SEX DETERMINATION IN BEES. 11. ADDITIVITY OF MALENESS GENES IN APIS MELLIFERAI JOSk CHAUD-NETT0 Departamento de Gendtica da Faculdade de Medicina de Ribeircio Preto 14100-RibeirHo Preto, Scio Paulo, B r a d Manuscript received February 4, 1974 Revised copy received July 18, 1974 ABSTRACT
Twenty-two randomly taken morphological characters were used i n order to estimate the Mahalanobis generalized distance between diploid males, diploid workers, haploid males and triploid workers. It was found that adult diploid males are metamales and triploid females are slightly masculinized. These facts indicate that the maleness genes are slightly additive.
HE first hypothesis to explain sex determination in Hymenoptera, within the Tframe of genic balance, states that there are femaleness genes with additive effect (genes F ) and maleness genes without additive effect (genes M ) , the balance being such that in haploids the effect of M>F (= male) and in diploids 2 F>M (= female) (CUNHAand KERR1957). KERR (1969; 1974), after studying larvae of diploid drones (Apis mellifera) ,modified slightly this initial proposition considering that the maleness genes could be either non-additive or slightly additive; that is, sex in honeybees would be produced by an interaction between major overdominant femaleness genes (z alleles), minor femaleness genes with additive effect (f alleles), and maleness genes without or with small additive effect ( m alleles). The objective of this paper is to elucidate the details of the balance between the sex genes in Apis mellifera, using an accurate statistical method. MATERIALS A N D METHODS
Four groups of honeybees (Apis mellifera adansonii) were used in this experiment: twentynine haploid males (16 chromosomes), twenty-nine diploid workers (32 chromosomes), sixteen diploid males ( 3 2 chromosomes) and twenty-one triploid workers (48 chromosomes). The honeybees from the first thre- groups were sons of the same queen (129) and were cousins of the triploid workers. Twenty-two external morphological characters were used in order to estimate the generalized Mahalanobis distances between the four groups of honeybees (see Table 1 ) . The measurements were made with a micrometric occular i n a Zeiss stereoscope. The techniques of measure(1970). ment followed the one described by GON~ALVES This paper is part of the M.Sci. thesis of the author and was financed by FL4PESP (Processes: 70/73$ and 71/9(38) and CNPq (Process: 14-138). Genetics 79: 213-117 February, 1975.
214
J O S k CHAUD-NETT0
TABLE 1
Means and siandard errors (in mm) of imnty-two randomly taken morphological characters of Apis mellifera adansonii
Anterior wing width Hind wing length Radial cell length Total thorax length Head length Anterior wing length Head width Hind wing width Radial cell width M.L. femur length M.L. tibia length M.L. tibia width M.L. femur width H.L. femur length H.L. tibia length H.L. tibia width H.L. tarsus length Flagellum length Scape length Pedicel length H.L. basitarsus width H.L. femur width
Haploid males
Diploid males
Diploid workers
Triploid workers
3,89 f 0,12 8,Ol f 0,W 4,40f 0,lO 6,43 t 0,27 3,95 f 0,14 11,76 f 423 4,46 t 0,m 3,06 f 0,m 467 f 0,04 2,88 f 0,08 2,34 t 0,07 0,34 & 0,03 0,4Q f 403 3,29 f 0,09 4,08 k 0,09 1,17 f 0,06 4,13 f 0,15 3,98 f 0,11 1,08 f 0,M 0,25 k 0,M 1,W f 0 , s 0,63 0,05
4,07 f 412 8,2l t 0,15
2,96 f 0,06 5,98 t 0,lO 3,017 f 0,06 4,35 t 0,14 3,48 f 0,12 8,61 f 0,12 3,77 t 0,IO 1,71 t 0,M 0,45 t 0,03 2,12 f 0,07 1,91 0,05 0,47 t 0,W 0,55 f 0,06 2,44 -C 0,cw 2,96 t 0,12 1,lO f 0,05 3,56 f 0,14 2,601 +- 0,05 1,22 f 0,w 0,22 f 0,03 1,W f 0,05 0,58 +- 0,M
3,12 t 0,06 6,26 I 0,19 3,09 I 0,07 4,46 I 0,08 3,57 0,m 9,% t 0,17 3,81 f 0,W 1,81 k 0,07 O,@ 401 2,16 t 0,05 1,96 t 0,M 0,46 t 0,08 0,54 t 0,04 2,48 t 0,05 3,02 +- 0,08 1,07 t 0,04 3,67 t 0,ll 2,52 +- 0,M 1,19 t 0,02 0 , s t 0,03 1,03 f 0,M 0,57 f 0,05
*
Note: M.L. = middle leg; H.L.
