SEX DETERMINATION IN BEES. IV. GENETIC CONTROL OF JUVENILE HORMONE PRODUCTION IN MELIPONA QUADRZFASCZATA () .

WARWICK ESTEVAM KERR2, WKIO AKAHIRAS AND CONCEICAO A. CAMARGO

Department of Genetics, Faculdade de Medicina, Universidade de Srlo Pau1~14100- Ribeirrlo Preto, S.P.,Brazil Manuscript received June 27, 1974 Revised copy received July 17, 1975

ABSTRACT Cell number and volume of corpora a2lata was determined for 8 phases of development, the first prepupal stage to adults 30 days old, in the social Apidae Melipona guadrifasciata. In the second prepupal stage a strong correlation was found between cell number and body weight (r = 0.651**), and cell number and corpora datavolume in prepupal stage (r = 0.535*), which indicates that juvenile hormone has a definite role in caste determination in Melipona. The distribution of the volume of corpus allatum suggest a 3:1 segregation between bees with high volume of corpora allata against low and medium volume. This implies that genes za and zb code for an enzyme that directly participates in juvenile hormone production. It was also concluded that the number of cells in the second prepupal stage is more important than the weight of the prepupa for caste determination. A scheme summarizing the genic control of sex and caste determination in Melipona bees in the Frepupal phase is given.

REVIOUS publications of this series on the sex determination in bees have demonstrated that: ( 1) sex in bees is produced by an interaction between the genes determining the male and female characters, since aneuploids caused normal, intersex and hermaphrodite bumble bees (GAR~FALOand KERR1974) ; (2) genes determining male characters are slightly additive in Apis mellifera (CHAUD-NETTO1975); and (3) there are two sets of sex genes: one, whose action is in the beginning of embryo development acd the other in the beginning of pupal development in the prepupa (KERR1974a, 1975). In this second phase, the experiments of WIRTZ(1973) in Apis mellifera strongly suggest that juvenile hormone plays an important role in the determination of the female external morphological characters, derived from imaginal discs. The work described in this paper was carried aut to obtain information on the role of the corpora allata in the determination of castes in Melipona quadrifascita. In this two genes, xa and xb, with two alleles each, play a decisive fuflction in caste deter-

This paper has been supported by the State of SZo Paulo Research Foundation (FAPESP), the National Research Council (CNPq) and the Japanese Foundation of Sciences. Present address, Director, Instituto Nacional de Pesquisas da Amazonia, P.O. Box 478, 69000 Manaus, Amazonas, Brazil. a Present address: Kushiro Branch, Hokkaido University of Education, Kushiro, Hokkaido, Japan 085.

Genetics 81 : 749-756 December, 1975 750 W. E. KERR, Y. AKAHIRA AND C. A. CAMARGO mination (KERR1948, 1969; KERR, STORTand MONTENEGRO1966; KERRand NIELSEN1966) turning on or off the female-determining genes (KERR1974a, 1975).

MATERIALS AND METHODS

The prepupae, pupae and adults were collected from a hive of anthidioides Lep. originally coming from Pocos de Caldas, Minas Gerais, Brazil, but actually bred in our Department. Bees in the following stages of development were used: Prepupa-1: These are prepupae immediately after they finish spinning; the legs are developed to the median line; yellow or brown excrements still present in their intestines; (20 samples). Prepupa-2: These are near the pupal stage; the legs begin taking the final shape but in this phase are still folded; (20 samples). Pupa-3: The pupal stage was divided into four phases; pupa-3, one of these phases, have black ryes; external epithelium beginning chitinization; (5 queens and 20 workers were sampled). Recently emerged adults: Five queens and 20 workers were sampled. Bees 5 days old: Five virgin queens and 20 workers were sampled. Bees 15 days old: Five virgin queens and 20 workers. These workers were feeding the brood (KERRand SANTOSNETO 1956). Bees 20 days old: Five virgin queens. Bees 30 days old: Two virgin queens and 20 workers, The workers were beginning to be field bees, that is, beginning to be engaged in collecting pollen, nectar, mud ,resins, or water. Carnoy fixative was used. Bees were preserved in 70% alcohol. An aceto-carmine stain diluted to 50% was used. Bees were fixed in Carnoy for 24 hours and left in 70% alcohol. In order to carry out the

TABLE 1

Auerage of number of cells and volume of corpora allata, average weight of prepupae and pupae, and coefficients of correlation, in Melipona quadrifasciata

