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The genetic basis of the production of the active form of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)
J. stored Prod. Res. Vol. 22, No. 3, pp. 115-123, Printed in Great Britain
1986
0022-474X186
$3.00 + 0.00
Pergamon Journals Ltd
THE GENETIC BASIS OF THE PRODUCTION OF THE ACTIVE FORM OF CALLOSOBRUCHUS MACULATUS (F.) (COLEOPTERA: BRUCHIDAE) Isuzu SANO-FUJII* Entomological Laboratory, College of Agriculture, Kyoto University, Kyoto, Japan (Received 16 January 1986)
Abstract-An experimental study was conducted to determine the hereditary factor in the production of the active form of Callosobruchus muculafus (F.). Through artificial mass selection for or against the active form of C. macularus, the proportion of the active form could be increased or decreased over several generations. A crossing experiment between the selected line created by selection for active forms and the stock line showed that the percentage of the active form among the offspring was 0% in all stock x stock crossings, whereas it was more than 40% in selected x selected crossings. Crosses between stock and selected lines exhibited intermediate values. Whether the parents came from lines which had been subjected to selection or not, played a more significant role than the actual form of the parent in determining the percentage of the active form among the offspring. There was no significant effect of selection on such biological properties as the number of eggs oviposited by a female in her lifetime. However, a modest shift was observed in adult longevities. From the present experiments, it can be concluded that the ability to become an active form in C. maculutus is heritable and that external factors (e.g. larval population density) only stimulate this potential to develop into active forms. The repeated mass selection for active form in the present experiment increased this ability to such a level that some active forms emerged even with no external stimulus.
INTRODUCTION
The southern cowpea weevil, Cuflosobruchus maculutus (F.), exhibits 2 adult forms which are called “flight form” and “flightless form” (Utida, 1954), or “active form” and “normal form” (Caswell, 1960). The forms differ distinctly in morphology (Utida, 1954, 1967, 1972; Southgate et al., 1957; Caswell, 1960), behavior (Taylor and Agbaje, 1974), and physiology (Utida and Takahashi, 1958). Such differences seem to be related to adaptation to divergence in life style. The adult oviposits on the mature crop and the larvae feed in the field or on stored beans in the warehouse. The active form is apparently adapted to life in the field, and the normal form to life in the storage house. This dimorphism may be analogous to phase polymorphism in migratory locusts, or wing polymorphism in aphids or leaf hoppers (e.g. Kennedy, 1956; reviewed by Harrison, 1980). Several environmental factors have been considered responsible for the production of the active form. These include larval density, temperature, water content of the bean, photoperiod, and so on during the preimaginal stages (Utida, 1954, 1965, 1969; Sano, 1967; Sano-Fujii, 1979, 1984). The genetic regulation has not yet been established. Caswell (1960) showed some indirect evidence for genetic regulation of active form production. The proportion of the active form in laboratory cultures decreased with culture age despite the high frequency of the active form in new cultures under identical culture condition. Utida (1954, 1970) has obtained similar results. This paper reports on several experiments designed to provide straightforward evidence for hereditary control of the production of active form. Experiment I concerns the results of artificial selection of active and normal forms. In Experiments II and III, the results of crossing between a stock culture line and the artificially selected active form line are presented. The biological characteristics of the two forms in the two lines are also investigated. MATERIALS
AND
CONDITIONS
OF
EXPERIMENTS
The strain of C. ,maculutus used in the present experiments has been reared in the Entomological Laboratory, Kyoto University, for more than 20 years and is the same strain as that used in my *Present address: 2-815-I Azuma, Sakura-Mura,
Ibaraki, 305 Japan 115
116
Isuzu
SANO-FUJII
orevious studies (Sano, 1967; Sano-Fujii, 1979, 1984). Stock cultures of the weevils have been maintained at 30°C and 75% r.h., using azuki bean (Vigna angularis, variety Dainagon) with water content of about 16%. The same environmental conditions used for stock cultures were employed throughout the experiments. EXPERIMENT
I
Method
