Isolation and primary structures of neuropeptides of the AKHRPCH family from various termite species

Peptides, Vol. 16. No. 4, pp. 559-564, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All tights reserved 0196-9781/95 $9.50 + .OO

0196-9781(95)00012-7

Isolation and Primary Structures of Neuropeptides of the AKWFWCH Family From Various Termite Species WALTER *Zoology

LIEBRICH, *l ROLAND

Department,

fEuropean

KELLNERt’

AND

GERD G;iDE*3

of Cape Town, Rondebosch 7700, South Africa and Biology Laboratory, D-69012 Heidelberg, Germany

University

Molecular

Received

8 August 1994

LIEBRICH, W., R. KELLNER AND G. GADE. Isolation and primary structures of neuropeptides of the AKH/RPCHfamilyfrom various termite species. PEPTIDES 16(4) 559-564, 1995.-We have isolated neuropeptides of the AKH/RPCH family from extracts of whole heads of four termite species (Mastotennes darwiniensis, Microhodotermes viator, Hodotermes mossambicus, and Trinervitermes trinervoides) using the effect of mobilizing lipids in Locusta migratoria for bioassay. Isolation was essentially achieved by two steps of reversed-phase chromatography (on phenyl-support followed by C- 18). The peptides were identified by Eklman degradation after deblocking with oxoprolyl peptidase. Each termite species contained only one AKIURPCH family member. The primary structure in M. darwiniensis and T. trinervoides is pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH*, a peptide previously found mainly in cockroaches and code named Pea-CAH-I. The peptide from M. viator has the primary sequence pGluIle-Asn-Phe-Thr-Pro-Asn-Trp-NH*; it is a novel member of the family and is code-named Miv-CC (Microhodotermes viator corpus cardiacum peptide). Phylogenetic relations between the known cockroach and mantid AKI-hRPCH octapeptides and the termite peptides from this study could be revealed employing the parsimony method. Based on a computer analysis, using PAUP 3.1.1.) we concluded that termites arc plesiomorphic with regard to cockroaches, and mantids arc the sister taxon to the termite/cockroach group. Termites Mastotefmes Amino acid sequence

ainwiniensis

Trinervitermes

trinervoides

and hypertrehalosemic neuropeptides are insect peptides that ensure the availability of metabolites (mainly lipids and carbohydrates, but possibly also amino acids) during such energy-demanding processes as flight. These peptides have been isolated from the corpora cardiaca of the major insect orders and, functioning as a chromatotropin, from the sinus gland of some crustaceans (7,ll). They are grouped into the adipokinetic hormone/red pigment-concentrating hormone family (AKH/ RPCH family). The peptides are characterized as 8 to lo-mers with blocked termini (Soxoprolyl-2-carbonic acid at the N-terminus and a carboxyamide at the C-terminus) having aromatic amino acids at positions 4 and 8, a Gly residue at position 9, and all but two members are not charged (7,20); one member occurs in a modified form at the Trp residue (19). Previously, we had studied the isolation and primary sequences of AKH/RPCH family members in mantids (9) and cockroaches (6,17,18). These groups together with the termites (Isoptera) form the insect order Dictyoptera, which is generally believed to be a monophyletic taxon (24). Both to extend our knowledge of structural data to another insect group and to con-

Microhodotermes

viator

AKH/RPCH family

tribute to the ongoing discussion on the phyletic relationship between Mantodea, Blattaria, and Isoptera (2,20,29), we have isolated and characterized AKH/RPCH family members from various termite families that are believed to belong to more “primitive” or “advanced” groups of Isoptera.

