. In agreement with current phylogenetic scenarios of termites as an internal monophyletic clade nested within cockroaches (Blattaria) in the proximity of the family Blattidae, our phylogenetic analysis of the AKH precursor sequences (in the absence of data for the Cryptocercidae) placed the Blattidae (Periplaneta americana) as a sister group of termites and the AKHs of other cockroach families (Blattellidae and Blaberidae) were more divergent from those of termites. Representatives of the basal termite families Mastotermitidae and Archotermopsidae (but also one rhinotermitid genus Prorhinotermes) occurred separately from the phylogenetically advanced lineage (Rhinotermitidae and Termitidae), and Neotermes (Kalotermitidae) was sister to all other termites included.


INTRODUCTION
Adipokinetic hormones (AKHs) are well-known insect neuropeptides that have an important role in the control of metabolism.The AKHs are specifi ed as mobilizing factors for a variety of energy reserves, such as carbohydrates, lipids and certain amino acids (Gäde et al., 1997;Gäde & Auerswald, 2003).AKHs are also involved in various physiological and biochemical processes such as stimulation of locomotion, enhancement of immune responses, activation of antioxidant mechanisms and inhibition of RNA synthesis (reviewed in Kodrík, 2008;Bednářová et al., 2013).AKHs are structurally similar to the red pigment concentrating hormone (RPCH) of crustaceans, which enables them to adopt the background colour (Fernlund & Josefsson, 1972;Carlsen et al., 1976;Rao, 2001), and together belong to a widespread group of neuropeptides called the AKH/RPCH family.
AKHs are synthesized, stored and released predominantly from the paired endocrine retrocerebral glands, corpora cardiaca (CC), and there are small amounts of AKH reported in the brains of some insect species (Moshitzky et al., 1987;Kodrík et al., 2015).AKH is synthesized as a preprohormone consisting of a hydrophobic signal peptide, analyzed using agarose gel electrophoresis, cloned into pGEM-T Easy vector (Promega) and sequenced.

5´ RACE PCR
The 5´ ends of AKH transcripts were isolated using the FirstChoice RLM-RACE Kit (Invitrogen) with 10 μg of the corresponding RNA according to manufacturer's instructions.Reverse transcriptions were carried out with gene specifi c primers derived from the previously sequenced 3´ RACE PCR products (Table 1).PCR reactions were performed with 5´ RACE Outer Primer of RLM-RACE Kit and specifi c AKH reverse primers (Table 1).Subsequently, nested PCR was performed using an aliquot of purifi ed product of the fi rst round PCR, 5´ RACE Inner Primer of RLM-RACE Kit and nested AKH reverse primer specifi c for each termite species (Table 1).All RT-PCR products were cloned into pGEM-T Easy vector (Promega) and sequenced.

Sequence analyses
The sequencing data for the transcriptomes of in silico analyzed termites were accessed from the public source of the NCBI Sequence Read Archive (SRA) using the SRA accession codes as follows: SRR863603.sra(Zootermopsis nevadensis), SRR789255.sra(Macrotermes natalensis), DRR013047.sra (Nasutitermes takasagoensis), SRR528715.sra(Odontotermes formosanus), SRR700381.sra(Reticulitermes fl avipes), DRR013046.sra(Reticulitermes speratus).The raw data fi les were processed as described previously (Jedličková et al., 2015).Briefl y, after conversion to a FASTQ fi le using fastq-dump utility in the SRA Toolkit (SRA Software page), the sequence read quality was monitored using FastQC and all reads trimmed using Trimmomatic software version 0.27 (Bolger et al., 2014).The previously cloned AKH cDNA sequence of Coptotermes formosanus (KT805304) was used as a reference and the resulting alignment process was completed using Bowtie2.1.0(Langmead et al., 2009) and SAM/BAM processing (SAMtools; Li et al., 2009).broad set of closely related species.Termites are a suitable taxon in this respect.They are a large and diversifi ed group of hemimetabolous insects consisting of more than 3000 species (Krishna et al., 2013).Recent phylogenetic studies indicate that this group of eusocial insect is an ingroup of the cockroaches (Blattaria) and the subsocial wood-feeding genus Cryptocercus is its sister group (Lo et al., 2000;Klass & Meier, 2006;Inward et al., 2007;Bourguignon et al., 2015).Two AKH bioactive octapeptides were previously found in termites: Micvi-CC in Microhodotermes viator, which seems to be unique for Isoptera, and Peram-CAH-I in the termites Mastotermes darwiniensis and Trinervitermes trinervoides (Liebrich et al., 1995), which also occurs in various species of cockroaches and beetles (Coleoptera).
The aim of this study was to produce an overview of the AKH diversity in the phylogeny of Isoptera.We combined classical molecular biology approaches (5´ and 3´ RACE technique and cloning of cDNA) with bioinformatic analyses of massive parallel sequencing data to obtain AKH sequences for major lineages of both lower and higher termites.The predicted bioactive peptides and the complete preprohormonal sequences were subsequently used in comparative analyses within and between termites and their closest relatives, the cockroaches.