4,sf 0,w 6,32 -C 0,27 3,97 f 0,18 12,13 f 0,34 4,43 f 0,12 3,28 f 0,12 0,65 f 0,05 2,77 f 0,11 2,st 0,lO 0,39 k 406 0,52 f 0,M 3,19 f 0,12 3,79 f 0,15 1,12 f 0,07 3,93 f 0,w 3,85 t 0,12 1,oo f 0,M 428 f 0,M 1,26 t 0,08 0,62 +- 0,oS
*
*
*
= hind leg.
The four groups of honeybees were compared in pairs, the respective values of D2 being with some modifications introduced by calculated using the method described by MAHALANOBIS, RAO(1952). This method can be summarized as follows:
If X ijk is the kma observation of the imo character in individuals of the j”” group, for i = 1.2, ..., 22 (number of characters that were measured) i = 1,2,..., 4 (number of groups) k = 1,2,..., until 29 (the number of honeybees in each group varied from 16 to 29), then the sum of squares matrix and “within” sum of products is defined as:
(i,h = l,2, ... ,22) Where,
xii
xi
is the average of the ima variable for the j“J group and is the average of the ima variable for all the groups. This matrix cmsists of elements representing sum of squares and sum of prJducts of the deviations from all observations with relation to the respective averages in each group. Considering now the dispersion matrix, obtained from the sum of squares matrix and “within” sum of products, dividing each element by its freedom degrees number. It is a matrix of variance and co-variance. Let a i h be the dispersion matrix and aih its inverse, d i = the vector of difference between the averages of two groups in the ima variable (i = 1,2, ..., 22),
SEX D E T E R M I N A T I O N IN BEES
Then the Mahalanobis generalized distance 0 2 between two groups can be defined as: D? = Z Z aih di dhr for i,h = 1,2,..., 22. i
h
215 (1)
The differences between two compared groups are examined simultaneously in all the characters that can be correlated. This indicates that the information given by one character possibly is not independent from that furnished by the others. Th. numerical value of the possible major separation between any two groups is called generalized distance between them and measures, in a scale independent of that used for the measurements, the clearness of disjunction between them (PISANI 1969). Therefore, the value 0 2 between two groups is a pure number with properties of common distance and measures the extension with which they differ in shape and size (BARRACLOUGH and BLACKITH1962; BLACKITH and ALBRECHT 1959). The program for a n IBM 1130 was made by MR. LUISANTONIO F. BEZERRAunder the in this Dept. of Genetics. supervision of DR.F. A. M. DUARTE RESULTS A N D DISCUSSION
Figure 1 is an approximated graphic representation of the generalized distances of Mahalano3bis between the four groups of honeybees. The diagram was constructed in such a way that the basal line is the distance between haploid males and diploid workers, which constitute the normal sexual individuals in an Apis mellifera colony. The sexual constitution of the honeybees from each group are represented as follows. in which m, and mz mean the maleness genes and X , , X , and f the femaleness genes: a ) Haploid drones are m, mz > X I f. b) Diploid drones are 2m, f 2m, > X , X , ff. c) Diploid workers are X , X , 4-ff > 2ml 4-2m2. d) Triploid workers are X,X,X, f fff > 3m, 3m,. The genes X and f have high additivity since the effect of X,X2 ff is much greater than the effect of X , f, while the genes m have small additivity (the distance between 2n males and 2n workers is only 8% greater than the distance between n males and 2n workers). An examination of Figure 1 shows that maleness genes ( m ) are slightly additive in the diploid drones, since these males have characteristics of metamales,
+
+
+
+
64,78
dn FIGURE 1.-Configuration showing the generalized Mahalanobis distances, considering twenty-two external morphological characters of haploid and diploid drones (left) and diploid and triploid workers (right), Scale is arbitiary; value used is D, instead of D2 in order to make the drawings easier to represent the data.