ADIPOKINETIC

METHOD

Insects

Colonies of Hodotermes mossambicus (Hodotermitidae) were obtained from Professor R. Crewe (Zoology Department, University of the Witwatersrand, Johannesburg) and from the Plant Protection Research Institute (Pretoria) and kept at 25°C. Individuals of all castes of the species Mcrohodotermes uiator (Hodotermitidae) and Trinervitermes trinervoides (Termitidae) were collected in the field near Van Rhynsdorp and Citrusdal (both in the Cape Province), respectively, and were only held in the laboratory for about 2 days until all preparations were done (see below). Heads from Mastotermes ahwiniensis (Mastotermitidae), preserved in 80% methanol, came from Dr. J. A. L. Watson

’ Present address: Department of Medicine, University of Cape Town, Observatory, South Africa. ’ Present address: Institute for Physiological Chemistry and Pathobiochemistry, D-55099 Mainz, Germany. ’ Requests for reprints should be addressed to Professor Gerd Gade.

559

LEIBRICH, KJZLLNER AND GiiDE

560

(Commonwealth Scientific and Industrial Research Organization, Canberra, Australia). For heterologous bioassays (see below) 1525-day-old male migratory locusts, Locusta migratoria, and adult male cockroaches, Periplaneta americana, were used, which were kept and fed as described previously (10,12).

the enzyme was purchased from Sigma Chemical Co.; the deblocked peptide was separated from the undigested one by RPHPLC. Sequencing of the deblocked peptides was performed using a model 477A pulsed-liquid phase sequencer with integral model 120A phenylthiohydantoin amino acid analyzer (both from Applied Biosystems, Foster City, CA).

Bioassays

Hemolymph sampling, injection of material, and hyperlipemic (in acceptor locusts) and hypertrehalosemic (in acceptor cockroaches) bioassays are outlined elsewhere (4). Increases in the levels of hemolymph carbohydrates and lipids upon treatment with active material or fractions were compared to water-injected controls by using Student’s t-test. Isolation and Purification of Peptides

Due to the small size of the termites, corpora cardiaca could only be dissected from the workers of M. viator and H. mossambicus, which were treated for methanolic extraction as described previously (14); the material was used for some pilot experiments on C- 18 reversed-phase liquid chromatography (RP-HPLC). For bulk extraction of peptides, whole heads from all species were collected into liquid nitrogen and then stored at -70°C. Batches of up to 300 frozen heads of each species were separately crushed in liquid nitrogen, the powder suspended in 4 ml of 80% methanol, vigorously mixed, and centrifuged at 6000 X g for 10 min at 4°C. After reextracting the pellet twice with 3 ml of 80% methanol, sonification, and recentrifugation, the combined SUpematants were dried by vacuum centrifugation and taken up in 0.5 ml of distilled water. For lipid removal this extract was partitioned with an equal volume of chloroform and the water phase was loaded onto a Sep-Pak C-18 RP cartridge (Millipore Corporation, Milford, MA). This was washed with 18%, 30%, and 100% acetonitrile containing 0.1% trifluoroacetic acid (TFA). The 30% fraction, which was shown in pilot experiments to contain active material, was dried by vacuum centrifugation and used for RP-HPLC. In the case of h4. viator and H. mossambicus either the corpus cardiacum material or the material from heads (only M. viatur) after the Sep-Pak step was subjected to RP-HPLC on a Nucleosil 100 C-18 column as described previously (5). One-minute fractions were collected and used for bioassays in acceptor locusts. For head material of M. aiwwiniensis and T. trinervoides it had been shown in pilot experiments that, when the extract after the Sep-Pak step was applied to C-18 RP-HPLC, no pure fraction could be obtained. Therefore, in these cases the prepurified material was further purified in two steps. First the material was applied to a PBondapak phenyl column (0.39 x 30 cm, 10-b particles), which was developed isocratically (3 min) at 25% solvent B (0.1% TFA in 60% acetonitrile, solvent A was 0.11% aqueous TFA; flow rate 0.9 ml/min) followed by a linear gradient of 25% B to 45% B in 30 min. One-minute fractions were collected into tubes containing 0.1 mg bovine serum albumin to prevent lossess due to unspecific binding to the plastic during vacuum drying. Using an aliquot of the dissolved material, active fractions were detected by the locust bioassay. In a second step, this active material was applied to a Nucleosil 100 C-18 column as previously described (5) and biologically active material used for structural analyses. Sequence Determination

