Identification and quantification of juvenile hormones from different developmental stages of the cockroach Nauphoeta cinerea

Life Scieaoes Vol . 16, pp" 1271-1284 Priated in the II.3 .A.

Pergamon Press

IDENTIFICATION AND QUANTIFICATION OF JUVENILE HORMONES FROM DIFFERENT DEVELOPMENTAL STAGES OF THE COCTCROACH NAUPHOETA CINEREA Beatrice Lanzrein *, Masashi Hashimoto**, Vinka Parmakovich, Roji Nakanishi Department of Chemistry, Columbia University, New York New York 10027, USA and Rita Wilhelm and Martin Lflacher Division of Animal Physiology, University of Bern Engehaldeatrasse 6, CH-3012 Bern, Switzerland

(Received in final form March 17, 1975)

Summary By the combined use of high-pressure liquid chromatography, Ga1T is bioassay and qas chromatography/ chemical ionization/mass spectrometry we were able to isolate and identify the three known natural juvenile hormones (JHs) from haemolymph extracts of larval and adult females of the cockroach N__a~~u h~ce~ta cinerea . This is the first demonstration imu~taneous occurrence of the three JHs in the same insect and the first time JH I and II have been identified in a hemimetabolous insect . Quantitative investigations show that the composition of the three JHs is different at different developmental stages . The haemolymph of larvae contains a high percentage of JH I and II, whereas the haemolymph of adult females in the oocyte maturation stage contains mostly JH III . This suggests more juvenilizing functions for JH I and II and more gonadotropic functions for JH III . The endocrine function of the corpora allata (c .a .), i .e . the secretion of juvenile hormone (JH), has been established for

several insect orders for a long time (review, see 1), and it has

*

Leave of absence from University of Bern, Present address : Zoecon Research Laboratory, 975 California Avenue Palo Alto, California 94304, USA Leave of absence from Fujisawa Pharmaceutical Co ., Osaka

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been shown that transplants of adult c .a . into allatectomized (2 ,3) and intact larvae

(4 ,5) can duplicate the action of larval

c .a . At present, three JHs have been identified from insects (6-8), and all three induce juvenilizinq effects when applied to

last instar larvae or pupae and gonadotropic effects when applied

to allatectomized adult females of several insect species . On the basis of these findings it has been assumed that the JHa secreted by the c .a . of larval and adult insects are identical .

However, recent experiments in our laboratory (B . Lanzrein, unpublished) using the cockroach Nauphoeta cinerea have indicated that JH extracts from larval or adult haemolymph elicit quantita

tively different ratios of activity when compared on a juvenilizinq (Galleria wax test) versus a gonadotropic (Nauphoeta vitellogenin teat) bioassay . This suggested that not only may the different JHs have distinct physiological roles, but that the titres of these JHS may vary during the life history of Nauphoet a .

in this report we describe a combination of techniques which

permitted the identification and quantification of the three JHs in haemolymph of Nauphoeta at different developmental stages .

COOMe

Rt=Rs=Et

IJH II

Rt=Et, Rs=Me

tJH III

Rt=Rs=Me

IJH IIII

Material and Methods Animals :

Nauphoeta cinerea was raised at 26°C and 50-60$ r .h . on dog flakes and water . Under these conditions the second last larval instar lasts about 20 days and the first oocyte maturation cycle lasts 12-13 days . For the investigations presented here female second last instar larvae various ages were used .

(7 days old) and adult females of

Extraction and preliminary purification :

In order to reduce

clotting and to increase the yield of extractable haemolymph, the insects were injected 100 ul of Insect-Ringer solution 3 hrs

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The haemolymph was collected directly into

diethyl ether/ethanol 5 :1 V/v by cutting away one leg in the case of larvae and by piercing the extruded uterus in the case of adult females . We estimate that about 1/5 of the total haemolymph content can be extracted by this procedure . The ether/ethanol layer was removed and pooled with 3 ether extracts of the water layer . Subsequently the pooled organic phases were dried over Na 2304 and evaporated to dryness under N2 on a rotatory evaporator . The crude lipid extract was purified on preparative thin layer chromatography (TLC) plates of 0 .5mm thickness, freshly prepared

from Si11ca Gel H (Merck) . Extracts from 30 insects were combined and applied to a 20cm x 20cm plate and developed with 68 ethyl acetate benzene . The zone of Rf 0 .3-0 .5 containing 85-908 of the biological activity was scraped off and eluted with ether .