216
J O S d CHAUD-NETT0
being about 8% more masculine than the haploid males. The diploid drones differ from diploid workers because they possess the inactive X , X , pair (the X alleles work only when they appear in heterozygosis). The greater maleness of the diploid drones results from the mJm, ;m2/m2genes; since the diploid drones are very similar to the haploid drones it can be concluded that 2ml 2m? have a maleness determinant effect greater than the femaleness determinant effect produced by f f , that is, this can only happen if 2m, 2m, are additive. Triploid workers are closer both to haploid drones (D= 62, 96) and diploid drones (D= 66, 2 8 ) , than the diploid workers ( D = 64, 78 and D = 68, 48, respectively). This fact demonstrates a slight masculinity of the triploid females when compared with the diploid females. An explanation could be formulated if what happens here is the same that happens in triploid chickens (ABDELHAMEED 1972). I n these birds there are some indications that the three chromosome sets are genetically active; the triploidy increases the content of both RNA and DNA per erythrocyte. If this same thing happens with the triploid workers of Apis mellifera, then an increase of masculinity would not occur because the bee is X I / X 1 / X z ;that is, the X , / X , pair would function as in diploid bees (66% of the RNA being produced by X I and the rest by X , ) . The differences between triploid workers and diploid workers were very minute, indicating that the functioning of the femaleness genes is the same in both types. The slight masculinity demonstrated by triploid workers must therefore be from the small additivity of the maleness genes. This confirms the results obtained f o r the diploid drones. According to the hypothesis of CUNHA and KERR (1957), the triploid bees should be meta-females because the X alleles of each bee (X,/X,/X2, for example) would have a feminine effect of 2 F since X , / X , are not additive. The effect of the other femaleness genes (f/f/f) could be equal to, f o r instance. 3 X 0,4 = 1.2 F (if f is a minor gene it should have a value smaller than 1 ) . The maleness genes (ml,m,, ma,etc.) would have an effect equal to M because they have no additivity. So the triploid workers, in any real case considered, would be slightly more female than the normal females. According to KERR (1974), in the triploid workers, the effect of the major femaleness genes (X,/X,/X,) would be less than 2 F (1,8, for instance) because X , / X , are not additive at all (since they are complementary genes) and X J X , are not totally additive. The effect of the minor femaleness genes (f/f/f) would be less than 1,2 F (for instance, 1,O) because they are not totally additive. So we would have 3,2 F-3d, where d is a small absence of additivity of f genes for each haploid set. The maleness genes would have a factor b of lack of additivity and then we would have 3M-3b ( b would be greater than d ) . Then the sexual 3M-3b. This scheme (or balance of the triploid workers should be 3,2 F-3d any other similar) implies that triploid workers may be slightly masculinized when compared with diploid workers. The results obtained in this experiment demonstrate that the maleness genes of Apis mellifera have a small additivity. They also strengthen the hypothesis that X alleles are femaleness genes.
+
+
+
SEX DETERMINATION I N BEES
21 7
LITERATURE CITED
ARDEL-HAMEED, F., 1972 Hemoglobin concentration in normal and intersexes triploid chickens: genetic inactivation or canalization? Science 178 (4063) :864-865. BARRACLOUGH, R. and R. E. BLACKITH,1962 Morphometric relationships in the genus Ditylenchus. Nematologica 8 : 51-58.
R. E. and F. 0. ALBRECHT, 1959 Morphometric differences between the Eye-stripe BLACKITH, polymorphs of the red locust. Scien. Joui. Royal Coll. Scien. 27: 13-27. CUNHA,A. B. and WARWICK E. KERR,1957 A genetical theory to explain sex-determination by arrhenotokous parthenogenesis. Forma et Functio l ( 4 ) : 33-36.
L. S., 1970 A n & e genktica do cruzamento entre Apis mlilfera ligustica e Apis GONCALVES, mellifera adansonii. Tese de doutoramento, 142 pgs. KERR,W. E., 1969 Genbtica e melhoramento de abelhas. pp. 263-297. XIV. Capitulo do livm “Melhoramento e Genktica.” Edit. Univ. S. Paulo e Edic6es Melhoramentos. -, 1974 Advances in cytology and genetics of bees. Annual Review of Entomology, 19: 253-268.