Corre- Corre- lation (r) lation (r) between .4verage Correlation (r) between volume of number of Average between number corpus cells per volume of Body number of of cells in allatum Phase of corpus corpu6 weight cells and c.a. and and weight development allatum allatum in mg volume of c.a. body weight of body N - Prepupa 1 80.85 41 2560.19 84.60 0.126 0.140 0.0861 20 Prepupa 2 78.90 304431.27 78.10 3.582** 0.651** 0.535* 20 Pupa 3-worker 72.20 182528.08 80.95 0.001 0.287 0.332 20 Pupa 3-queen 100.60 2135652.29 80.00 -0.276 -0.624 0.279 5 0 days-worker 83.33 178098.64 - 0.194 - - 20 0 days-queen 112.60 1406495.74 - 0 344 - - 5 5 days-worker 84.95 207957.10 - 0.730** - - 20 5 days-queen 208.30 2269779.44 - -0.378 - - 5 15 days-worker 71.25 178489.93 - 0.390 - - 20 15 days-queen 220.70 1193053.27 - 0.685 - - 5 20 days-queen 94.70 871236.53 - 0.652 - - 5 33 days-worker 73.13 171296.19 - 0.053 - - 20 30 dgys-queen 98.00 898525.48 - - - - 2 All adul t-queen 205.34 11 59085.25 - 0.578** - - 22

Values in italics are for queens. * Significant at the 5% level. ** Significant at 1 % level. SEX DETERMINATION IN BEES 75 1

- - - WEIGHT

-__ VOLUME __ NUMBER AV ERABE fOLUME

I I7 1800000 a W I04 I600000 m 5 3 z 91 1400000 w (3 Q 78 1200000 a W > a 65 1000000

I 52 800000 +- I (3 39 600000 $

W 26 400000 a: W > 13 200000 a

I 2 3 4 5 6 I 8

STAGES FIGURE1.-Number of cells of the corpora allata, average volume of both corpora alZata, weight of prepupa and pupa for queens and workers of Melipom qzcadrifasciata (Apidae) . The vertical lines denote the 95% confidence intervals. In order that one confidence interval would not be superimposed on another, the data for weight, cell number and corpora allata volume were drawn slightly to one side of their real positions.

dissection each bee was put in a petri dish with 70% alcohol and cotton. The head was opened on the dorsal side and the brain was removed, revealing the esophagus, the corpora cardiaca, the corpora allata, and the first ganglion. The esophagus with the corpora allata were left for 1 minute in an aceto-carmine solution diluted to 50% with acetic acid, and then transferred to 95% alcohol. Both the esophagus and the corpora allata were placed on a slide in a drop of glycerin. They were now separated with a thin needle; the esophagus removed, the drop containing the complete corpora a2kaia was covered by a small coverglass, and observed with an oil immersion lens. Camera lucida drawings were made of the cells and nuclei of both corpora allata, and the larger and smaller diameters of each corpus allatum were recorded. Volume was estimated assuming that the corpora allata approximate the shape of a prolate spheroid. The tables, calculations and graphs were carried out with a small computer-type Hewlett Packard 9810A; we thank MISSLIBEXTAD MARIA NALIfor the programming. 752 W. E. KERR, Y. AKAHIRA AND C. A. CAMARGO Queens and workers can be easily differentiated by the size of their heads (greater in workers), eyes (much greater in workers), hind legs (queens have no corbicula), and abdomen (greater in queens, and with different shape). Male pupae, when present, can be distinguished from the workers by the characteristics of the genitalia. Queens 5, 15, 20, and 30 days old were maintained alive in small cages 10 x 10 x 3 centimeters; each cage contained one queen and 5 workers (which were substituted by recently emerged ones every 7 days). Each cage received an aliquot of pollen, 50% sugar syrup and water daily.

RESULTS

The results are presented in Table 1 and Figure 1, which summarizes the data. Units are the direct readings of the micrometer. The full data are in 13 tables filed in the department of the senior author. Weight of imago is not recorded since it is not reliable; it varies according to the food in the stomach and intestines, or with the water it lost through evaporation. The vertical lines in each point of the graph indicate the 95% confidence limits.