The potential for creating laboratory strains with high and low production of active forms was investigated using artificial selection. Selection for active form. A culture was established with 54 active form females and 37 active form males, and 162 beans. Three beans per female were provided to give high larval densities (1 active form female can oviposit up to 30 eggs in her lifetime, though the actual number is much less at high adult density). The procedure for selection was as follows. Newly emerged active forms (aged O-l day) from the stock culture were introduced into a plastic container (7.5 cm in dia and 3 cm in height) and kept there until they all died. The number of emerged offspring of each form was scored daily and normal forms were discarded. Active forms were immediately transferred into a new plastic container with new beans in proportion to the number of females among them (3 x number of females). A maximum of 70 (usually 50-65) pairs of active forms were placed in the new plastic container. When more active forms than were required for 1 container emerged in one generation, additional plastic containers were set up. They were treated as different subculture(s). This procedure was foilowed for 10 generations. Selection for normal form. A normal form culture was established at the same time as the active form culture was established. Approximately 8 beans per female were supplied to permit larval densities comparable to those in the active form culture (1 normal form female can oviposit up to 80 eggs). The initial culture contained 21 pairs of weevils. The procedure for selection was as follows. Newly emerged normal forms (aged O-l day) were transferred from the stock culture into the plastic container and kept there until they all died. The number of emerged offspring of each form was scored every l-3 days. When 21 pairs of the normal form emerged in 1 day (usually the 2nd or 3rd day after the first adult emergence), they were transferred to a new container with the same number of beans as used initially, and were kept until they all died. The same procedure was repeated each generation. Results
In selection for the active form, the proportion of this type increased gradually, stabilizing at around 55% in the 7th and following generations (Fig. 1). The increase of the proportion of active form was independent of the population density after the first few generations. The sequential establishment of subcultures created 38 lines of the active form. These differed in relative emergence time. This temporal displacement among subcultures continued from generation 5 to generation 9. However, comparison of subcultures of earliest vs latest emergent active forms exhibited no significant differences in the proportion of active form offspring. In all cultures, the earliest emerging individuals were all normal form. The active form began emerging several days later. The proportion of the active form increased with time and the majority of late-emerging adults were active form in all cultures. The results of selection for the normal form are shown in Fig. 2. The proportion of the active form varied during the first severai generations depending upon the popuIation density measured by the number of adults emerged per bean. Thereafter, the proportion decreased and remained low independent of population density. EXPERIMENT
II
Method
Reciprocal crossing experiments using selected and unselected lines were conducted to analyze the effect of parental type on the production of active forms. The main culture of active form selection in Experiment I was used as the “selected line” and the stock culture was used as the
Genetic basis of the C. maculufus active form
-
60 t
117
2400 ‘I.
-
TIII
III 1
3
2
2000
0 J
111
4
5
6
7
8
9
Generation
Fig. I. Result of artificial selection of active form (data pooled from main and all subcultures). Upper; the changes in the proportion of the active form (solid line) and total number of adults emerged (broken line). Lower; changes in mean number of adults emerged per bean.
“unselected line”. Several biological properties of the 2 forms in the selected and unselected lines were also investigated. These included the proportion of reproductive pairs, the longevity of male and female adults, the preoviposition period of female, the number of eggs oviposited by a female in her life and of eggs hatched among them, larval mortality, and the number of emerged offspring. Beans with eggs oviposited by weevils of selected and unselected lines were separated into small vials. Newly emerged virgin females and males were removed from the vials and each pair was introduced into a plastic container with 40 beans for a normal form female, and with 20 beans for an active form female. They were kept in the container until their deaths. Data involving percentages were analyzed after arcsine transformation. The means and 95% confidence intervals were presented after retransformation, instead of means and standard deviations as in other cases. All statistical decisions were made at 5% significance level.
Results The percentages of active form offspring in the crossing experiments are shown in Table 1. When the 16 combinations were classified into 4 major groups, the order of the percentages of active form emergents was as follows; crosses within selected line > crosses between selected and unselected
I
11”
T’ 1
2
” 3
4
5
6
”
1’ 7
6
9
10
’ 11
12
Generation
Fig. 2. Results of artificial selection of normal form. For explanation, see Fig. 1.
Isuzu
118
Table
I.
The
percentages
SANO-FUJII
of active
form
emerged
Form Stock
w
line
ForIll
AS
0.0%
0.0%
(OsM.0)
(0.0-0.0)
Selected
~_
line SAC?
0.0%
I I .4% (0.0-45.8)
0.9%
form
from
stock
and SA: active
line,
2.2%
5.7%
(0.&9.1)
(0.5-16.3)
form
from
form
selected
(2
7)
(6) 47. I %
I .9-59.6)
(0-C
100.01
(5) 55.2% (35.4-74.
(10)
confidence
A: active
16.4% (I.741
40.0%
(O&-12.4)
(10)
(6)
(5)
(14)
(9) Top line: mean; Second line: 95%
(12)
(0.0-0.0)
(0.1-5.5)
SNZ
I .O% (0.0 -7.4)
(0.7-5.9)
0.0%
1.7%
male
SA$
3.0%
(0.0-0.3)
..
line
SN:’
(5)
(9)
of
Selected A:
and line
line
Ni
(281
_
Stock
in the offspring
and line of female
limit;
from
Bottom
stock line.