PISANI. J. F., 1969 Anilise estatistica multidimensional em biologia. Ci&nciae Cultura 21 (3): 6 19-61 3. RAO,C. R., 1952 Advanced Statistical Methods in Biometric Research. John Wiley & Sons, Inc., London. Corresponding editor: D. SUZUKI
Twenty-two randomly taken morphological characters were used i n order to estimate the Mahalanobis generalized distance between diploid males, diploid workers, haploid males and triploid workers. It was found that adult diploid males are metamales and triploid females are slightly masculinized. These facts indicate that the maleness genes are slightly additive.
HE first hypothesis to explain sex determination in Hymenoptera, within the Tframe of genic balance, states that there are femaleness genes with additive effect (genes F ) and maleness genes without additive effect (genes M ) , the balance being such that in haploids the effect of M>F (= male) and in diploids 2 F>M (= female) (CUNHAand KERR1957). KERR (1969; 1974), after studying larvae of diploid drones (Apis mellifera) ,modified slightly this initial proposition considering that the maleness genes could be either non-additive or slightly additive; that is, sex in honeybees would be produced by an interaction between major overdominant femaleness genes (z alleles), minor femaleness genes with additive effect (f alleles), and maleness genes without or with small additive effect ( m alleles). The objective of this paper is to elucidate the details of the balance between the sex genes in Apis mellifera, using an accurate statistical method. MATERIALS A N D METHODS
Four groups of honeybees (Apis mellifera adansonii) were used in this experiment: twentynine haploid males (16 chromosomes), twenty-nine diploid workers (32 chromosomes), sixteen diploid males ( 3 2 chromosomes) and twenty-one triploid workers (48 chromosomes). The honeybees from the first thre- groups were sons of the same queen (129) and were cousins of the triploid workers. Twenty-two external morphological characters were used in order to estimate the generalized Mahalanobis distances between the four groups of honeybees (see Table 1 ) . The measurements were made with a micrometric occular i n a Zeiss stereoscope. The techniques of measure(1970). ment followed the one described by GON~ALVES This paper is part of the M.Sci. thesis of the author and was financed by FL4PESP (Processes: 70/73$ and 71/9(38) and CNPq (Process: 14-138). Genetics 79: 213-117 February, 1975.
214
J O S k CHAUD-NETT0
TABLE 1
Means and siandard errors (in mm) of imnty-two randomly taken morphological characters of Apis mellifera adansonii
Anterior wing width Hind wing length Radial cell length Total thorax length Head length Anterior wing length Head width Hind wing width Radial cell width M.L. femur length M.L. tibia length M.L. tibia width M.L. femur width H.L. femur length H.L. tibia length H.L. tibia width H.L. tarsus length Flagellum length Scape length Pedicel length H.L. basitarsus width H.L. femur width
Haploid males
Diploid males
Diploid workers
Triploid workers
3,89 f 0,12 8,Ol f 0,W 4,40f 0,lO 6,43 t 0,27 3,95 f 0,14 11,76 f 423 4,46 t 0,m 3,06 f 0,m 467 f 0,04 2,88 f 0,08 2,34 t 0,07 0,34 & 0,03 0,4Q f 403 3,29 f 0,09 4,08 k 0,09 1,17 f 0,06 4,13 f 0,15 3,98 f 0,11 1,08 f 0,M 0,25 k 0,M 1,W f 0 , s 0,63 0,05
4,07 f 412 8,2l t 0,15
2,96 f 0,06 5,98 t 0,lO 3,017 f 0,06 4,35 t 0,14 3,48 f 0,12 8,61 f 0,12 3,77 t 0,IO 1,71 t 0,M 0,45 t 0,03 2,12 f 0,07 1,91 0,05 0,47 t 0,W 0,55 f 0,06 2,44 -C 0,cw 2,96 t 0,12 1,lO f 0,05 3,56 f 0,14 2,601 +- 0,05 1,22 f 0,w 0,22 f 0,03 1,W f 0,05 0,58 +- 0,M
3,12 t 0,06 6,26 I 0,19 3,09 I 0,07 4,46 I 0,08 3,57 0,m 9,% t 0,17 3,81 f 0,W 1,81 k 0,07 O,@ 401 2,16 t 0,05 1,96 t 0,M 0,46 t 0,08 0,54 t 0,04 2,48 t 0,05 3,02 +- 0,08 1,07 t 0,04 3,67 t 0,ll 2,52 +- 0,M 1,19 t 0,02 0 , s t 0,03 1,03 f 0,M 0,57 f 0,05
*
Note: M.L. = middle leg; H.L.