ANALYSIS AND DISCUSSION One must bear in mind that since prepupae and pupae do not eat, their weights are a direct measure of what they have eaten in the larval life. Therefore, an unusual feature of these data is that there is no correlation between weight and number of cells in the corpora allata nor in Prepupae-1 (r= 0.14) (beginning of caste differentiation) nor in Pupae-3 (r = 0.29 for workers and r = -0.62 for queens) where castes are already differentiated. However, in the crucial phase for caste determination in Melipona, that is Prepupae-2, a strong positive corre- lation was found (r = 0.65**, significant at 1% level). In this same phase a correlation also exists between the number of cells and the volume of the corpora allata (r = 0.583**), and between this volume and prepupa weight (r = 0.535’) as well. Also, looking at Figure 1, there is no difference in Pupa-3 stage either between the pupae weight or the number cells of the corpora allda of queens and workers; however, significant differences exist between the vol- umes of the corpora allata and cell numbers. This indicates that the number of cells in the corpora allata is important just in the beginning of caste differentia- tion, and their function (measured by the corpora allata volume) is important from phase Prepupa-2 on. Figure 2 shows the distribution of the corpora allata volume for the first and second prepupal stages. Both phases indicate a 3:l segregation betweeri 15 or 16 bees with low or medium volume, and 4 or 5 with high volume. Although similar bimodal distributions do not exist for cell number and weight, there are nevertheless strong correlations between the volume of the corpora allata and the number of cells and its weight. This strongly suggests that an enzyme (provisionally called “juvenile hormone synthetase”) is the final product of the battery of genes that contains xa and xb. Therefore, the proposed scheme for sex and caste differentiation in Melipona bees in the pupal stage is the one of Figure 3. SEX DETERMINATION IN BEES 753

VOLUME OF THE CORPORA ALLATA,PREPUPA- I 14 : 6

VOLUME OF THE CORPORA ALLATA, PREPUPA- 2 12 : 8

FIGURE2.-Upper histogram: Volume of corpora aZZata for 20 Prepupa-1 bees. ~Lm histogram: volume of corpora allata of 20 Prepupa-2 bees.

FOO C I. NIJMEER OF CELLS IN THE CORPORA ALLATA I.I XP lZ.7- L=

PRECURSOR JH CL., E::- +JUVENILE HORMONE ___ BATTERY I SYNTHETASE - JUVENILEI H3RM3NE 6 8910

h THRESHOLD -RF PF ACTIVATOR RNA - FEMALENESS PRODUCT -RP BATTERY 3 CR- ,* MALENESS PRODUCT FIGURE3.-Scheme proposed for gene/environment interaction in the determination of sex (and caste) in Melipona quadrifasciata. See text for explanation. 754 W. E. KERR, Y. AKAHIRA AND C. A. CAMARGO The scheme of Figure 3 shows the following factors in accordance with the proposal of BRITTENand DAVIDSON(1969, 1971) ; previous work (KERR1975) has shown that this conjecture can be applied to the sex and caste determination in bees: a) Food: With the right amount of food many cells in the corpora allata are produced; with a small amount of food the corpora allata has a smaller number of cells. b) Number of cells in the corpora allata: below a certain threshold not enough juvenile hormone will be produced, the genes of female characters will not be turned on, and the bee will become a worker. In this respect it is interesting to note that KERR(1974a, 1975) showed that workers of Melipona resemble more the males than the females-they somehow are females inside and males on the outside. c) Buttery I: It contains the sex alleles xa and xb as Integrator genes (for literature see KERR 1969, 1975); we suggest that the double heterozygote (xd/xa2,xbl/xbP) is necessary to produce a complete activator RNA that acti- vates the receptor gene RXafaband the gene PJHswhich produces the “juvenile hormone enzyme.” This enzyme would act in a “juvenile hormone precursor” and juvenile hormone will be produced (in our case, measured through volume of corpora data).In case any of the two alleles of xa and/or xb are homozygous, a smaller amount of the enzyme would be produced. This is a modification of an earlier hypothesis (KERR1975), which indicated an “all or none’’ scheme. With a small amount of enzyme a small amount of juvenile hormone is produced, unable to trigger Battery 2. d) Battery 2: The sensor gene of battery 2 is triggered by a given amount of juvenile hormone. If it is in an amount superior to the threshold it puts PF into work. Thus, a scheme equal, or similar, to the one proposed by KERR(1974b) would be in action: the genes determining female characters are additive, or almost additive, and the genes determining male characters are non-additive or slightly additive. e) Battery 3 is the battery for the male genes, which, according to the findings we have up to this moment are, in practice, always derepressed.