Experimental animals
The termite species Hodotermopsis sjöstedti and Termes hospes were obtained from laboratory colonies of D. Sillam-Dussès, Institut de recherche pour le développement, Paris, France.Termites Coptotermes formosanus and Mastotermes darwiniensis were provided by R. Plarre, Federal Institute for Materials Research and Testing, Berlin, Germany.Species Coptotermes gestroi, Neotermes castaneus and Prorhinotermes simplex were provided by J. Šobotník, University of Life Sciences, Prague, Czech Republic.The other species were analyzed in silico and respective transcriptomic data were accessed from the public source of the NCBI Sequence Read Archive (see chapter Sequence analyses).

Nucleic acid isolation
Total RNA was extracted using TRI reagent (Sigma) following the manufacturer's protocol.RNA isolates were treated with TURBO DNase (Ambion) to remove traces of contaminating DNA.

3´ RACE PCR
To obtain 3´ end of AKH transcripts, we performed reverse transcription using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) and 2 μg of respective template RNA with an oligo-dT primer.The resulting cDNA was amplifi ed in a subsequent PCR reaction using a 1 μl aliquot of the RT reaction as a template, a reverse primer from the oligo-dT adapter and the degenerate forward primer AKH-3RACE (Table 1).Forward primer was designed based on the known amino acid composition of AKH bioactive peptide of Blatella germanica (FJ943774.1)and Periplaneta americana (AY622321.1).The PCR profi le included initial denaturation (2 min at 94°C), 35 cycles of denaturation (94°C for 1 min), primer annealing (1 min) and extension (72°C for 2 min), followed by fi nal extension at 72°C for 10 min.The annealing temperature was 63°C in the fi rst cycle and was decreased by 1°C in each subsequent cycle down to 55°C, which was used for the rest of the reaction.The RT-PCR products were The resulting cDNA sequences were translated into proteins in silico and the respective ORFs (i.e. the preprohormonal AKH sequences) determined.Multiple sequence comparison was performed using CLUSTALW (Thompson et al., 1994) and consequent phylogenetic analysis using the neighbour-joining and maximumlikelihood methods in the SeaView program (Gouy et al., 2010).Bootstrap values were calculated based on 1000 replications in both analyses.The resulting phylogenetic trees were edited using FigTree software (version 1.4.2.; http://tree.bio.ed.ac.uk/software/fi gtree/).

