The hypertrehalosaemic peptides of cockroaches: A phylogenetic study

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

75,287-300

(1989)

The Hypertrehalosaemic Peptides of Cockroaches: A Phylogenetic Study GERD GADE Institut fur Zoologie IV der Vniversitiit Diisseldofl, Vniversitiitsstrasse Federal Republic of Germany

1, D4ooO Dusseldorf I,

Accepted January 10, 1989 Hypertrehalosaemic peptides from the corpora cardiaca of 14 different species were compared with respect to phylogenetic relationships within the insect suborder Blattaria (cockroaches). Gland extracts from members of the family Blattidae (Periplaneta americana, P. brunnea, P. australasiae, P. fuliginosa, and Blatta orientalis) contain two hypertrehalosaemic octapeptides with identical properties to the recently sequenced peptides M I and M II from the American cockroach, whereas corpora cardiaca from members of the families Blaberidae and Blattellidae (Nauphoeta cinerea, Leucophaea maderae, Blaberus discoidalis, B. trapezoideus, Diploptera punctata, and Gromphadorhina portentosa) possess one hypertrehalosaemic decapeptide with identical properties as the peptide recently sequenced from B. discoidalis and N. cinerea. A member of the family of Polyphagidae (Polyphaga aegyptiaca), placed at the origin of the phyletic tree of Blattaria, has two hypertrehalosaemic factors in its corpus cardiacum which are each diierent from M I, M II, and HTH. 0 1989 Academic Press,Inc.

Many peptide hormones of vertebrates are placed into so-called families according to similarities in their biological functions and chemical structures. Such relationships are also found among peptide hormones isolated from invertebrates. In recent years, much attention has been focused on structurally related but functionally diverse compounds from arthropods (insects and crustaceans) belonging to the so-called adipokinetic hormone/red pigment-concentrating hormone (AKIYRPCH) peptide family isolated from the (insect) neurosecretory corpora cardiaca as well as the (crustacean) neurosecretory X-organ/sinus gland complex of the eyestalk (for reveiws, see Greenberg and Price, 1983; Schaffer, 1986; Orchard, 1987; Wheeler et al., 1988). Up to now, 11 members of this family are fully sequenced and some of their functions are known (for a recent review, see for example, Gfide, 1988a). Grouping such peptides into a distinct family may reflect evolutionary and genetic relationships. It was

shown, for example, that such closely related species as Locusta migratoria, Schistocerca gregaria, S. nitans, and Romalea microptera, all belonging to the Acrididae, possess already genus-specific neuropeptides of the AKHmPCH-family (see Table 1). Moreover, in the suborder Blattaria (cockroaches), three structurally related but not identical neuropeptides that are capable of elevating hemolymph trehalose levels in the donor species have been found in the corpora cardiaca (although two of the peptides were also isolated on the basis of their myotropic action or their cardioacceleratory properties; G&de, 1984, 1985a, b; Witten et al., 1984; Scarborough et al., 1984; Siegert and Mordue, 1986; Glide and Rinehart, 1986; Hayes et al., 1986; G&de, 1988b). These hypertrehalosaemic hormones are the octapeptides M I (pGluVal-Asn-Phe-Ser-Pro-Asn-Trp-NH& and M II (pGlu-Leu-Thr-Phe-Thr-Pro-AsnTrp-NH& from the American cockroach, Periplaneta americana, and the decapep-

287 0016-6480/89 $1.50 Copyright All rights

0 1989 by Academic Press, Inc. of txpmduction in my form reserved.

288

GERD

GjiDE

TABLE 1 PRIMARY STRUCTURES OF NEUROPEPTIDES FROM THE CORPUS CARDIACUM OF DIFFERENT SPECIES FROM THE FAMILY ACIUDIDAE Source

Ntltlle

Structure

Ref,

Locusra migratoria Schisrocerca gregaria

AKH I

pGlu-Leu-Asn-Phe-Thr-Pro-AstwTrp-Gly-Thr-NH2

L. migraroria

AKH II-L

pGlu-Leu-Asn-Phe-Ser-Ala-Gly-TrpNH,

S. gregaria S. &tans

AKH II-S

pGlu-Leu-Asn-Phe-Ser-Thr&ly-Trp-NH2

Stone et al (1976)

Siegert et al. (1989 Siegert el al. ww G&de er al. m-w Siegert er al. (1985) G&de et al. (1986)