Chromatography and spectroscopy ; High-pressure liquid chromatography (LC) separations were performed on a Waters

Associates instrument equipped with a Model 6000 pump, a variable wavelength UV detector (Schceffel Instrument Corp . SF-770/GM-770) adjusted to 215nm (maximum absorbance of JH) and two 30cm x 4mm

(i .d .) columns packed with uPorasil (Waters Associates) . Separations were carried out using 5$ ether hexane with a flow rate of 2 .5 ml/min .

Chemical ionization (CI) mass spectra were obtained on a

Finnigan 3300 mass spectrometer equipped with a Finnigan 9500 gas chramatograph (GC) with a 5' x 2mm i .d . glass column packed with 3$ OV-1 on 60/80 Gas Chrom Q . The samples were introduced into the mass spectrometer via the GC inlet using methane or isobutane

ae carrier and reactant gas . The mass spectra were recorded both on an oscillographic recorder and on magnetic tape with subsequent computer processing . The mass spectrometry conditions were the following : source pressure, 1 .0 Torr . ; source temperature, 150°C;

electron energy, 120 eV ; filament current, 600 mA ; electron multiplier, 2 .6 RV . Solvents : Ether (Mallinckrodt) and n-hexane (Mallinckrodt, spectrograde) were redistilled before use . Bioassay : JH activity was determined by means of the Galleria wax test (9) . In every assay 25-56 insects were treated for each concentration . Under the conditions employed, a 508 positive

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response was obtained with 0 .8-1 .6 pg of JH I (t,t,c, Eco-Control) and JH II (t,t,c, Zoecon), and 60-80 pg per pupa of JH III (t,t, Zoecon) .

Results Identification of JH I, II - and III The three JHs can be readily separated under the LC conditions described above . Prior to injection of the insect extracts, the retention times of the JHs were checked by injection of about 200 ng of JH I, II and III . The OD2 15 absorbance trace of the reference compounds is shown in Fig . 1 above the trace of the TLC

S

nm

W

mm ~i

,_

Wm,WUVU~s

-o

FIG . 1 LC separation of a mixture of JHs (ca . 200 ng each) as reference (top) and of TLC purified haemolymph extract from 300 adult females (bottom) . Time scales are identical for both traces . purified haemolymph extract from 300 adult females (7-8 day old) . Some fast eluting peaks visible in both traces are due to solvent and septum material and were used as markers to check the reproducibility of the separation conditions . Fractions indicated by the double-headed arrow in Fig . 1 were collected and assayed for

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JH activity by the Galleria wax test . Less than 58 of the total activity could always be observed in the forerun and in the fraction after the JHs but this material was not further investigated . Fractions 2 and 3 with a retention time similar to JH I showed some activity but in an amount far less than that suggested by the size of the visible peak . Fractions 5 and 6 having the retention time of JH II were also JH active ; however, the highest activity was observed in fraction 12 containing the peak with a retention time corresponding to that of JH III . The amount of biological activity corresponded to 900 ng of JH III and this is correlated well with the peak size on the LC trace . A portion of this fraction 12 was then further analyzed by GC mass spectrometry .

.w rouT

FIG . 2 Mass spectra (GC/CI, methane) of 50 ng of JH III and of 208 of LC fraction 12 (see Fig . 1) . Recording of peak intensities are doubled above m/e 237 . Methane was first used as both carrier and reactant gas and the spectrum obtained with 50 ng of JH III is shown in Fig . 2 (top) . JH III is characterized by nine peaks in this method of GC/CI (methane gas)/ms : a_ m/e 307 (M+C 3 H5 ) + , b_ 295 (M+C2H5 ) + , c_ 267 (MH+), d_ 249 (MH +-H2 0), e_ 235 (MH +-CH30H), _f 217 (MH +-H2O-CH30H), 189 (MH+-H2O-CH30H-CO), h_ 153 and _i 135 (see 10 for discussion on peaks h and i), One-fifth of LC fraction 12 was analyzed under

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267 235

FIG . 2a Mass spectrum (GC/CI, isobutane) of 50 nq of JH III . the same conditions and the GC total ion current trace revealed two peaks, one identified as dibutylphthalate, a plasticizes, and the other one as JH III (Fig . 2, bottom) . The above experiments conclusively prove that the most abundant JH in 7-8 day old adult females is JH III .