73.2% I)
(63.1 +Q.?)
(81 line: numbers SN:
normal
WI
of replicates. form
from
N: normal
selected
Ime.
line.
lines (both reciprocals) > crosses within unselected line. The reciprocal selected x unselected crosses were not significantly different (by Student-Newman-Keuls (SNK) multiple comparison test [Sokal and Rohlf, 19691) from each other, suggesting that any genetic factor(s) involved in the production of the active form were not sex-linked. Four values within each major group of crosses were not significantly different from each other, except in the crosses within the selected line where a SNK multiple comparison test showed that SN$! x SNg was significantly different from the rest and SAQ x SAd was again significantly different from the rest. Overall, there were significantly more active form offspring in the progeny of selected line females than in stock line females. There was no significant difference between forms within each line (multiple comparison by S-method, Scheffe, 1959). The same conclusions also apply to males. These data analyses can be summarized as follows: The selected line produced significantly more active forms than the unselected line and the potential to produce the active form was similar between normal and active forms within the selected line. When the percentages of active form offspring were calculated for each sex within each crossing combination (not shown in Table 1), there were no significant differences between sexes in all 16 combinations. The results of the biological properties investigated are shown in Table 2. Not all data are available for all replicates due to such events as the accidental escape of one sex of the overlooking of the exact date of death or initiation of oviposition, So sample sizes are provided when necessary in Table 2. Over all crosses, there was a significantly higher percentage of non-reproductive individuals in active form females than in normal form females (column 1). However, there was no significant difference between selected and unselected females of the same form (multiple comparison by S-method). Over all crosses, there was no significant difference between females with active and females with normal male mates. Most non-reproductive pairs in all combinations were those which produced no eggs at all. This suggests that reproductive success seems to depend primarily on the female. Column 2 shows female adult longevity (based on reproductive pairs only). The selected active form female (SA?) has a significantly longer adult life than the 3 other types of female. Although the effect of the form of the male mate was a significant factor in a 2 way analysis of variance test, there was no male significantly different from the others according to multiple comparisons by the S-method. Non-reproductive females usually lived longer than reproductive females in each female form and line (but the differences were not statistically significant). The forms and lines of the female mate had no significant effect on male adult longevity (column 3). Normal form males had shorter lives than active form males within each line. When all forms of both lines are compared, the following order is established at the 5% significance level; normal form male of unselected line (Nd) < active form male of the unselected line (Ad) = normal form
*Based on adults ti’ercentage and IMean, standard §Mean and 95%
Form and line of female
0.0 0.0 9.1 25.0 50.0 38.5 54.6 31.3 1.7 25.0 10.0 38.5 57.1 22.2 60.0 10.7
(28) (IO) (II) (12) (10) (26) (1 I) (16) (13) (8) (20) (13) (14) (9) (IO) (28)
%Nonreproductive pairs?
in reproductive pairs only. sample size (in parentheses). deviation and sample size (in parentheses). confidence interval of mean.
Form and line of male (27) (9) (9) (7) (5) (13) (5) (8) (12) (6) (16) (7) (6) (7) (4) (22)
(28) (IO) (IO) (9) (5) (16) (5) (II) (12) (6) (18) (8) (6) (6) (4) (25) 78.9 f 21.1 (27) 54.3 f 19.4 (IO) 71.2521.6 (9) 70.0 + 3 I .8 (9) 42.6 + 22.0 (5) 31.8+ 13.3 (13) 27.0 + 13.8 (5) 37.1 f 13.8 (9) 81.9 f 21.6 (12) 70.8 f 16.7 (6) 69.8 _+ 18.9 (16) 66.3 + 23.4 (8) 53.8 f 21.7 (6) 47.4 f 10.5 (5) 39.0 f 7. I (2) 44.9 f 13.1 (23)
(27) (9) (9) (9) (5) (15) (4) (7) (12) (5) (16) (8) (6) (7) (4) (24)
0.0 + 0.0 0.5 f 0.8 0.0 f 0.0 l.2k2.1 I.6 + 0.5 l.9+ I.5 I .4 f I .7 2.7 *_I.7 0.1 f 0.3 0.2 + 0.4 0.1 + 0.2 0.0 f 0.0 0.8 f 0.4 I.3 f 0.5 I.3 * I.9 1.7 f I.1
II
7.9 + I .7 12.0 + 4.0 9.7 + 2.0 14.1 + 5.1 7.8 + 0.8 9.4 + 3.6 ll.6k2.7 15.1 + 3.6 7.9 + 1.0 13.5 + 5.1 9.8 fr 3.4 13.9 + 5.2 7.5 + I .8 13.0+ 1.5 IO.5 rfI 2.1 15.1 + 3.7
of crosses in Experiment
8.6 + I .7 1.7 + 1.9 7.1 f 2.0 9.7 + 2.8 II.8 k4.3 10.3 + 3.4 7.3 _+ I.0 10.3 f 3.9 8.3 f 1.7 8.4 1: I.1 9.0 + 3.1 9.3 f 2.4 ll.8f5.2 11.7k3.8 I I .O ?r 3.2 14.5 + 5.0
combinations Total no. of eggs oviposited per femalef
in various
Male adult longevity (days)?
