Optimization of insect juvenile hormone bioassays on Tribolium confusum (DuV.) (Coleoptera: Tenebrionidae) by application of central composite rotatable designs

0022-474X/82/010021-05$03 00,O CopyrIght 0 1982 Pergamon Press Ltd

OPTIMIZATION OF INSECT JUVENILE HORMONE BIOASSAYS ON TRIBOLIUM CONFUSUM (DuV.) (COLEOPTERA: TENEBRIONIDAE) BY APPLICATION OF CENTRAL COMPOSITE ROTATABLE DESIGNS X. BELL@ Institute de Quimica Bio-Or&mica (C.S.I.C.). Jorge Girona Salgado S/N. Barcelona-34. Spain (Received

injinalform

6 November

1981)

Abstract --The simultaneous influence of age and temperatureon the morphogeneticactivity of farnesyl methyl ether (FME) on pupae of Tribolium confusum (DuV.) using the mathematical method of central composite rotatable designs of Box and HUNTER is studied. The post-applica-

tion temperature has little influence on the activity. Conversely, the specimens age represents a decisive factor which can alter drastically the results; maximum activity being attained in the youngest pupae. Surprisingly, with the same dose of FME the whole range of morphogenetic activity can be elicited within the first 36 hr of pupal life. These results show the convenience of a strict systematization of the morphogenetic bioassays, specially as far as the definition of the specimen age is concerned. in order to obtain reproducible results.

INTRODUCTION

SINCEthe systematization of the quantitative assays for the evaluation of morphogenetic activity of the insect juvenile hormones or their bioanalogues (KARLSON and NACHTIGALL, 1961; BOWERS and THOMPSON,1963; WIGGLESWORTH,1963; STAAL, 1972), increasing attention has been paid to several experimental factors that can influence this activity (BENSKIN and VINSON, 1973; REDDY and KRISHNAKUMARAN,1973; KRISHNAKUMARAN, 1974; SCH~~NEVELD and WIEBENGA, 1974). However, to date incomplete standardization of these assays has often precluded the comparison of results from different laboratories. In this communication, the optimum conditions for the morphogenetic activity of farnesyl methyl ether (FME) on pupae of Tribolium conjiisum (DuV.) are established, as a function of two variables, specimen age and post-application temperature, using the method of central composite rotatable designs (Box and HUNTER, 1957). FUNDAMENTALS

OF THE MATHEMATICAL

METHOD

Although there is a fair amount of literature on experimental designs (DAVIES, 1956; Box and HUNTER, 1957; PENG, 1966; BARELLA,1977), a survey of the mathematical basis of the present study will be given here. In an experimental design, if the functional relation y = 4(x1,x2) between response and variables is a smooth one, it can be approximated sufficiently closely by a polynomial. Moreover, assume an experimental design to study the response surface and also assume that the perfectly known experimental point u is designated by the row vector (x,~, x,J. The set of these vectors form the design matrix D:

where the number of experiments (N) must be equal to or greater than the number of coefficients to be fitted. By conveniently enlarging the matrix D a matrix X is obtained for the polynomial to be fitted. 21

X. BELLS

22

TABLE 1. MATRIX ROTATABLE DESIGN

D

OF

FOR

SECOND

CENTRAL COMPOSITE TWO

Experiment

1 Factorial

VARIABLES

x,

x2

-1

2 3

-1

1

-1

1

+1

-1

4

1

6 I

- 1.414 1.414 0

8

0

5

Centre

AND

DEGREE

0 0 - 1.414 1.414

9 10

0 0

0 0

11 12 13

0 0 0

0 0 0

It can be shown (DAVIES,1956) that the vector 8 corresponding the polynomial fitted by the method of least squares is given by:

to the coefficients of

where 2 is the transposed matrix of X, ($ X)- ’ is the inverse matrix of (% X), and Y is the response vector. For two variables, the corresponding polynomial will take the following form: _G,=

60+ i 6iXiu+ i 6iXf”+ i=l

i=l

1 iijXi"Xj" i>j

which represents a quadratic surface. The analysis of this surface can be simplified by reducing it to its canonical form. As shown in Table 1, the central composite rotatable design of Box and HUNTER(1957) involves 13 experimental combinations distributed as follows: (i) four combinations corresponding to the factorial 2’; (ii) four combinations in star; and (iii) five replicates at the centre for estimating the experimental error and determining the precision of y at and near the centre. In this way, the results of these 13 experiments allow the plotting of the whole surface of response within the experimental range chosen.