Biologically active fractions after the last RP-HPLC step from and M. darwiniensis were separately cleaved with oxoprolyl-peptidase according to (15) except that

M. viator, T. trinervoides,

Analysis of the Phylogeny of Dictyopteran Octapeptides

AKH/RPCH

For the analysis of phylogenetic relations between the dictyopteran octapeptides we employed the parsimony method (28). An optimization was achieved with ACCTRAN (accelerated transformation) using the PAUP 3.1.1. computer program (27). After an exhaustive search the program found four shortest trees (seven steps; consistency index 0.714), which could not be fully resolved. A strict consensus tree was drawn, which was rooted on Pab-RPCH (see the Discussion section). Synthetic Peptides The adipokinetic peptide of Locusta migratoria, Lom-AKHI, and the hypertrehalosemic peptide of Periplaneta americana, Pea-CAH-I, came from Peninsula Laboratories (Belmont, CA). The hypertrehalosemic peptide of Tenebrio molitor, which is also found in the cockroach Polyphaga aegyptiaca, Tern-HrTH, and the novel termite peptide were synthesized on a multiple solid-phase instrument (AMS 422, Abimed, Germany) employing fluoren-9-ylmethoxycarbonyl (Fmoc) chemistry. The synthetic peptide was purified by RP-HPLC and its identity verified by amino acid analysis and mass spectrometry [MALDI-MS, Bruker REFLEX spectrometer; as outlined in (23)]. RESULTS

Presence of Biological Activity in Termite Corpus Cardiacum and Head Extracts

Methanolic extracts of corpora cardiaca from H. mossambicus or of heads from the other species were injected into acceptor locusts and/or cockroaches, which are both widely used as heterologous bioassay systems to check for the presence of AKH/ RPCH peptides (7). As depicted in Table 1, one head equivalent from M. danviniensis and 2.5 head equivalents from M. viator were able to elicit maximum adipokinetic responses in locusts compared to injection of 10 pmol Lom-AKH-I, which results in the maximal possible response in this insect (13). However, the high doses used of H. mossambicus (2.5 corpora cardiaca equivalents) and of T. trinervoides (10 head equivalents) caused only 50% of the maximal possible response in locusts. When these latter materials were bioassayed in cockroaches, the maximal possible hypertrehalosemic response was achieved (Table 1). For convenience, the locust bioassay was chosen as routine test. We also attempted to use nymphs of the two Hodotermitidae species (H. mossambicus, M. viator) for conspecific bioassays. Due to the small size of the insects and the fast clotting of the hemolymph in other, marginally larger castes, it was extremely difficult to handle these species. We quantified the hemolymph lipid concentration as 6.4 + 2.7 mg/ml (mean 2 SD; n = 13), and the hemolymph carbohydrate concentration was 21.8 + 5.4 mg/ml (n = 14). These resting, basal values for both hemolymph metabolites did not significantly change when insects were exposed to stress (making them walk for 10 min), starvation (no food for up to 5 days, but access to water), or when they were neck ligated (up to 24 h) to prevent release of a putative AKHlike neuropeptide from the head region. We also measured the activity of the glycogen phosphorylase in the abdominal fat body

AKH/RPCH

NEUROPEPTIDES

561

IN TERMITES

TABLE

1

EFFECTS OF INJECTION OF CORPORA CARDIACA AND/OR HEAD EXTRACrS PROM VARIOUS TERMITE SPECIES ON HEMOLYMPH LIPID AND CARBOHYDRATE CONCENTRATIONS IN ACCEPTOR LOCUSTS AND COCKROACHES

Acceptor Insect

Control (10 ~‘1Dist. Water)