4,sf 0,w 6,32 -C 0,27 3,97 f 0,18 12,13 f 0,34 4,43 f 0,12 3,28 f 0,12 0,65 f 0,05 2,77 f 0,11 2,st 0,lO 0,39 k 406 0,52 f 0,M 3,19 f 0,12 3,79 f 0,15 1,12 f 0,07 3,93 f 0,w 3,85 t 0,12 1,oo f 0,M 428 f 0,M 1,26 t 0,08 0,62 +- 0,oS
*
*
*
= hind leg.
The four groups of honeybees were compared in pairs, the respective values of D2 being with some modifications introduced by calculated using the method described by MAHALANOBIS, RAO(1952). This method can be summarized as follows:
If X ijk is the kma observation of the imo character in individuals of the j”” group, for i = 1.2, ..., 22 (number of characters that were measured) i = 1,2,..., 4 (number of groups) k = 1,2,..., until 29 (the number of honeybees in each group varied from 16 to 29), then the sum of squares matrix and “within” sum of products is defined as:
(i,h = l,2, ... ,22) Where,
xii
xi
is the average of the ima variable for the j“J group and is the average of the ima variable for all the groups. This matrix cmsists of elements representing sum of squares and sum of prJducts of the deviations from all observations with relation to the respective averages in each group. Considering now the dispersion matrix, obtained from the sum of squares matrix and “within” sum of products, dividing each element by its freedom degrees number. It is a matrix of variance and co-variance. Let a i h be the dispersion matrix and aih its inverse, d i = the vector of difference between the averages of two groups in the ima variable (i = 1,2, ..., 22),
SEX D E T E R M I N A T I O N IN BEES
Then the Mahalanobis generalized distance 0 2 between two groups can be defined as: D? = Z Z aih di dhr for i,h = 1,2,..., 22. i
h
215 (1)
The differences between two compared groups are examined simultaneously in all the characters that can be correlated. This indicates that the information given by one character possibly is not independent from that furnished by the others. Th. numerical value of the possible major separation between any two groups is called generalized distance between them and measures, in a scale independent of that used for the measurements, the clearness of disjunction between them (PISANI 1969). Therefore, the value 0 2 between two groups is a pure number with properties of common distance and measures the extension with which they differ in shape and size (BARRACLOUGH and BLACKITH1962; BLACKITH and ALBRECHT 1959). The program for a n IBM 1130 was made by MR. LUISANTONIO F. BEZERRAunder the in this Dept. of Genetics. supervision of DR.F. A. M. DUARTE RESULTS A N D DISCUSSION
Figure 1 is an approximated graphic representation of the generalized distances of Mahalano3bis between the four groups of honeybees. The diagram was constructed in such a way that the basal line is the distance between haploid males and diploid workers, which constitute the normal sexual individuals in an Apis mellifera colony. The sexual constitution of the honeybees from each group are represented as follows. in which m, and mz mean the maleness genes and X , , X , and f the femaleness genes: a ) Haploid drones are m, mz > X I f. b) Diploid drones are 2m, f 2m, > X , X , ff. c) Diploid workers are X , X , 4-ff > 2ml 4-2m2. d) Triploid workers are X,X,X, f fff > 3m, 3m,. The genes X and f have high additivity since the effect of X,X2 ff is much greater than the effect of X , f, while the genes m have small additivity (the distance between 2n males and 2n workers is only 8% greater than the distance between n males and 2n workers). An examination of Figure 1 shows that maleness genes ( m ) are slightly additive in the diploid drones, since these males have characteristics of metamales,
+
+
+
+
64,78
dn FIGURE 1.-Configuration showing the generalized Mahalanobis distances, considering twenty-two external morphological characters of haploid and diploid drones (left) and diploid and triploid workers (right), Scale is arbitiary; value used is D, instead of D2 in order to make the drawings easier to represent the data.