Romalea

microprera

Ro I Ro II

Giide et al. wm

pGlu-Val-Asn-Phe-Thr-Pro-Asn-T@ly-Thr-NHz pGl+Val-AsrbPhe-Ser-Thr-Gly-Trp-NH2

tide HTH (pGlu-Val-Asn-Phe-Ser-ProGly-Trp-Gly-Thr-NH,) from the cockroaches Blaberus discoidalis and Nau-

from as many different cockroach species as possible, isolate, and (partially) characterize their hypertrehalosaemic peptides to phoeta cinerea. get clues of the phylogenetic distribution in The probable phylogenetic relationships one particular suborder of insects. Cockof families and subfamilies of the Blattaria roaches are easy to rear, are used for teachare well known. A dendrogram based on ing and research in many laboratories, and morphological and physiological character- are also used as test insects in screening istics was constructed by M&&rick (1W) processes for insecticides by pharmaceutiand is represented in a slightly modified cal companies. Therefore, we were able to form in Fig. 1. The aim of the present study obtain and investigate 14 different species was to collect corpus cardiacum material belonging to 4 (out of 5) families and 7 (out

Wobcmid

complex

2Epilomproid

complex

Pikwtcrinae-Dipkgtera

punctato

Periploneta

Bhtta

FIG. 1. Evolutionary tree of cockroach families (modified after M&&rick, species used for the present study are given.

americano

orientalis

1964). Note that the

HYPERTREHALOSAEMIC

of 20) subfamilies (see Fig. 1). The data show clearly that all species investigated contain hypertrehalosaemic peptides in their corpora cardiaca; the family of Blattidae possesstwo peptides indistinguishable by chromatographic behavior and amino acid composition data from M I and M II; the families of Blaberidae and Blattellidae possess one peptide identical in all investigated parameters to HTH; and in the family of Polyphagidae, two peptides are present which are different from those of other cockroach families. MATERIALS

289

PEF’TIDES IN COCKROACHES

AND METHODS

1. Insects. Specimens of adult cockroaches were gifts from the following sources: Dr. G. Seelinger (Universitlt Regensburg, FRG), Bfatta orientalis, Periplaneta P. fuliginosa,

americana, P. australasiae, P. brunnea, and Gromphadorhina portentosa; Professor Dr. H. Bohn (Universitat Miinchen, FRG), P. brunnea, P. australasiae, Supella longipalpa, Diploptera punctata, Polyphaga aegyptiaca, G. portentosa, and Blaberus discoidalis; Dr. B. Lanzrein (Universitat Bern, Switzerland), Nauphoeta cinerea and Leucophaea maderae; Professor Dr. H. Kayser (Ciba Geigy AG, Basel, Switzerland), B. trapezoideus; Hoechst AG (Frankfurt, FRG), Blattella germanica and B. discoidalis; Thompson Co. (Dilsseldorf, FRG), P. americana. All animals were kept in our insectary

at ca. 25” with a LD 14:10-hr light cycle and were reared as described previously (Glde and Scheid, 1986). 2. Preparation of extracts from corpora cardiaca, and synthetic peptides. Whole corpora cardiaca were

removed from the different cockroach species under a stereomicroscope. Methanolic extracts of the glands were prepared for bioassay and reversed-phase highperformance liquid chromatography (RP-HPLC) as described previously (Gade et al., 1984). The synthetic hypertrehalosaemic peptides from P. americana (trade names: M I and M II) and N. cinerea were gifts from Peninsula Laboratories (Belmont, CA). 3. Bioassay. Hypertrehalosaemic activity was measured by the injection of an aliquot of corpus cardiscum extract from the different cockroaches into adult male acceptor P. americana as outlined earlier (G&de, 1980). Concentrations of total hemolymph carbohydrates (anthrone-positive material) were analyzed before the injection and 120 min thereafter according to Spik and MontreuiI (1964). 4. Reversed-phase HPLC. Methanolic extracts of corpora cardiaca from the different cockroach species

were dried down at reduced pressure (Speed-Vat, Savant) and dissolved in 25% solvent B (0.1% trifluoroacetic acid in 60% acetonitrile) for application onto a Nucleosil C-18 column (i.d., 4.6 mm; length, 250 mm; 5 k particle size). Details of the equipment used and the conditions applied are given elsewhere (G&de, 1985c; see also legend to Fig. 2). In brief, the aqueous solvent was 0.11% trifluoroacetic acid (solvent A), and solvent B was as given above. The solvents were applied as a linear gradient (43-53% in 20 min; flow rate 1 ml/mm), and the eluant was monitored at 210 nm. Peak fractions were collected, dried in vacua as above, resuspended in 100 4 of distilled water, and an aliquot was used for bioassay in P. americana by the injection of a lO-~1 dose into at least five assay insects. 5. Amino acid analysis. HPLC-purified samples of active fractions were dried in vacua, transferred to Pyrex tubes, and hydrolyzed with 5.7 M HCl. Derivatization and amino acid analysis on a Waters Associates Pica-Tag system were according to standard procedures supplied by the manufacturer and were custom-made by Dr. S. Kyin (Biotechnology Center, University of Illinois at Urbana, Champaign, IL).