Since the Galleria wax test results indicated the presence of only very small amounts of JH I and II in haemolymph extracts of adult females, optimal GC/MS conditions had to be worked out in

order to allow the identification of the expected ng quantities of JH I and II in the extracts . Isobutane turned out to be more suitable than methane for use as a carrier and reactant gas

because it gave less fragmentation and showed a strong MH+ peak with 4 ng of JH I (Fig . 4, top ; also compare Figs . 2 and 2a for 50 ng of JH III) .

The JH active LC fractions in the regions corresponding to JH I (2 and 3) and II (5 and 6) from three different LC separa-

tions (each from 300-400 adult females, 7-8 day old) were pooled and repurified by LC . However, these fractions still contained

some impurities, which in the case of JH II could not be completely

removed by GC for mesa spectral analysis .

For this reason, iden-

tification of JH II from haemolymph extracts rests on selected mass chromatograms (for an explanation of this technique see 11)

of the three main mass peaks of JH II : 1 m/e 281 (MH+), _ k 263 (MH+- H2 0), _1 249 (MH +-CH30H), see Fig . 3 . GC fractions 85 to 100 correspond to the retention time of JH II under the GC conditions used, and the simultaneous appearance of the three characteristic mass . peaks in these spectra indicates the occurrence of JH II in the haemolymph of 7-8 day old adult females .

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Juvenile Hormonen o! Nauphoeta

~-i-r--

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sr-r-

FIG . 3 Mass chromatograms (GC/CI, isobutane) of mass 281 (j), 263 (k), and 249 (1), of 20 ng of JH II (left column) and o~ the LC fraction with the JH II retention time from haemolymph of adult females (right column) . In the case of the pooled LC fractions 5 and 6 with a retention time corresponding to JH I, the contaminants were separable by GC and thus a clear mass spectrum could be obtained (Fig . 4) . JH I is characterised by three peaks : m_ m/e 295 (MH+), n_ 277

(MH+-H20), o_ 263 (MH+-CH30H) . As shown in Fiq . 4, the haemolymph JH I LC fraction gave a spectrum closely resembling that of synthetic JH I . Herewith the occurrence of JH I together with JH II and III in the haemolymph of adult females is demonstrated .

All three JHs could also be isolated and identified from the haemolymph of second last instar larvae (7 day old) through usage of the same techniques . Quantification of JH I, II and III The findings presented above permitted the quantification of the three JHs in haemolymph extracts from Nauphoeta at different developmental stages . The TLC purified extracts were separated by LC as described before and the quantitative determinations are

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vvemrta~ oF mra °~

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re~aExr~ ff rom

FIG . 4 Mass spectra (GC/CI, isobutane) of 4 ng of JH I (top) and of the LC fraction with the JH 2 retention time from haemolymph of adult females (bottom) . based on extensive Galleria wax tests : every LC fraction was assayed in 2-3 concentrations in order to allow appropriate calculation of the 50B positive response . In the case of JH III, the peak area served as an additional means to estimate the amount of

JH .

Since preliminary investigations suggested a different composition of JHs in larval and adult females, haemolymph extracts from female second last instar larvae with supposedly active c .a . (12) were submitted to a quantitative determination of the JHs . Fig . 5 shows the OD 215 absorbance trace of extract from 349 second

last instar larvae together with the JH references . The peak with the retention time of JH 222 represents approximately 90 ng, a quantity which is confirmed by bioassay results . A comparison with Fig . 1 where ca . 900 ng of JH III was detected from 300 adult females (7-8 day old) demonstrates that the larval haemolymph JH 222 content is about one-tenth that of 7-8 day old adult females . In the case of JH 2 and I2, quantitative investigations indicate that about 20 times more JH I and about 3 times more JH II is present in the larvae as compared to adult females . Thus,

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c ô

waien+ .ew

mowmnucom

FIG . 5 LC separation of TLC purified extract from haemolymph of 349 second last instar larvae (7 day old) (bottom) and of the three JHs as a reference (top) . in 7-8 day old adult females, the JH I and II content is only ca . 18 of the total JH content ; in contrast it is ca . 458 in second last instar larvae .