BIOLOGICAL TESTS range of O-40 hr for the age of pupae of T. temperature (x2) was fixed. After codification, the values of both variables were: In the present case an experimental

confusum (DuV.) (x1) and 20-35°C for the post-application

-1.414 - l.ooo o.ooo 1.000 1.414 Pupae were time, thus the the first level, 10 pg of FME

x1 (hr) 0 5.9 20 34.1 40

x2 (“C) 20 22.2 27.5 32.8 35

collected every 2 hr and aged at rearing temperature (30°C) for the desired true age of the pupae is + 1 hr (REDDYand KRISHNAKUMARAN, 1973). For pupae were collected every 30min and used immediately. A solution of in 0.5 ~1 of acetone was administered topically on the posterior sternites of

Hormone bioassayson Tribolium TABLE 2. MORPH~GENETIC ACTIVITIESOF FME

23

confusum (DuV.) ON Tribolium confusum (DuV.)

Experimental combinations

Age (X,, hr)

Temperature (X,. “C)

1st rep.

2nd rep.

3rd rep.

Average

1 2 3 4 5 6 I 8 9 10 11 12 13

5.9 34.1 5.9 34.1 0 40 20 20 20 20 20 20 20

22.2 22.2 32.8 32.8 21.5 21.5 20 35 21.5 21.5 21.5 21.5 21.5

4.40 0.27 4.20 0.60 5.00 0.10 2.08 3.16 3.00 2.43 2.43 2.50 3.10

4.15 0.25 4.41 0.84 4.92 0.21 1.66 3.20 3.50 2.56 2.56 2.66 2.90

3.94 0.37 3.57 0.84 4.66 0.08 1.66 3.00 2.15 2.71 2.69 2.83 2.90

4.16 0.29 4.06 0.76 4.86 0.13 1.80 3.12 3.08 2.56 2.56 2.66 2.96

each specimen. After treatment, the pupae were maintained at the required temperature in a culture oven, within a temperature range of &-1°C. Quantitative evaluation of morphogenetic activity was carried out according to previously established activity tables (REDDY and KRISHNAKUMARAN, 1972; BELLES et al., 1980). Three separate replicates of each bioassay were performed, with 10 specimens each, and compared with a standard set treated only with acetone. The activities observed for each experimental combination are summarized in Table 2. RESULTS

AND DISCUSSION

The average activities shown in Table 2 gave the coefficients of the equation of response surface: Y = 2.764 - 1.732~~ + 0.279~~ - 0.174~: - 0.192x; + 0.142x,x2 and the variance analysis is shown in Table 3. Figure 1 illustrates the graph corresponding to such surface. The contour for activity is shown in this graph plotted in front of age (xi) in abcissae and post-application temperature (x2) in ordinates. The post-application temperature has less influence on the morphogenetic activity than that previously reported by SCHOONEVELD and WIEBENGA (1974) in a study with JH-I and 6,7-epoxy-3,7-dimethyl-1-(3,4-(methylenedioxy)phenoxy)-2-nonene, on larvae of Adoxophyes orana (Lep. Tortricidae). Conversely, the specimens age represents a decisive factor which can alter drastically the results observed; maximum activity being attained in the youngest pupae. This result agrees with those reported for other Tenebrionidae (CRITCHLEY and CHAMPION, 1971; EDWARDS, 1976). REDDY and KRISHNAKUMARAN(1973) when studying the influence of age of Tenebrio molitor L. on the activity of four juvenile hormone analogues, found optimum results with pupae 12-36 hr old, the activity decreasing rapidly after that time. On the other hand, they point out slight differences depending on the compound tested. TABLE 3. VARIANCE ANALYSIS

Source of variation 1st Degree 2nd Degree Lack of fit Error * Significant

terms terms

Degrees of freedom

Mean square

F

2

12.31577

212.04838

3 3 4

0.16525 0.12125 0.05808

2.8452 2.08764

at the l”,, level.