RESULTS I. Presence of Hypertrehalosaemic Activity in the Corpora Cardiaca Various Cockroach Species

of

In order to check the various extracts of corpora cardiaca from the different cockroach species for their ability to cause hypertrehalosemia’ in P. americana, 0.2 gland equivalents were injected into this species. The data summarized in Table 2 show that all extracts are potent in elevating hemolymph carbohydrates in the American cockroach and, thus, contain hypertrehalosaemic material. 2. Separation Compounds

of Hypertrehalosaemic by RP-HPLC

Chromatography of an extract of 10 corpora cardiaca from P. americana revealed the typical elution pattern as observed pre‘Although total blood sugar was measured, the specific term “hypertrehalosemic” is used throughout this study because in P. americana it is the main blood sugar, trehalose, that is affected by the injection of corpus cardiacum extract (see Goldsworthy and G&de, 1983).

290

GERJI GADE

HYPERTREHALOSAEMIC

ACTIVITY

OF EXTRACTS DIFFERENT

TABLE 2 OF Comru COCKROACH

CARDIACA SPECIES

(0.2 GLAND

EQUIVALENTS)

FROM

Hemolymph carbohydrate concentration (mgknl)” Source

II

Distilled water P. americana P. brunnea P. fuliginosa P. australasiae B. orientalis P. aegyptiaca S. longipalpa B. germanica N. cinerea L. maderae G. portentosa B. discoidalis B. trapezoideus D. punctata MI

6 8 8 8 7 8 8 8 5 8 8 7 8 7 7 8

Omin 14.8 15.4 13.4 16.0 15.8 15.4 14.1 13.2 14.3 15.7 15.4 15.6 15.3 14.8 16.3 15.7

f 3.5 f 2.6 f 2.3 -’ 1.5 2 3.2 2 1.0 2 3.0 2 1.6 AI 2.2 k 2.4 2 3.2 +- 2.3 t 1.5 f 1.9 2 2.2 f 2.3

120 min 14.6 35.3 36.7 38.9 33.8 37.7 37.3 31.7 35.5 34.9 41.8 39.3 38.6 30.0 32.7 40.2

k f 2 ” 2 -’ 2 f a f _’ ” f k 2 +

2.6 3.9 8.6 6.5 4.9 6.7 7.6 2.8 1.0 7.1 5.4 3.2 4.9 5.3 4.9 5.3

Change in concentration -0.2 19.9 23.3 22.9 18.0 22.3 23.2 18.5 21.2 19.2 27.5 23.7 23.3 15.2 16.4 24.6

2 + + f + + + ” + + + f ” + f +

I.26 4.8 7.6 5.8 2.6 7.2 4.8 2.0 2.6 6.1 5.4 2.9 4.3 4.2 4.7 5.9

a The mean and SD are given ’ Not significant, Student’s t test; all other changes significant, P < 0.001.

viously (Gade, 1984, 1985a): hypertrehalosaemic activity was associated with two prominent absorbance peaks labeled 1 and 2, with retention times of 10.9 and 18 min, which represent the two hypertrehalosaemic hormones M I and M II; no other absorbance peak assayed had bioactivity (Fig. 2A). The separation of extracts of corpora cardiaca from P. fuliginosa (28 glands; Fig. 2B) and P. austrulusiue (19 glands; Fig. 2C) revealed almost identical chromatograms to those obtained from P. umericuna. Again, two peaks with retention times of 10.9 and 18 min were the only ones showing hypertrehalosaemic activity. The HPLC chromatograms of the corpus cardiscum material from P. brunneu (20 glands, Fig. 2D) and from B. orient& (12 glands; Fig. 2E) gave more distinct absorbance peaks than the former extracts. However, there were absorbance peaks with the retention times of 10.9 and 18 min present (labeled 3 and 6) and, in both species, these peaks were very potent in causing carbohydrate elevation. Although the peaks labeled

4 (both chromatograms) and 5 (for B. orientulis in Fig. 2E) showed a higher absorbance at 210 nm as the pronounced peak with bioactivity (labeled 3), their ability to mobilize carbohydrates was only small. Future studies must show whether this bioactivity is due to a peptide. In contrast to the members of the Blattidae, all species belonging to the Blaberidae and Blattellidae showed quite different HPLC chromatograms. First, we again seg arated the extract of corpora cardiaca from N. cinerea (10 glands; Fig. 3A) and obtained the typical elution pattern previously observed (G&de and Scheid, 1986; Giide, 1987): there is one very pronounced absorbance peak with a retention time of 8.9 min with hypertrehalosaemic activity which represents HTH. The separation of extracts of corpora cardiaca from some other members of the Blaberidae/Blattellidae, G. portentosa (8 glands; Fig. 3E), D. puncrufu (10 glands; Fig. 3F), and B. germanica (75 glands; Fig. 3H), also had only one prominent absorbance peak at 8.9 min which con-