observed
Female adult longevity (days)*t
nronerties Female preoviposition period (days)*t
Table 2. Biolonical
78.9 88.9 84. I 84.3 64.8 75.8 64. I 16.7 84.8 87.5 75.0 78.3 81.0 82.1 45.9 66.0
(75.6-82.0) (83693.2) (8 I .5-86.5) (73.s92.6) (52.2-76.4) (64.6-85.5) (48.9-78. I) (69.9-82.9) (81.1-88.2) (79.2-93.9) (66.8-82.3) (66.9-87.9) (69.3-90.5) (59.8-96.6) (t&100) (60.2-71.5)
% Egg hatchability
12.4 13.6 9.6 16.4 6.8 9.2 13. I 24.4 13.0 19.1 18.2 17.8 21.0 21.6 36.8 30.9
(9.2-l 5.9) (9.tH9.1) (2620.3) (11.8-21.6) (0.3-20.7) (2.6-19.3) (0.&50.5) (17.C32.6) (6920.6) (11.628.0) (9.628.8) (3.140.9) (12.9-30.4) (5957.6) (f&100) (25.7-36.4)
%Larval mortality§
Isuzu SANO-FUIII
120
male of selected line (SN$) < active form male of selected line @AS). The longevity of the male was not affected by whether it was paired with reproductive or non-reproductive females. Normal form females of both lines had, in most cases, no preoviposition period, whereas active form females of both lines had preoviposition periods of l-2 days (column 4). Multiple by the S-method showed the following order in preoviposition period; comparisons SN9 = NO < SA9 < A$?. The male mate was a significant factor in the preoviposition period according to a 2-way analysis of variance. There was a tendency for preoviposition period of females to be longer when selected active form males (SAJ) were the mates, except in selected normal form females (SN?). There was no clear relationship between preoviposition period and female adult longevity. The three characteristics, the number of eggs oviposited per female in her lifetime, the numbers of eggs hatched, and the numbers of emerged offspring showed a similar tendency. The numbers of eggs oviposited are shown in column 5. Normal form females (NO and SNQ produced significantly more eggs, more hatched eggs and more emerged offspring than active form females (A’$ and SA?). However, there was no significant difference between females of the same form. As with the previous properties, the numbers of eggs oviposited and hatched were independent of the form or line of the male mate. However, there was a significant effect of the male mate on the number of emerged offspring. The number of offspring was significantly higher when an unselected normal form male (NS) was the mate. The percent egg hatchability of active form females of the unselected line was significantly lower than those of the 3 other types of females (column 6). There was significantly higher larval mortality in selected active form females than in the other types (column 7), resulting in the production of more offspring by normal form females than by active form females in both lines. There was no significant effect of male parent on hatchability or on larval mortality. No clear trends were detected in the relationship between female longevity and number of eggs oviposited per female, or between preoviposition period and number of eggs oviposited per female.