Hemolymph Metabolites

L. migratoria

lipids

P. Americana

carbohydrates

0 min 90 min difference n 0 min 120 min difference n

13.1 % 2.7 12.2 5 2.2 0.9 + 1.8 13 19.6 t_ 4.8 21.0 k 4.5 1.4 2 1.3 6 Control (IO ~1 Dist. Water)

L. migratoria

lipids

P. americana

carbohydrates

L. migratoria

0 min 90 min difference n 0 min 120 min difference n

11.7 2 25.4 -c 13.7 2 6 15.0 + 33.4 2 18.4 -t 6

1.3 2.8 3.2*

T. trinervoides

2.2 3.8 2.0 2.9 7.4 6.1*

0 min

12.2 k 3.2

90 min difference

13.5 3 3.6 1.3 + 1.3

16.9 k 4.5 51.2 % 10.7 34.3 2 6.7*

6

6

Control (10 pl Dist. Water)

(1 Head Equivalent)

0 min

22.3 2 6.7

90 min difference

25.0 2 7.1 2.7 + 3.1

lipids

n

8

14.0 2 6.3 36.0 k 13.9 22.0 -t 8.4*

12.2 2 4.1 58.4 t- 14.9 46.2 rt 12.3* 12 16.1 2 2.8 34.1 2 8.8 18.0 2 8.8* 6

M. victor Nymph (2.5 Head Equivalents)

lipids

Lam-AKH-I/Pea-CAH-I ( IO pmol)

6

(I Head Equivalent) 11.1 + 14.6 k 3.5 + 7 15.1 k 30.5 + 15.4 + 8

H. mossambicus (2.5 CC Equivalents)

3.3 9.4 7.9*

Control (10 ~1 Dist. Water)

n

L. migraioria

10.6 2 2.2 10.9 + 2.6 0.3 k 1.9 15 16.3 2 3.3 17.0 2 3.5 0.7 % 2.0 14

H. mossambicus (1 CC Equivalent)

T. trinervoides (10 Head Equivalents) 10.4 k 33.2 % 22.8 2 7 16.9 ? 40.6 t 23.7 i 6

2.3 8.7 7.0* 3.0 8.9 9.6*

U. viaror Soldier (2.5 Head Equivalents) 19.0 k 62.1 + 43.1 +

3.1 7.9 7.4*

Lom-AKH-IPea-CAH-I ( 10 pmol) 10.2 2 3.3 45.3 2 10.2 35.1 t 8.3* 16 2.0 16.8 t 39.1 t 7.6 22.3 + 7.2* 15

Lam-Am-1

(IO pmol)

11.3 t 49.5 2 38.2 t

2.1 8.3 6.6* 6

7

M. danviniensis

17.6 2 4.5 55.0 2 15.2 37.4 z 11.0’ 7

Lam-AKH-I (10 pm@ 11.3 2 52.5 2 41.2 2

2.7 4.4 3.2* 5

Values are mean 2 SD of total hemolymph lipid or carbohydrate concentration (mg/ml). * Hemolymph metabolite increases are significantly different compared to water-injected controls, p < 0.001.

[according to (S)]; the enzyme was up to 50% in the active aform and stress (see above) did not increase the active form. Injections of water and synthetic peptides into the abdomen were attempted but were not successful due to practical limitations (see above). Thus, in all these experiments we could not find any indication of an endogenous effect ascribed to AKIURPCH-type neuropeptides. Peptide Puri$cation and Characterization hf. viator and H. mossambicu.s. The W absorbance profile of a methanolic extract of 50 corpora cardiaca from il4. viutor shows only a few peaks on RP-HPLC (Fig. 1, inset). After testing ail fractions for hyperlipemic activity in locusts, only the fraction at 13.3 min was active. This fraction corresponded to a distinct W absorbance peak and had the same retention time as synthetic Tern-HrTH when run under the same conditions. An almost identical profile was