HYPERTREHALOSAEMIC

PEPTIDES

IN

291

COCKROACHES

- LO is

-208s -0 0 Retention

0

time

i0 Retention

Retention

20 time

io

(mini

0 Retention

b

20 time

lminl

20

13

(mini

$F a-

time

(mini

3

12

h

0

10 Retention

6

20 time

lminl

FIG. 2. Separation of extracts from corpora cardiaca of different Blattidae species using reversedphase hi-performance liquid chromatography (RP-HPLC) . The chromatograms show (A) Pen&rem americana, 10 corpora cardiaca; (B) P. fufiginosa, 28 corpora cardiaca; (C)P. australasiae, 19 corpora cardiaca; @) P. brunnea, 20 corpora cardiaca; (E) Blatta orientalis, 12 corpora cardiaca. The analyses were performed on a Nucleosil C-18 column which was eluted with a linear gradient of 0.11% trifhroroacetic acid (solvent A) and 0.1% tritkoroacetic acid in 60% acetonitrile (solvent B). The gradient ran from 43 to 53% B within 20 mm at a flow rate of 1 ml/mm and started immediately with injection. The elution was monitored at 210 nm. Fractions of absorbance peaks (labeled with arabic numbers) were collected, dried in vacua, dissolved in 100 pl of double distilled water, and an aliquot was used for bioassay in American cockroaches (at least tive insects assayed). The increase in blood carbohydrate levels is given as means 2 SD in the histogram.

292

GERD GdDE

i Retention

Ib time

Retention

3

20

10 Retention

time

time

(mid

E R a .02L-

1

0

20

10

0

2.0 (mid

0

Imid

IO Retention

time

20 lminl

.OL8-E

.nw0

20

10 Retention

time

(mid

0

20

10 Retention

time

Iminl

FIG. 3. Separation of extracts from corpora cardiaca of different Blaberidae and Blattellidae species on RP-HPLC. The chromatograms show (A) Nuuphoeta cinema, 10 corpora cardiaca; (B) Blaberus trapezoideus, 8 corpora cardiaca; (C) B. discoid&, 10 corpora cardiaca; (D) Leucophaeu muderue, 10 corpora cardiaca; (E) Gromphudorhimzportentosa, 8 corpora cardiaca; (F) Diploptera punctutu, 10 corpora cardiaca; (G) Supellu longipulpu, 26 cmpora cardiaca; (H) Bluttelia germanica, 75 corpora cardiaca. For farther details, see legend to Fig. 2.

tained the biological activity. However, the extract of corpora cardiaca from B. trapezoideus (8 glands; Fig. 3B), B. discoidalis (10 glands; Fig. 3C), and S. iongipalpa (26

glands, Fig. 3G) had in addition to the most active peak (retention time: 8.9 min, Iabeled 2) another distinct absorbance peak (labeled 3, retention time 15 min) which

HYPERTREHALOSAEMIC

10 Retention

PEPTIDES

20 time

0

(mini

293

IN COCKROACHES

Retention

10 time

20 (mini

FIG. 34ontinued

caused some hypertrehalosaemia (exception: S. longipalpa). As yet no clear decision can be made whether this activity is due to peptidic material. Separation of the corpora cardiaca extract from L. maderae (10 glands; Fig. 3D) revealed two distinct absorbance peaks (labeled 2 and 3) in addition to the most prominent one at 8.9 min (labeled 1) which had biological activity. However, neither of these additional peaks were active in eliciting significant increases in blood carbohydrates in Periplunetu, but were not tested conspecifically. A totally different HPLC profile was obtained when an extract of 13 corpora cardiaca from the Polyphagidae P. aegyptiaca was analyzed on the C-18 column (Fig. 4). Two prominent absorbance peaks were obtained, not with retention times of 8.9 min (representative for HTH) or 10.9 and 18 min (representative for M I and M II), but with 14.1 and 15.4 min (labeled 4 and 5). Material of both peaks gave good responses in cockroaches, but the peak 4 material appeared to be more active. Having established that all of the species of Blattidae investigated contained two hypertrehalosaemic compounds that had apparently identical retention times to the hypertrehalosaemic hormones M I and M II from the corpora cardiaca of the American cockroach, an experiment was conducted to confirm this chromatographic behavior.

For this, extracts of corpora cardiaca from P. brunnea, P. australasiae, P. fuliginosa, and B. orient&s were run separately on

HPLC and material equivalent to about 0.03 absorbance units full scale (aufs) with a retention time of 10.9 min (designated peak I) and equivalent to 0.01 aufs with a retention time of 18 min (designated peak II), respectively, were collected. The peak I material from the four species was combined and “spiked” with 100 pmol of synthetic M I, whereas the combined peak II material from the four species was spiked with 100 pmol of synthetic M II: HPLC chromatography of these two batches is depicted in Fig. 5B and 5C. The chromatograms clearly show distinct single large absorbance peaks at 10 min (Fig. 5B) typical for M I and at 16 min (Fig. 5C) characteristic for M II. The same method was used for the hypertrehalosaemic compound of the species of Blaberidae/Blattellidae which all contained a peak with bioactivity eluting at around 9 min as the HTH of N. cinerea. Material equivalent to about 0.02 to 0.03 aufs was collected from separate HPLC runs of corpora cardiaca from B. discoidalis, B. trapezoideus, L. maderae, G. portentosa, B. germanica, D. punctata, and S. fongipalpa, combined and spiked with 100

pmol of synthetic HTH. The HPLC chromatogram of this mixture is shown in Fig.