Significance * NS NS

24

X. BELLBS

25

IO

20 xI ’

30

40

hr

FIG. 1. Contours for activity plotted as a function of age (x,) and post-application (x2).

temperature

With our test insect, the optimum activity of FME is found with pupae CL1 hr old, the values decreasing progressively with age. Most striking is the fact that, as can be seen, with the same dose of the product the whole range of morphogenetic activity can be elicited, within the first 36 hr of pupal life (Fig. 1). These results show the convenience of a strict systematization of the morphogenetic bioassays, especially as far as the definition of the specimen age is concerned, in order to obtain reproducible results. Work is in progress with other juvenile hormone analogues to check the dependency of activity on age for different structural types of compounds, as well as optimization of bioassays for three variables (pupal age, post-application temperature and product dose). We feel that these optimization studies by apili-cation of maihematical designs could bk useful for pest control planning with juvenile hormone bioanalogues. Acknowledgements-The author thanks Dr A. BARELLAand Mr J. P. VIGO of the Instituto de Tecnologia Quimica y Textil. and Drs F. CAMPSand J. COLL of the Instituto de Quimica Bio-OrgAnica for helpful discussions.

REFERENCES BARELLA,A. (1977) Principios de diseiio de experiencias y optimizacidn de procesos industriales. Manuales TCcnicos A.I.T.A. 15, Barcelona. BELL&S, X., CAMPS,F., COLL,J., MESSEGUER, A., SEBA,M. E. and ROCA,A. (1980) Biomimeticos de la Hormona Juvenil de insectos. Sintesis y actividad biolbgica. EFCE Pub/. Ser. 1980, 12 (Proc. Congr. Nat. Quim. 3), pp. 467-472.

BENSKIN.J. and VINSON,S. B. (1973) Factors affecting juvenile hormone analog activity in the tobacco budworm. J. econ. Ent. 66, 15-20. BOWERS,W. S. and THOMPSON, M. J. (1963) Juvenile hormone activity. Effects of isoprenoid and straight-chain alcohols on insects. Science 142, 1469-1470. Box, G. E. P. and HUNTER,J. S. (1957) Multifactor experimental designs. Ann. math. Statist. 28, 195-241. CRITCHLEY,B. R. and CHAMPION,D. G. (1971) Effects of a synthetic iuvenile hormone and a iuvenile hormone analogue methyl farnesoate dihydrochloride on pupal development of the yellow mealworm Tenebrio molitor L. Bull. ent. Res. 61, 293-297. DAVIES,0. L. (1956) Designs and analysis of industrial experiments. Oliver & Boyd, London. EDWARDS,J. P. (1976) Age-related susceptibility of Tribolium castaneum (Herbst.) to synthetic Cls juvenile hormone. J. stored Prod. Res. 12, 71-76. KARLSON,P. and NACHTIGALL,M. (1961) Ein biologischer test zur quantitativen bestimmung der Juvenilhormon-aktivitat von insektenextrakten. J. Insect Phqlsiol. 7, 21&215. KRISHNAKUMARAN, A. (1974) Factors influencing morphogenetic activity of juvenoids. Marathwada Unio. J. Sci. 12, 81-86. PENG, K. C. (1966) The Design and Analysis of Scientific Experiments. Addison Wesley, Reading. REDDY.G. and KRISHNAKUMARAN, A. (1972) Synergistic effect of ecdysterone on morphogenetic activity of juvenile hormone analogues. Life Sci. 11, 781-792.

Hormone bioassays on Tribolium

conjiisum (DuV.)

25

and KRISHNAKUMARAN,A. (1973) Changes in the morphogenetic response of Tenebrio molitor pupae to juvenile hormone in relation to age. J. Insect Physiol. 19, 773-780. SCHOONEVELD,H. and WIEBENGA, J. (1974) Temperature-dependent juvenile hormone effects on pupation of Adoxophpes orana. J. econ. Ent. 67, 711-715. STAAL, G. B. (1972) Biological activity and bioassay of juvenile hormone analogues. In Insect Juoenile Hormones: Chemistrv and Action (Edited bv MENN.J. J. and BEROZA, M.). pp. 69-94. Academic Press, New York. WIGGLE&•RTH. \;. B. (1963) Tde juvenilk hormone effect of farnesol and some related compounds: quantitative experiments. J. Insect Physiol. 9, 105-l 19. REDDY, G.