RESULTS AND DISCUSSION
Termites are a species-rich group of social insects that was traditionally classifi ed at the taxonomic rank of order (Isoptera).However, in the past decade, evidence of a sister-group relationship between termites and the wood roaches of the genus Cryptocercus have accumulated (Lo et al., 2000;Inward et al., 2007;Legendre et al., 2008;Cameron et al., 2012;Djernaes et al., 2012).Thus, termites are now considered as "highly advanced social roaches" and classifi ed as a subgroup Isoptera within the order Blattaria (e.g., as an infraorder in Krishna et al., 2013).Within termites, there are currently nine families containing extant species: Mastotermitidae, Hodotermitidae, Archotermopsidae, Stolotermitidae, Kalotermitidae, Stylotermitidae, Rhinotermitidae, Serritermitidae and Termitidae (Engel et al., 2009).Nevertheless, recent phylogenetic studies based on morphological and/or molecular characters yield tree topologies slightly different from one another (reviewed in Krishna et al., 2013) and the phylogenetic relationships within termites are currently widely debated (Bourguignon et al., 2015;Legendre et al., 2015).Liebrich et al. (1995) used mass spectrometry to identify AKH bioactive peptides in representatives of three termite families, Mastotermes darwiniensis (Mastotermitidae), Microhodotermes viator (Hodotermitidae) and Trinervitermes trinervoides (Termitidae).A novel type of neurohormone was detected in M. viator (Micvi-CC; pQINFTP-NWamide), whereas in M. darwiniensis and T. trinervoides the sequence of Peram-CAH-I (pQVNFSPNWamide) was recorded.This bioactive peptide is also recorded in representatives of the family Blattidae, in two families of Coleoptera and in the hemipteran Locris arithmetica (re-viewed in Gäde, 2009).We detected a sequence of the Peram-CAH-I peptide in other termite families, namely in Kalotermitidae and Rhinotermitidae (Table 2).The Emppe-AKH (pQVNFTPNWamide), previously reported in Mantodea (Gäde, 1991), also occurs in the termite family Archotermopsidae (Table 2).All peptides only differ at positions 2 (isoleucine/valine) and 5 (serine/threonine).
In termites, we detected a single form of AKH in each of the species studied and all predicted bioactive peptides consisted of 8 amino acids.In this respect, there is a greater variety in the cockroaches, the closest relatives of termites.In two families, Blaberidae and Blattellidae, a single AKH (Bladi-HrTH) consisting of 10 amino acids was found, while in families Blattidae and Polyphagidae, two AKH octapeptides were recorded in one species, i.e.Peram-CAH-I and -II in Blattidae, and Polae-HrTH and Tenmo-HrTH in Polyphaga aegyptiaca (Polyphagidae; reviewed in Gäde, 2009; see Table 2).The occurrence of two bioactive peptides in one species might indicate a different role for each AKH in the metabolism as in Locusta migratoria and Pyrrhocoris apterus (Bártů et al., 2010;Tomčala et al., 2010).
The most frequent form of AKH recorded in termites is Peram-CAH-I (Table 2).It is thought to be the ancestral type of AKH peptide in Chrysomelidae, in which there are two other types of AKH peptides (Emppe-AKH and Peram-CAH-II).The phylogenetic scenario of Gäde & Marco (2011) proposes a replacement of nucleotide T in position one of the genetic code for serine in Peram-CAH-I by A to code for threonine in Emppe-AKH; subsequent nucleotide changes then lead to the formation of Peram-CAH-II peptide.Peram-CAH-I also appears to be the ancestral peptide  Fig. 2. Alignment of AKH preprohormones in termites.Conserved residues are boxed with the color black, identical with dark grey, and similar with light grey background.In the preprohormonal sequence, the initial part represents a signal peptide that precedes the AKH bioactive peptide (underlined with a solid line).The processing site is underlined with a dotted line followed by the AKH-associated peptide. in termites; a point mutation leading to the substitution of serine in Peram-CAH-I to threonine in Emppe-AKH is obvious from the DNA data shown in Fig. 1.A subsequent single nucleotide substitution may have lead to the formation of Micvi-CC, a third type of AKH detected in termites (Fig. 1).Interestingly, Peram-CAH-I is not shared with the phylogenetically closest family of cockroaches, Cryptocercidae (Table 2).Nevertheless, Tenmo-HrTH (pQLNFSPN-Wamide) detected in C. darwini differs only in the occurrence of leucine at position 2 in comparison to valine in Peram-CAH-I (pQVNFSPNWamide); a single nucleotide substitution (C/G in position one of the genetic code) may have lead to the replacement of this amino acid.
The sequences of AKH bioactive peptides were used to investigate phylogenetic relationships in Dictyoptera and the results are in agreement with contemporary views of the phylogeny of this taxon (Liebrich et al., 1995;Roth et al., 2009).To test whether the sequence of a large AKH precursor protein will be similarly suitable for the reconstruction of phylogenetic relationships we compared preprohormonal sequences of representatives of all major phylogenetic lineages of termites.The sequences obtained by both cloning and bioinformatic analysis are available from GenBank (accession numbers KT805304-KT805316).
The alignment of AKH preprohormones of the termite species examined is shown in Fig. 2. The amino acid (aa) similarity, recorded in representatives of fi ve families of termites, was at least 84%.Congeneric species showed either identical aa sequences (R. fl avipes and R. speratus) or only one aa substitution (C.formosanus and C. gestroi).The latter case was recorded also in species of different genera, namely in T. hospes and N. takasagoensis (Termitidae).Preprohormones of termites compared to those of Periplaneta americana (Blattidae, for which there is only a whole preprohormonal sequence available for Peram-CAH-II), Blaberus discoidalis (Blaberidae, Bladi-HrTH) and Blatella germanica (Blattellidae, Bladi-HrTH), share from 74% to 89% aa similarity.In general, the within-order similarity of AKH precursor sequences greatly varies among individual orders.For example, AKHs in representatives of different families in Diptera and Hemiptera share generally 50%-70% of aa similarity.The aphids represent the exception in Hemiptera with an 81% aa similarity detected between representatives of Aphididae (Acyrthosiphon pisum and Sitobion avenae) and a member of Adelgidae (Dreyfusia spp.).Among dipteran species, the highest value (78%) was detected between AKHs of Musca domestica (Muscidae) and Drosophila melanogaster (Drosophilidae; see Material and methods for species studied and their GenBank accession numbers).Thus, the high level of similarity found in termite AKH precursors resemble more the values detected within the rank of family than whole orders in other insect groups.Nevertheless, more data of the whole preprohormones in different taxa would be necessary to depict any trends in AKH sequences.
The phylogenetic analysis of all of the AKH preprohormones was carried out in order to reveal relationships within termites and also between termites and cockroaches .
Since we did not obtain data of representatives of all termite families, the simplifi ed view of phylogenetic relationships within termites presented by Thompson et al. (2000) and Legendre et al. (2008) was accepted as a plausible hypothesis for comparison (Fig. 3).Using two different methods of reconstructing trees (neighbour-joining and maximum-likelihood analyses) the termites (in the absence of data for the Cryptocerc idae) clustered as a sister group of Blattidae (represented by P. americana), whereas members of the other cockroach families (Blattellidae and Blaberidae) were assigned to more distant clades.This pattern is in accordance with the recent classifi cation of Blattaria (Legendre et al., 2008;Djernaes et al., 2012).The clustering within termites based on the AKH precursors revealed some divergences from the views of termite phylo geny based on morphological traits (Engel et al., 2009) and/ or nucleotide sequences (Fig. 3C; Legendre et al., 2008).Mainly, the AKH of N. castaneus (Kalotermitidae) differed the most from that of the preprohormones of other species (Fig. 3A, B) and also from its expected phylogenetic position (compare with Fig. 3C).In addition, P. simplex (Rhinotermitidae) was segregated from other termite species in the phylogenetic tree reconstructed using the neighbourjoining method (Fig. 3A), whereas this species was placed in the Mastotermitidae/Archotermopsidae cluster in the maximum-likelihood tree (Fig. 3B).The remaining AKH sequences were placed in two separate clusters by both methods.The fi rst cluster also included the phylogenetically primitive termites, M. darwiniensis (Mastotermitidae), and H. sjoestedti and Z. nevadensis (Archotermopsidae), whereas the second cluster encompassed the monophylum Neoisoptera, which included the families Rhinotermitidae (except Prorhinotermes) and Termitidae (Fig. 3).This cluster had the highest bootstrap value and the phylogenetic position of the termites was as in the recent classifi cation (Krishna et al., 2013).In short, with a few exceptions, the phylogenetic analysis based on AKH precursors is similar to the common termite phylogeny at the family level.