yielded when a methanolic extract of 40 corpora cardiaca of H. mossambicus was applied to the column (results not shown). To purify sufficient material for structural work, 150 heads of M. viutor (700 heads for total purification) were chromatographed on the C18 column after prepurification as outlined in the Method section. The biologically active material that was eluted at 29.5 min again had an identical retention time to Tern-HrTH (Fig. 1); the material was sufficiently pure for enzymatic deblocking. This procedure yielded the deblocked peptide, which was sequenced as Ile-AsnPhe-Thr-pro-Asn-Trp (Table 2). We synthesized the new peptide (including the C-terminal amide) and found that it was coeluted on RP-C18-HPLC with native peptide material isolated from M. vziztor and H. mossumbicus (results not shown). This chromatographic behavior shows that the correct C-terminus was assigned, because a peptide with a free carboxyl-terminus would have a diffiint retention time [see (12,21)].

562

LEIBRICH, KELLNER AND GADE

TABLE 2 AMINO ACID SEQUENCE ANALYSES OF TERMITE HEAD PEPTIDES

n

~~ 0 ,

time

(min)

. * .

,I

10

30

20

Retention

time

40

bin)

FIG. 1. Purification of an active neuropeptide from M. viaior heads (150 head equivalents) using C-18 RP-HPLC. Inset: Separation of a methanolic corpora cardiaca extract of M. riotor (50 CC-equivalents). Nucleosil 100, C-18, 5 p, 0.46 X 25 cm; 1 ml/min; solvent A: 0.11% TFA, B: 0.1% TFA, 60% acetonitrile; linear gradient as indicated in the figures; fractions active in the locust bioassay marked with a square; retention times of synthetic peptides determined under the same conditions; UV absorbance is monitored at 214 nm.

T. trinervoides and M. darwiniensis. Pilot experiments with 150 and 90 head equivalents from T. trinervoides and M. darwiniensis, respectively, prepurified as outlined in the Method section, had shown that the biological activity was eluted at 22 min on the C-18 column, but was buried in a region with other major absorbance peaks (data not shown). Synthetic Pea-CAH-I had an identical retention time on this system. Therefore, we decided to first apply those extracts on a phenyl column [Fig. 2(A,B)]. The absorption profiles of 150 head equivalents (for total purification 550 heads were used) of T. trinervoides [Fig. 2(A)] and 300 head equivalents (total 500 heads) of M. darwiniensis [Fig. 2(B)] showed numerous peaks; the active material was identified by bioassays in locusts and was coeluted with synthetic Pea-CAHI under the same conditions. These materials were rechromatographed on the C-18 column and gave single absorbance peaks with biological activity, which again were coeluted with PeaCAH-I [see insets in Fig. 2(A) and (B)]. After separately treating the materials with oxoprolyl-peptidase, Edman degradation of the deblocked peptides in each case yielded the following sequence: Val-Asn-Phe-Ser-Pro-Asn-Trp (Table 2). The identical chromatographic behavior of native and synthetic peptide material (not shown) again showed that the correct structure was assigned (see above).

Residue (pmol)

Edman Cycle

M. viator

1 2 3 4 5 6 I

T.trinervoides

Ile 162 Asn 26 Phe 68 Thr 45 Pro 25 Asn 43 Trp 18

Val Asn Phe Ser Pro Am

M. danviniensis

44 11 34 8

Val Asn Phe Ser

113 61 54 25 Pro49 Asn 36 Tip 20

II I

Trp5

small for dissecting corpora cardiaca or because of logistical reasons, when we were given head material of M. darwiniensis. Starting with such crude whole-head material made additional purification steps necessary, such as removal of lipids and prepurification on Sep-Pak cartridges. Sufficiently pure material.