CONCLUSION These results indicate that juvenile hormone, the main product of the corpora allata cells, has an important role in caste differentiation in bees. The queens of Melipona are the double heterozygote xa1/xa2;xbl/xbP while workers are homo- zygous for any of the xa anci/or xb alleles (KERR1969, 1974b). A model for gene action was suggested by KERR(1974a, 1975). The present paper decisively implicates juvenile hormone as an integral part of the gene control for caste determination in Melipona; also, the histogram indicates that an enzyme, provisionally called juvenile hormone synthetase, is the final product of the battery of genes containing xa and xb, which transforms a precursor into the juvenile hormone. In turn the juvenile hormone activates the sensor gene of the SEX DETERMINATION IN BEES 755 battery of genes, making the control of sex genes slightly more complicated than originally proposed by KERR (1974a, 1975). The new proposition is explained in Figure 3. It is possible that sex determination in Apis and also fallows the same scheme, without mutations in the gene xa and xb. As a by-product of this research, a strong correlations was found between cell number and volume of corpora allata (0.730**) in adult workers five days old, indicating juvenile hormone synthesis in this phase-that is, a phase five days earlier than that in which worker bees normally lay eggs. This same fact was found by BEIGand BALDISSERA(1974) using a quite different method. Also, if all adult queens are pooled together a correlation of r = 0.58** (P < 0.01) was found between corpora allata volume and cell number, indicating juvenile hormone synthesis in all or almost all cells.

Note: After this paper was sent for publication CAMPOS,VELTHUIS and VELTHUIS(1975) and CAMPOS(1975), in this laboratory, published that painting prepupae of Melipona quadrifasciuta with adequate amount of juvenile hormone analog makes 100% of the pupae become workers. This supports the scheme proposed in Figure 3.

LITERATURE CITED

BEIG,D. and S. BALDISSERA,1974 Controle end6crino nos meliponineos. I. Atividades dos corpora ullata e desenvolvimento dos ovdrios de Meliponn quudrifasciaia Lep. (Hym. Apidae). Cibncia e Cultura 26(12): 1155-1160. BRITTEN,R. J. and E. H. DAVIDSON,1969 Gene regulation for higher cells: a theory. Science 165: 349-357. -, 1971 Repetitive and non-repetitive DNA sequences and a specula- tion on the origins of evolutionary novelty. Quart. Rev. Biol. 46:111-133. CAMPOS,L. A. DE OLIVEIRA,1975 DeterminaGo de castas no gbnero Meliponn (, Apidae): Papel do Horm6nio Juvenil. Master of Science Thesis, 4.1. pp., Fac. Medicina, University of SBo Paulo, RibeirGo Prbto, Brazil.

CAMPOS,L. A. DE OLIVEIRA,F. M. VELTHUIS-KLUPPWand H. H. W. VELTHUIS,1975 Sex determination in Bees. VII. Juvenile Hormone and caste determination in a . Natunvissenschaften 62 : 198-99. CHAUD-NETTO,J., 1975 Sex determination in bees. 11. Additivity of maleness genes in Apis mellifera. Genetics 79: 213-217. GAR~FALO,C. and WARWICKE. KFXR, 1975 Sex-determination in bees. I. Balance between femaleness and maleness genes in Bombus atrutus Franklin (Hymenoptera, Apidae). Genetica 45: 203-209. KERR,WARWICK E., 1948 Estudos sobre o g&neroMelipona. An. Esc. Sup. Agr. Luiz de Queiroz (Piracicaba, Brasil) 5: 181-276. -, 1969 Some aspects of the evolution of social bees (Apidae). Evolutionary Biol. 3 : 119-175. -, 1974a Sex determination in bees. 111. Caste determination and genic control in Melipona. Insectes Sociaux 21: 357-368. -, 1974b Geschlechts-und Kastendetermination bei stachellosen Bienen. pp. 336-349. In: Socialpolymorphismus bei Insekten. Edited by G. H. SCHMIDT.Wissenschaftliche Verlags- gesellschaft, MBH, Stuttgart. __ , 1975 Evolution of the population structure in bees. Proc. XIIIth Int. Cong. Genet. (Berkeley). Genetics 795: 73-84. KERR,WARWICK E. and G. R. SANTOSNETO, 1956 ContribuiQIopara o conhecimento da bionomia dos Meliponini. 5. DivisBo de trabalho entre as operdrias de Melipona quadrifnsciata anthidioides Lep. Insectes Sociaux 3: 423-430. 756 W. E. KERR, Y. AKAHIRA AND C. A. CAMARGO GRR,WARWICK E. and Ross NIELSEN,1966 Evidences that genetically determined Melipona queens can become workers. Genetics 54: 859-866. ICERR, WARW~CKE., A. C. STORTand M. J. MONTENEGRO,1966 Importtcia de alguns fatores ambientais na determinasiio das castas do genero Melipona. An. Acad. Bras Cihncias 38: 149-168. WIRTZ,P., 1973 Differentiation in the honeybee larva. A histological, electron-microscopical and physiological study of caste induction in Apis mellifera melligera L. VIII, 155p. Depart- ment Entomology, Agric. Univ. Wageningen, Holland. Corresponding editor: R. W. ALLARD