294

GEFtD GjiDE

.CC I, 0 Retention

10 time

20 IminI

FIG. 4. Separation of an extract from 13 corpora cardiaca of Polyphaga aegyptiaca on RP-HPLC. For further details, see legend to Fig. 2.

5A: there is a single large absorbance peak at 8.8 min characteristic for HTH. To characterize the hypertrehalosaemic material from the corpora cardiaca of P. aegypriaca further, the peak 4 material (see Fig. 4), now designated PO I, was spiked with about 70 pmol of synthetic adipokinetic hormone from Munduca sextu and synthetic hypertrehalosaemic factor II from Curuusius morosus (AKH-M and HTF II) and rechromatographed. As shown in Fig. 6A, the POI material eluted with HTF II in the way that the peak was broadened at the trailing edge (Fig. 6A vs 6B). The peak 5 material (see Fig. 4) of P. uegyptiucu, now designated PO II, was spiked with about 70 pmol of synthetic adipokinetic hormone II from L. migrutoriu and S. greguriu (AKH II-L and AKH II-S) and rechromatographed. Compared to the elution pattern of AKH II-L and AKH II-S alone (Fig. 7B), it is obvious that the POII material coeluted exactly with AKH II-L (Fig. 7A). 3. Amino Acid Composition of Hypertrehulosaemic Peptides

Pooled peak I and peak II material obtained from the four different species of

Blattidae (see above) and pooled hypertrehalosaemic material from the seven Blaberidae/Blattellidae species (see above) were used for amino acid analyses. Representative data after hydrolysis in HCl are given in Table 3. The analyses indicated the following compositions: Blattidae, peak I material, Asx~,,,Glx(,,, Seq,,, F’y,,, V&,9 and Phe(,,; Blattidae, peak II material, A=(,), Glx(,,, Thq,,, Prql), Leu(,), and Phe(,,; Blaberidae/Blattellidae material, A=(I), G&I), Ser(l), G~Y(zI,Th), I’pt), V+, and Phql). Furthermore, amino acid analyses were made from the hypertrehalosaemic material from the species G. portentosu (peak 2, Fig. 3E), B. germanica (peak 1, Fig. 3H), and L. maderue (peak 1, Fig. 3D). The following composition data were revealed for all analyses (Table 4): Ser(,,, GUY,,,, Thq,,, Pq,, Val(,,, and Phq,,; unfortunately, Asx and Glx, although present in each analysis, were not very well separated and, thus, the amount present could not be calculated. Although Trp was not determined by this method, its presence in all peptides analyzed was deduced from spectrophotometric data. Thus, when an aliquot of each purified peptide was chromatographed in the same HPLC system as before but monitored with a fluorometer (excitation: 276 nm, emission: 350 nm; Shimadzu fluorescence spectromonitor RF530), two distinct peaks (retention times: 10.9 and 18min) were seen with the Blattidae extract and one peak (retention time: 8.9 min) with the extract from Blaberidael Blattelidae (results not shown). Fluorescence at 276 nm indicates the presence of either Tyr or Trp but Tyr, which is not destroyed during acid hydrolysis, was not detected by amino acid analysis. Therefore, the presence of a Trp residue in each of the three peptides can be inferred by elimination. Thus, corpora cardiaca of Blattidae contain two octapeptides and members of the Blaberidae/Blattellidae contain one decapeptide with hypertrehalosaemic activity.

HYPERTREHALOSAEMIC

PEPTIDES

295

IN COCKROACHES

4 HT

53

\

-

0

10

0 Retention

10 tme

0

8

16

hml

FIG. 5. HPLC chromatograms of (A) material (equivalent to about 0.02 to 0.03 absorbance units full scale) with hypertrehalosaemic activity and retention time of about 9 mitt from the eight Blaberidae/ Blattellidae species combined and “spiked” with 100 pmol of synthetic hypertrehalosaemic hormone from Nauphoeto cinerea (HTH); (B) material (equivalent to about 0.03 absorbance units full scale) with hypertrehalosaemic activity and retention time of about 11 min from the five Blattidae species combined and spiked with 100 pmol of synthetic hypertrehalosemic hormone I from Periplanera americana (M I); (C) material (equivalent to about 0.01 absorbance units full scale) with hypertrehalosaemic activity and retention time of about 18 min from the five Blattidae species combined and spiked with 100 pmol of synthetic hypertrehalosaemic hormone II from P. americana (M II). Conditions were as given in the legend to Fig. 2.