The calculated values for each JH (expressed

in JH content per insect) are summarized in Table 1, The values obtained for larvae and for 7-8 day old adult females are both

based on three different LC separations and subsequent quantification .

The difference observed in the JH composition between the second last instar larvae and the adult females in the oocyte maturation stage suggests that the three JHs might stimulate different responses . Therefore, the titres of the three JHs of adult females at different stages in the first oocyte maturation

cycle were determined . Several JH dependent processes are involved in oocyte maturation : vitellogenin synthesis in the fat body (13) and probably the haemocytes, release of vitellogenin from the fat body (13), uptake and incorporation of Vitellogenin

1280

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Juvenile Hormones of Nauphoeta TABLE 1

Titres of JH I, II and III per Insect* larvae 2nd last instar 3 day old 7 day old

JH I JH II JH III Total

0 .7-0 .8 ng 0 .4-0 .5 ng 1 .3-1 .5 ng 2 .4-2 .8 ng

0 .04 ng 0 .005 ng 0 .9 ng 0 .95 ng

adult females 5 day old

7-8 day old

0 .04 nq

0 .04 nq

5 .85 ng

16 .17 ng

0 .01 ng 5 .8 ng

0 .13 ng 16 ng

12 day old 0

0 .005 ng 2,2 ng 2 .2 ng

*In order to estimate the JH content per insect, the values obtained per bled insect were multiplied by 5, because only about 1/5 of the haemolymph of an insect was extractable by the method applied . by the oocytes (14), stimulation of the left colleterial glands (Zanolari, 1974, unpublished) . In 3 day old adult females vitellogenin is synthesized and released into the haemolymph, but is not yet incorporated into the oocytes, and the left colleterial glands are still inactive . For the determination of the JH composition at this stage, freshly moulted adult insects were collected and 303 females were bled 3

The results of JH analyses given in Table 1 show the occurrence of JH I (ca . 58), JH II (ca . 0 .58) and JH III (ca . 95$) . days later .

At the age of five days, the c .a . volume is still increasing and vitellogenin synthesis and release into the haemolymph continue, but incorporation of vitellogenin into the oocytes has

begun . The left colleterial glands seem only slightly stimulated . For the JH determination 303 freshly moulted adult females were collected and kept together with males in order to allow copula-

tion which takes place on days 2-4 and activates the c .a . Five days after collection the females were bled and the JH levels estimated . The data in Table 1 indicate a marked increase in JH III, a slight increase in JH II and no increase in JH I between days 3 and 5 .

JH III now represents 998 of the total JH content .

In 7-8 day old adult females the c .a . volume maximum, oocyte growth occurs at its maximum rate colleterial glands are stimulated . The values in obvious increase in JH II and III after day 5 and tent of about 16 ng .

reaches its and the left Table 1 show an a total JH con-

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Ovulation takes place on day 12-13, and by this time the volume of the c .a . has already decreased . The JH levels were determined in extracts from 303 females bled immediately prior to ovulation . The data in Table 1 demonstrate that the titre of JH I has fallen to zero and that the JH II and JH III titres have markedly decreased since day 8 .

The quantitative analyses of the three JHs presented in Table 1 show that JH I and II contribute substantially to the

total JH content only in the larval stage, whereas JH III is by far the most abundant JH in the adult female during the oocyte

maturation cycle.