Retention

25%

time

(min)

B

!:I 10

0

B

30

20 Retention

time

40

bin)

II

,c

II

DISCUSSION

Isolation and Structure

For the isolation of biologically active material, a bioassay that is reliable and easy to perform is paramount in monitoring the success of purification throughout the various steps. Our isolation of termite peptides was guided by the heterologous hyperlipemic bioassay in locusts, because we were unable to show any metabolic effect of conspecific injections into termites, due to the small size and possibly to unknown age and physiological state of termites used. Previously we had developed a fast, essentially single-step purification method when starting with dissected corpus cardiscum material (14). Although this method would have been successful with termites as well, as shown for the corpora cardiaca of M. viator and H. mossambicus workers (Fig. l), it could not be used because most of the animals we worked on were too

0

10

20

Retention

time

30

40

bin1

FIG. 2. Purification of a T. trinervitermes (A) or a M. datwiniensis neuropeptide (B; 150 and 300 head equivalents, respectively) by phenyl RPHPLC (as described in the Method section). Active fractions (marked with a square) were then subjected to ClI-RP-HPLC (insets; conditions as in Fig. 1). Active material (squares) was separately used for sequence determination. The retention time of synthetic Pea-CAH-I was determined under the same conditions.

AKH/RPCH NEUROPEPTIDES

563

IN TERMITES

b) the only peptide isolated so far from mantids, Emp-AKH; here the Ile’ in Miv-CC is changed to Val’ (Fig. 3).

however, was obtained employing one (M. vialor) or two (M. durwiniensis, T. rrinervoides) RP-HPLC steps. This contrasts quite sharply with three or at least five chromatographic steps necessary to isolate the AKH/RPCH-members from whole heads of the cockroach Blaberus discoidalis (21) and the silkworm Bombyx mori (22), respectively. The pure material obtained from several hundred heads of the three termite species was sufficient to fully elucidate the primary structures. Each species only had one active peptide and each was a typical member of the AKH/ RPCH family, judged by the criteria given in the Introduction. Although not backed up by mass spectrometry in this study, the chromatographic behavior of the native peptides compared to their respective synthetic compounds unequivocally assigned them an amidated C-terminus as opposed to a free acid [see (12,21)]. Whereas M. danviniensis and T. rrinervoides contain Pea-CAH-I, a peptide previously found in members of blattid cockroaches (1,17,26,30) and in the Colorado potato beetle, Z_eptinotursa decemlineatu (16), the peptide from M. viator is a novel member of the AKH/RPCH family (Fig. 3). According to the nomenclature proposed by Raina and Gade (29, this peptide is designated by the acronym Miv-CC (Microhodotermes viator corpus cardiacum peptide). The most striking resemblance (88%) of the primary structure of Miv-CC occurs with

Evolution Although AKH/RPCH peptides are small and do not encode a great deal of genetic data, it is interesting and enlightening to use the current information of the six octapeptides sequenced from Dictyoptera (Fig. 3), including the results of this study, and speculate about molecular evolution taking only single-point mutations into account. The only dictyopteran decapeptide, BldHrTH, was left out from the following considerations because the different length of the sequence would have added another variable. It is obvious that the C-terminal tripeptide (. . . ProAsn-Trp-NHJ is identical in all octapeptides, and changes only occur at positions 2 (Val, Leu, Ile), 3 (Asn, Thr), and 5 (Thr, Ser). Single-point mutations can account for all the amino acid substitutions at these positions (20). Phylogenetic relations between the dictyopteran AKH/RPCH octapeptides (Fig. 3) can be revealed employing the parsimony method (28). This approach allows constructing a phylogenetic tree, which requires a minimum number of amino acid replacements. The resulting tree was rooted on the crustacean red pigment-concentrating hormone, Pab-RPCH (Fig. 3). It is the most closely related noninsect AKH/ RPCH peptide. This approach was also used previously (20). The consensus tree (Fig. 3) shows a group of peptides @&v-CC, Pea-CAH-II, Poa-HrTH, and EmpAKH) that have EmpAKH as their common ancestor. The other two peptides, Pea-CAH-I and

a) the only other Be’-containing AKH/RPCH peptide isolated from the corpora cardiaca of the cockroach P. aegyptiaca (Poa-HrTH); the latter peptide contains a Thr at position 3, whereas Miv-CC has an Asn residue there, and