DISCUSSION

The present study clearly shows that all investigated species of the Blattaria contain substances in their corpora cardiaca that cause hypertrehalosaemia in American cockroaches. Further investigations using the powerful separation technique of RPHPLC demonstrated unambiguously that certain species possess only one major chemical entity with biological activity, whereas others had two compounds elicting carbohydrate elevation. In most cases these compounds were unequivocally identified as peptides. Moreover, by combining the HPLC fractions of the two biologically active compounds of each member of the investigated

Blattidae, it was shown that they not only had the same retention time under the employed HPLC conditions as the well-known hypertrehalosaemic hormones M I and M II of the American cockroach, but that their amino acid composition data were also identical to those of M I and M II. Although no sequence data are known, it seems safe to conclude that the hypertrehalosaemic compounds of the investigated Blattidae are most likely identical (see Fig. 8). Using the same techniques as discussed above, the presence of one major hypertrehalosaemic peptide could be demonstrated in the corpora cardiaca of the members of the Blaberidae and Blattellidae analyzed that were indistinguishable by retention time and amino acid composition

296

GERD GADE

a

0

8

16

.02- g

.Ol.

so

0

i Retention

Our knowledge of the hypertrehalosaemic compounds from the only member of the family Polyphagidae investigated is still rudimentary. It is obvious from the HPLC data that the two fractions with hypertrehalosaemic activity have retention times quite different from the other cockroach hypertrehalosaemic neuropeptides M I, M II, and HTH. Furthermore, the HPLC data suggest that one of the factors from P. aegyptiaca, designated POI, elutes close to the stick insect decapeptide HTF II, whereas the second factor, designated PO II, had an identical retention time to the octapeptide AKH II from the corpora cardiaca of L. migrutoriu. Unfortunately, the available material from P. aegyptiaca was too little to attempt any amino acid analysis. We are now starting to rear this species

16 time (mid

FIG. 6. HPLC chromatograms of (A) the first peak with hypertrehalosaemic activity from the corpora cardiaca of Pofyphaga aegyptiaca (PO I; see also Fig. 4) plus about 70 pmol of synthetic AKH from Manduca sexta (AKH-M) and 70 pmol of the hypertrehalosemic factor II from the stick insect Carausius morosus (HTF II); (B) 70 pmol each of AKH-M and HTF II. Conditions were as given in the legend to Fig. 2.

data from the previously characterized decapeptide HTH. In this case, composition data were available from combined peptides as well as from the individual material from two Blaberidae (G. portentosa and L. ma&rue) and one Blattellidae (Ii. germanica) species. In addition, the hypertrehalosaemic peak material after HPLC from L. maderae was analyzed by fast atom bombardment tandem mass spectrometry and was shown to be identical to HTH (G. GMe and K. L. Rinehart, unpublished observations). Thus, it seemssafe to conclude that the hypertrehalosaemic compound of all of the studied species of the Blaberidae and Blattellidae are most likely identical and have the same primary sequence as HTH (see Fig. 8).

E c 0

0

a

16

AKH II-S,

0

Retention

8 time (min)

16

FIG. 7. HPLC chromatograms of (A) the second peak with hypertrehalosaemic activity from the corpora cardiaca of Poiyphuga aegypriacu (PO II; see also Fig. 4) plus about 70 pmol of synthetic AKH II from Schistocerca gregaria (AKH II-S) and from Locusm migraroria (AKH II-L); (B) 70 pmol each of AKH II-S and AKH II-L. Conditions were as given in the legend to Fig. 2.

HYPERTREHALOSAEMIC

PEPTIDES TABLE

AMINO

ACID

Asx GlX Ser GUY Thr

Molar ratio” 1.61 1.07 1.10 -b 0.99 0.91

pro

Val Leu Phe

3

COMPOSITION OF HYPERTREHALOSAEMIC PEPTIDES FROM PEELED MNEFUAL OF BLATTIDAE AND BLABERIDAEEILAITELLIDAE DETERMINED AFTER HYDROLYSIS IN HCl

Blattidae, peak I Amino acid residue

297

IN COCKROACHES

Blattidae, peak II

Number of residues present

Molar ratio”

2

0.87

1 1

1.25 1.90 0.99 1.00 1.00

1 1

1.00

1

BlaberidaelSlattellidae

Number of residues present

Molar ratio

1 1

0.91 1.21 1.10 1.85

2 1

0.98 0.96 0.99

1 1

1.00

peak

Number of residues present

n Values are given as molar ratios to Phe = 1 b Molar ratio smaller than 0.1

in order to produce sufftcient material over the next years to elucidate the structures of the two compounds PO I and II. The strikingly structural resemblance of many insect peptides with adipokinetic and hypertrehalosaemic activity has led to the assumption that they belong to a single peptide family (see for example G&de, 1988a).