CONCLUSIONS
We analyzed and compared the AKH bioactive peptides and preprohormonal sequences of 13 species from all major phylogenetic lineages of termites.Three types of AKH bioactive peptides were detected in Isoptera, with Peram-CAH-I the most frequent form.Phylogenetic analysis of AKH preprohormonal sequences revealed that most of the species studied were clustered according to their expected affi liation at the family level.The exceptions are the sequences of N. castaneus and P. simplex, which diverge most from the AKH precursors of other species.

Fig. 1 .
Fig. 1.Proposed model for the formation of the different adipokinetic peptides detected in termites.Arrows indicate the changes in amino acids; conserved residues are boxed with the color black, and similar with grey background.Differences in nucleotides are highlighted in boxes.For Micvi-CC, for which no DNA data are available, a hypothetic scenario for a point mutation leading to the substitution of amino acids (valine to isoleucine) is given.

Fig. 3 .
Fig.3.Phylogenetic analysis of preprohormonal AKH sequences of termites (Isoptera) and cockroaches (Blattaria).The neighbour-joining (A) and maximum-likelihood (B) methods were used and the resulting trees were rooted using the precursor of AKH-1 of Locusta migratoria (Orthoptera; CAA60494.1).Bootstrap values were calculated based on 1000 replications in both analyses and cited for each branch.(C) A view of the phylogeny of termites adopted fromThompson et al. (2000)  andLegendre et al. (2008).

Table 1 .
List of primers used for cloning AKH transcripts.

Table 2 .
AKH peptides of Blattaria and Isoptera.In Blattaria, one representative is shown for each genus for which several species have been studied.* -in E. capucina and T. petiveriana a single AKH (Tenmo-HrTH) was studied.