Miv-CC termites (Hodotemtitidae; this study) pClu-lie-Asn-Phe-Thr-Ro-Asn-TrgMi2

0

I I -Miv-CC

I

i

Pea-CAH-II cockroaches (Blattidae) [17, 26, 301 pGlu-h-Thr-Phe-Thr-Pro-Asn-Trp-NH2

1-Pea-HrTH I

1-Emp-AKH

2-

0

1

I

Pea-CAH-I

0

Poa-HrTH cockroaches (Polyphagidae) [18] pGlu-Be-Th-Phe-Thr-Pro-Am-Trp-NH2 Emp-AKH mantids 191 pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp-NH*

Pab-RPCH

Pea-CAH-I 0

cockroaches(Blattidae)11, 17, 26, 301 temtites (Mastotermitidae, Termitidae; this study) pClu-W-Asn-Phe-Ser-Pro-Asn-TrpNH1

1

I

0

Tern-HrTH cockroaches (Polyphagidae) [18] pClu-&-Asn-Phe-Ser-Pro-Am-Trp-NH2

Pab-RPCH crustaceans [3] pClu-Len-Asn-Phe-Ser-Pro-Cly-TrpNH2

FIG. 3. Strict consensus tree for dictyopterau AKH/EWCH octapeptides found by the computer pmgram PAUP using the parsimony principle. The tree was rooted with the crustacean red pigment-concentrating hormone Pab-RPCH. The numbers of amino acid substitutions are indicated in the branches. Amino acid substitutions with regard to the respective preceding ancestral peptides are underlined.

564

LEIBRICH, KELLNER AND GADE

Tern-HrTH, are probably very old, as they form an unresolved trichotomy with the “EmpAKH group.” All dictyoptemn AKH/RPCH octapeptides have Pea-CAH-I as a common ancestor. Note that PeaCAH-I and Pea-CAH-II or Poa-HrTH and Tern-HrTH occur, in blattid or polyphagid cockroaches respectively, in pairs (Fig. 3). According to this computer analysis based on the molecular evolution of AKH/RPCH octapeptides, we can draw the following conclusions about the phylogenetic relationship between the three main dictyopteran groups:

Our data on termite AKWRPCH peptides are not sufficient to draw conclusions about evolutionary relationships between the different termite groups (2). However, the octapeptide Pea-CAHI, which was isolated first from cockroaches (Fig. 3), was found in both, allegedly “primitive” (Mastotermitidae) and “advanced” (Termitidae) termite groups (Fig. 3), supporting our view that Pea-CAH-I is a relatively “old” AKH/RPCH peptide, which was retained in both of these termite groups.

1. Termites are plesiomorphic with regard to cockroaches. 2. Mantids are the sister taxon to the group consisting of termites and cockroaches.

ACKNOWLEDGEMENTS

This interpretation is not in agreement with other recent studies. A phylogenetic survey based on 70 morphological and behavioral characters (29) suggests a closer relationship between cockroaches and mantids, with termites forming a sister group to the cockroach/ mantid complex. These findings ate confirmed by a phylogenetic analysis of dictyopteran mitochondrial DNA genes (2).

We thank Miss M. C. Breton (EMBL) for her help with peptide synthesis and Dr. A. E. Channing (University of the Western Cape, Bellville, South Africa) for helping us with the analysis of the dictyopteran peptides with the PAUP computer program. Financial support was provided by a postdoc position (W.L.) on a grant (to G.G.) from the Foundation for Research Development (Pretoria. South Africa) and by a University of Cape Town staff award (to G.G.).

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