Such a concept, then, means that these peptides may have originated from the same ancestral molecule. Although the variations in amino acid residues seen in the different members of this family sequenced can be explained by single point mutations (see G&de et al., 1988), the limited number of structurally fully known

TABLE AMINO

ACID

COMPOSITION

OF HYPERTREHALOSAEMIC L. maderae DETERMINED

G. portentosa, peak 2 (Fig. 3E) Amino acid residue

Molar ratio”

Asx Glx Ser GUY Thr

0.98

pro

1.00

Val Leu Phe

0.98 -b 1.00

Number of residues present

G. portentosa, IN HCI

B. germanica, peak 1 (Fig. 3H)

Molar ratio

c

c

c

c

1.28 2.13

4

PEPTIDES FROM AFTER HYDROLYSIS

Number of residues present

B. germanica, AND

L. maderae, peak 1 (Fig. 3D)

Molar ratio

Number of residues present

c c

1

1.40

1

1.27

1

2

2.40 1.02 0.98

2 1

2.11 0.99

2

1 1

1.16 0.96 1.00

1 1 1 1

1.04 1.00

1

1 1 1 1

o,b See Table 3. c Asx and Glx were found in the analysis, but they were not very well separated and therefore were not calculated.

298

GERD GiiDE

N.cinerea L.modeme G.portentosa D.punctata

m

Pamericana F? fuliginosa ;;z;:siae

m

B.orientatis

HTH

: pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-Thr-NH2

MI

: pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2

M II

: pGlu-Leu-

Thr-Phe-Thr-Pro-Asn-Trp-NH2

FIG. 8. The putative hypertrehalosaemic

peptides of the investigated cockroach species.

peptides does not yet allow to make firm conclusions about the evolutionary relationships of these peptides. However, the present study, analyzing the hypertrehalosaemic peptides of one monophyletic group (the cockroaches), makes it worthwhile and interesting to discuss the results from a phylogenetic point of view, although it should be noted that the data base is small. McKittrick (1964) distinguishes two divergent phylogenetic lines of the Blattaria: The Blattoidea and the Blaberoidea. She acknowledges that the most primitive families, the Cryptocercidae and Polyphagidae, have certain similarities to the termites and to each other, but also have a high degree of special features. Therefore, she places the Crytocercidae together with the Bfattidae into the group of Blattoidea, and the Polyphagidae together with the Blattellidae and Blaberidae into the group of Blaberoidea. Our studies on hypertrehalosaemic neuropeptides are supportive of these relationships in the way that all of the Blattidae apparently contain the peptides M I and M II in their corpora

cardiaca and, on the other hand, the Blattellidae and Blaberidae are linked by the existence of the peptide HTH. However, the only primitive group studied by us, the Polyphagidae, do not have HTH (as should be the case according to McKittrick’s dendrogram). Hennig (1%9) argues on morphological terms of the specific designs of the hind wings that the Polyphagidae should be separated from the other Blattaria. Such a view is supported by our data. For future research, it will be most rewarding to unravel the structures of the P. aegyptiaca compounds and, if present, those of Isoptera (termites), which are thought to be the most related insects to those primitive cockroaches. ACKNOWLEDGMENTS The author gratefully acknowledges the generous gift of cockroaches by various people (see Materials and Methods), the expert technical assistance of Miss Beate Schumacher, the valuable comments and corrections of Professor Dr. G. J. Goldsworthy (Birkbeck College, London), and financial support by a grant from the Deutsche Forschungsgemeinschaft (Ga 241/ 6-2). The author is a recipent of a Heisenberg Fellow-

HYPERTREHALOSAEMIC

PEPTIDES

ship awarded by the Deutsche Forschungsgemeinschaft (Ga 241/T-2).

REFERENCES G&de, G. (1980). Further characteristics of adipokinetic and hyperglycaemic factor(s) of stick insects. J. Insecr Physiol. 26, 351-360. G&de, G. (1984). Adipokinetic and hyperglycaemic factors of different insect species: Separation with high-performance liquid chromatography. J. Insect Physiol. 30, 729-736. Gade, G. (198Sa). Amino acid composition of cockroach hypertrehalosaemic hormones. 2. Naturforsch. 4Oc, 424. G&de, G. (1985b). Hypertrehalosaemic hormones and myoactive factors from cockroach corpus cardiscum are very likely identical. Naturwissenschuften 72, 95-96. G&de, G. (198%). Isolation of the hypertrehalosaemic factors I and II from the corpus cardiacum of the Indian stick insect, Carausius morosus, by reversed-phase high-performance liquid chromatography, and amino-acid composition of factor II. Biol. Chem. Hoppe-Seyler 366, 195-199. GBde, G. (1987). Characterization and amino acid composition of a hypertrehalosaernic neuropeptide from the corpora cardiaca of the cockroach, Nauphoeta cinerea. Z. Naturforsch. 42c, 225230.