216
J O S d CHAUD-NETT0
being about 8% more masculine than the haploid males. The diploid drones differ from diploid workers because they possess the inactive X , X , pair (the X alleles work only when they appear in heterozygosis). The greater maleness of the diploid drones results from the mJm, ;m2/m2genes; since the diploid drones are very similar to the haploid drones it can be concluded that 2ml 2m? have a maleness determinant effect greater than the femaleness determinant effect produced by f f , that is, this can only happen if 2m, 2m, are additive. Triploid workers are closer both to haploid drones (D= 62, 96) and diploid drones (D= 66, 2 8 ) , than the diploid workers ( D = 64, 78 and D = 68, 48, respectively). This fact demonstrates a slight masculinity of the triploid females when compared with the diploid females. An explanation could be formulated if what happens here is the same that happens in triploid chickens (ABDELHAMEED 1972). I n these birds there are some indications that the three chromosome sets are genetically active; the triploidy increases the content of both RNA and DNA per erythrocyte. If this same thing happens with the triploid workers of Apis mellifera, then an increase of masculinity would not occur because the bee is X I / X 1 / X z ;that is, the X , / X , pair would function as in diploid bees (66% of the RNA being produced by X I and the rest by X , ) . The differences between triploid workers and diploid workers were very minute, indicating that the functioning of the femaleness genes is the same in both types. The slight masculinity demonstrated by triploid workers must therefore be from the small additivity of the maleness genes. This confirms the results obtained f o r the diploid drones. According to the hypothesis of CUNHA and KERR (1957), the triploid bees should be meta-females because the X alleles of each bee (X,/X,/X2, for example) would have a feminine effect of 2 F since X , / X , are not additive. The effect of the other femaleness genes (f/f/f) could be equal to, f o r instance. 3 X 0,4 = 1.2 F (if f is a minor gene it should have a value smaller than 1 ) . The maleness genes (ml,m,, ma,etc.) would have an effect equal to M because they have no additivity. So the triploid workers, in any real case considered, would be slightly more female than the normal females. According to KERR (1974), in the triploid workers, the effect of the major femaleness genes (X,/X,/X,) would be less than 2 F (1,8, for instance) because X , / X , are not additive at all (since they are complementary genes) and X J X , are not totally additive. The effect of the minor femaleness genes (f/f/f) would be less than 1,2 F (for instance, 1,O) because they are not totally additive. So we would have 3,2 F-3d, where d is a small absence of additivity of f genes for each haploid set. The maleness genes would have a factor b of lack of additivity and then we would have 3M-3b ( b would be greater than d ) . Then the sexual 3M-3b. This scheme (or balance of the triploid workers should be 3,2 F-3d any other similar) implies that triploid workers may be slightly masculinized when compared with diploid workers. The results obtained in this experiment demonstrate that the maleness genes of Apis mellifera have a small additivity. They also strengthen the hypothesis that X alleles are femaleness genes.
+
+
+
SEX DETERMINATION I N BEES
21 7
LITERATURE CITED
ARDEL-HAMEED, F., 1972 Hemoglobin concentration in normal and intersexes triploid chickens: genetic inactivation or canalization? Science 178 (4063) :864-865. BARRACLOUGH, R. and R. E. BLACKITH,1962 Morphometric relationships in the genus Ditylenchus. Nematologica 8 : 51-58.
R. E. and F. 0. ALBRECHT, 1959 Morphometric differences between the Eye-stripe BLACKITH, polymorphs of the red locust. Scien. Joui. Royal Coll. Scien. 27: 13-27. CUNHA,A. B. and WARWICK E. KERR,1957 A genetical theory to explain sex-determination by arrhenotokous parthenogenesis. Forma et Functio l ( 4 ) : 33-36.
L. S., 1970 A n & e genktica do cruzamento entre Apis mlilfera ligustica e Apis GONCALVES, mellifera adansonii. Tese de doutoramento, 142 pgs. KERR,W. E., 1969 Genbtica e melhoramento de abelhas. pp. 263-297. XIV. Capitulo do livm “Melhoramento e Genktica.” Edit. Univ. S. Paulo e Edic6es Melhoramentos. -, 1974 Advances in cytology and genetics of bees. Annual Review of Entomology, 19: 253-268.
PISANI. J. F., 1969 Anilise estatistica multidimensional em biologia. Ci&nciae Cultura 21 (3): 6 19-61 3. RAO,C. R., 1952 Advanced Statistical Methods in Biometric Research. John Wiley & Sons, Inc., London. Corresponding editor: D. SUZUKI