Discussion By the combined usage of TLC, high-pressure LC equipped with a variable wavelength UV detector adjusted to 215 nm, Galleria wax test, and GC/CI mass spectrometry, we have been able to isolate known JHs from haemolymph of larvae (second last larval instar) and of adult females in the oocyte maturation stage . This is the first report of the simultaneous occurrence of JH I, II and III in the same insect and of JH I and II in a hemi-

and identify the three

metabolous insect, suggesting a limited diversification of JHs during insect evolution . The identification of the minute amounts of JH I and II in adult females was made possible only by the accumulation and careful purification of large amounts of haemo-

lymph extracts and by analysis of the isolated JHs by GC/CI mass spectrometry with subsequent computer processing . Under optimal

conditions using isobutane as carrier and reactant gas, as little as 2 nq of JH could easily be detected and identified . The fact

that Trautmann e_t a_1 . who independently found JH III in adult Nauphceta (15) did not detect JH I and II is probably due to the lower sensitivity of their method (16) . Since the purpose of our investigation was identification of the Nauphceta JHa as well as determination of their relative concentrations at different developmental stages, we were forced to extract JH directly from the haemolymph of whole insects at selected stages, rather than obtaining the JH from c .a . cultures

(8) .

The quantitative analyses of JH I, II and III in Nauphoeta at different developmental stages demonstrate that not only the titre of total JH but also the relative concentrations of respective JHa

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The fluctuation of total JH titre in the adult female is well correlated with volume changes of the c .a, (17) and shows a correlation between the c,a . volume and the c .a . activity . The difference in the JH composition between change during development .

larval and adult females suggests that the three JHs might stimulate different responses at different stages of the life cycle .

JH III is by far the most abundant JH in adult females during

oocyte maturation . This suggests important gonadotropic functions for JH III . Its identification in reproductively active Tenebrio molitor , Leptinotarsa decemlineata , Blatta orientalis , Leucophaea maderae , Schistocerca gregaria (15) as well as in cultures of c .a, from reproducing Schistocerca vaga (18), Schistocerca gregaria (19), and Periplaneta americana (20) supports this view .

JH II is more abundant in larvae than in adult females and this could indicate a juvenilizing function for JH II in Nauphoeta . In addition, bioassay results indicate that JH II is more active than JH III in inducing larval/adult intermediates in

Nauphoeta (B . Lanzrein and M . Lflscher, in preparation) . Topical JH assays in Galleria (21), Tenebrio (21), and Rhodnius (22) also demonstrate a higher morphogenetic effectiveness for JH II than for JH III . However, since the JH II titre, although low, changes

during the oocyte maturation cycle and is highest when the oocyte growth is at its maximum, it seems likely that JH II also has a function in oocyte maturation,

JH I is about 20 times more abundant in second last instar larvae than in adult females . This suggests juvenilizing functions for JH I in Nauphceta and agrees with bioassay results in Nauphoeta (B . Lanzrein and M . L$scher, in preparation),

(22), Galleria (21), and Tenebrio (21) where JH I shows a higher morphogenetic effectiveness than JH III . It is not known whether the continuous presence of very small amounts of JH I in Rhodnius

vitellogenin synthesizing adult females is of any physiological significance . The concept that different JHs may have qualitatively different modes of action has precedence in earlier studies on the termite Ralotermes (23) . We now provide qualitative and

quantitative data to support this hypothesis for the first time .

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Juvenile Hormonaa of Nauphoeta

From the growing body of evidence concerning the broad distribution of JH I, II and/or III among insects, it is not improper to suggest that the differentiated functions for the three JHs as

indicated in Nauphoeta may be shared by other species . This in turn raises a whole series of new questions concerning regulatory mechanisms for the biosynthesis of the three hormones, their binding to carrier proteins, specific degradative enzymes, and differences in the nature of the receptors for each . Acknowledgements :

Thanks are due to Ruth Marti and Vroni Aebi

for the assistance in preparing the extracts, to Dr . Philippa

Solomon and Dr . Kenneth Judy for critically reading the manuscript, and to Dr . David A . Schooley for discussions . JH II and JH III were kindly supplied by Zoecon Corp ., Palo Alto .

Financial support

by Swiss National Science Foundation to B . Lenzrein (postdoctoral fellowship), and to M. Ldscher (Grant No . 3 .633,71) is gratefully acknowledged . AI 10187 .

The studies at Columbia were supported by NIH grant References

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