G&de, G. (1988a). New structures of insect neuropeptides. In “Endocrinological Frontiers in Physiological Insect Ecology” (F. Sehnal, A. Zabza, and D. L. Denlinger, Eds.), pp. 635-650, University of Wroclaw Press, Wroclaw. GBde, G. (1988b). On release and action of the hypertrehalosaemic hormone from the cockroach Nauphoeta cinerea. Z. Naturforsch. 43c, 108-l 16. Gade, G., Goldworthy, G. J., Kegel, G., and Keller, R. (1984). Single step purification of locust adipokinetic hormones I and II by reversed-phase high-performance liquid chromatography, and amino-acid composition of the hormone II. Hoppe Seyler’s Z. Physiol. Chem. 365, 393-398. Gade, G., Goldsworthy, G. J., Schaffer, M. H., Cook, J. C., and Rinehart, K. L., Jr. (1986). Sequence analyses of adipokinetic hormones II from corpora cardiaca of Schistocerca nitans, Schistocerca gregaria, and Locusta migratoria by fast atom bombardment mass spectrometry. Biochem. Biophys. Res. Commun. 134, 723-730. G&de, G., Hilbich, C., Beyreuther, K., and Rinehart, K. L. (1988). Sequence analyses of two neuropeptides of the AKI-BRPCH-family from the lubber

IN COCKROACHES

grasshopper, Romalea microptera.

299 Peptides, 9,

681488.

Giide, G., and Rinehart, K. L., Jr. (1986). Amino acid sequence of a hypertrehalosaemic neuropeptide from the corpus cardiacum of the cockroach, Nauphoeta cinerea. Biochem. Biophys. Res. Commun. 141, 774-781. Glde, G., and Scheid, M. (1986). A comparative study on the isolation of adipokinetic and hypertrehalosaemic factors from insect corpora cardiaca. Physiol. Entomol. 11, 145-157. Goldsworthy, G. J., and G&de, G. (1983). The chemistry of hypertrehalosaemic factors. In “Endocrinology of Insects” (H. Laufer and R. G. H. Downer, Eds.), pp. 109-119. A. R. Liss, New York. Greenberg, M. J., and Price, D. A. (1983). Invertebrate neuropeptides: Native and naturalized. Annu. Rev. Physiol. 43, 271-288. Hayes, T. K., Keeley, L. L., and Knight, D. W. (1986). Insect hypertrehalosaemic hormone: Isolation and primary structure from Blaberus discoidalis cockroaches. Biochem. Biophys. Res. Commun. 140, 674-678. Hennig, W. (1969). “Die Stammesgeschichte der Insekten.” Verlag W. Kramer, Frankfurt am Main. McKittrick, F. A. (1964). Evolutionary studies of cockroaches. Mem. Cornell Univ. Agric. Exper. Sr. 389, Ithaca, NY. Orchard, I. (1987). Adipokinetic hormones: An up date. J. Insect Physiol. 33, 451-463. Scarborough, R. M., Jamieson, G. C., Kalish, F., Kramer, S. J., McEnroe, G. A., Miller, C. A., and Schooley, D. A. (1984). Isolation and primary structures of two peptides with cardioacceleratory and hyperglycaemic activity from the corpora cardiaca of Periplaneta americana. Proc. Natl. Acad. Sci. USA 81, 5575-5579. Schaffer, M. H. (1986). Functional and evolutionary relationships among the RPCH-AKH family of peptides. Amer. Zool. 26, 997-1005. Siegert, K. J., and Mordue, W. (1986). Elucidation of the primary structures of the cockroach hyperglycaemic hormones I and II using enzymatic techniques and gas-phase sequencing. Physiol. Entomol. 11, 205-211. Siegert, K., Morgan, P., and Mordue, W. (1985). Primary structures of locust adipokinetic hormones II. Biol. Chem. Hoppe-Seyler 366, 723-727. Spik, G., and Montreuil, J. (1964). Deux causes d’erreur dans les dosages colorimetriques des oses neutres totaux. Bull. Sot. Chim. Biol. 46, 739-749.

300

GERD GdDE

Stone, J. V., Mot&e, W., Batley, K. E., and Morris, H. R., (1976). Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilisation during flight. Nature (London) 263, 207-2 11. Wheeler, C. H., G&de, G., and Goldsworthy, G. J. (1988). Humoral functions of insect neuropeptides. In “Neurohormones in Invertebrates”

@I. C. Thomdyke and G. J. Goldsworthy, Eds.), pp. 141-157. Cambridge Univ. Press, Cambridge. Witten, J. L., !&chaffer, M. H., O’Shea, M., Cook, J. C., Hemling, M. E., and Rinehart, K. L., Jr. (1984). Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry. Biochem. Biophys. Res. Commun. 124, 35&358.