EXPERIMENT
III
Method In Experiment II, 20 beans for an active form female and 40 beans for a normal female were provided for oviposition. With this experimental arrangement, there were some beans on which more than one egg was oviposited. High larval density facilitates the production of active form offspring. The following experiment was designed to eliminate this effect. Pairs of newly emerged virgins (NO x No’, A9 x A&, SNP x SNd, or SA? x SA6) were prepared in the same way as in Experiment II and liberated into a container with 150 beans. After the pair died and eggs hatched, beans with only 1 egg deposited were selected and kept until the offspring emerged. The number of emerged offspring of each form was scored daily. The numbers of replicates for each cross are shown in Table 3. Results
The results are summarized in Table 3. Active forms were found only in the offspring from the selected parents. There was no significant difference in the percentage of active form offspring between the 2 forms of the selected line parents. Table 3. The uercentaaes of active form emerned from beans with onlv one eaa
Crossing
Number of replicates
N?xN$
4
AP x Ad
5
SN9 x SNd
12
SAF x SAd
13
Mean number of beans used per replicate
Mean number of adults emerged per replicate
48.0 (Ck109.6) 42.8 (20.265.3) 51.1 (35.6-66.6) 54.8 (36.6-73.0)
45.5 (Q-102.9) 38.4 (15.7-61.1) 46.3 (31.1-61.4) 48.2 (30.4-66.0)
Mean percentage of aclive form (E) (E) 24.2% (15.3-34.5) 33.0% (23.U3.9)
The numbers in parentheses show 95% confidence limit. The symbols are the same as in Table
I
Genetic basis of the C. macularusactive form
121
DISCUSSION Experiment I demonstrates that the proportion of active forms in the laboratory Culfosobruchus can be increased or decreased by artificial selection, implying that there is an hereditary factor involved in the production of active form. The result seen in the proportions of active form offspring in the crossing experiments (II and III) confirms that the ability to produce active forms is heritable. Most importantly, the selected line (either active or normal form) could produce more active forms than the unselected line (i.e. stock culture). Normal form individuals from the selected lines produced a higher proportion of active form offspring than did crosses between active forms from stock cultures, and the potential they had to produce active forms was similar to that of active forms of the same line, i.e. selected active forms. It may be hypothesized that the external factors known to produce active forms release an internal genetic potential to develop into this form. Only those individuals who have this genetic potential would develop into active forms under such external stimuli. Those individuals with this internal ability would not emerge as active forms without such external stimuli. In the present experiments, larval population density was presumably the only environmental stress parameter. High larval population density generates a variety of environmental changes including sudden increase of temperature and/or water content in the heap of beans (Sano, 1967; Sano-Fujii, 1984). For the system to stimulate active form production, there must exist individuals which generate the environmental changes and serve as a source of stimulus. These individuals are generally those that develop faster or are oviposited earlier. During the artificial selection for the active form, some normal forms always emerged earlier than the active forms in each generation. This consistent phenomenon observed in every generation makes the above hypothesized mechanism plausible. The difference in emergence time between active and normal forms is, as stated above, also in part due to the slower development of active forms than normal forms, as clearly shown in Fig. 3. The results in Experiment III suggest that the internal ability to be the active form can be increased by selection to such an extent that active form development can take place with no apparent external stimulus. However, by comparing the mean percentages of active form offspring in Table 3 (with 1 egg on each bean) and those with density effects (Table 1), we notice that the latter values are twice as high (24.2% vs 40.0% and 33.0% vs 73.2%). It is evident that external stimulus (larval population density) still plays an important role in the production of active form. It remains to be seen whether a line consisting only of the active form (without any external stimulus) can be established by further artificial selection. However, in the present experiment, the percentage of the active form remained essentially the same (ca. 60%) after the 7th generation in spite of further selection. When the biological properties of the 2 forms in the selected and stock lines are compared, normal forms from the stock line (N) show most typical traits of the normal form, e.g. high fecundity, almost no preoviposition period, low percentage of non-reproductive females, and short adult life. All of these properties appear optimal for populations adapted to life in stored beans. The normal form of the selected line (SN) shows similar tendencies. Active forms of both lines, on the other hand, show low fecundity, long preoviposition period, high percentage of nonreproductive females, long adult life, and low viability (low hatchability in active forms from the stock line, and high larval mortality in active form from the selected line). Many of these properties
macularus population
Days after oviposit ion
Fig. 3. Cumulative percentage of the emergence of normal (solid line) and active (broken line) forms from beans with only 1 egg oviposited by the females of selected line. Left; from a replicate in the Sn9 x SNd cross in Table 3. Right; from a replicate in the SA? x SAg cross in Table 3. The numbers at the end of the lines show the total numbers of adults emerged.
122
Isuzu SANO-FIJJII
are related to the high capacity for flight and are definitely adaptive for migration and life in the field. It was expected that significant differences in the biological properties might appear between the same form in the selected and stock lines. However, the forms and the properties associated with them in stock culture are apparently tightly linked genetically and this association did not break down dramatically during mass selection. Nevertheless, there are some shifts in selected lines. For example, the male and female adult longevities of the active form from the selected lines are significantly longer than the three others. The longevities of the selected normal form are similar to those of stock active forms and different from those of stock normal form (although not significantly so in females). The results of Caswell(l960) and Utida (1970) which showed that the proportion of the active form in their laboratory populations decreased over a number of generations, could be interpreted from Experiment I as being due to an unintentional selection for normal forms under their experimental conditions (perhaps in the method of husbandry). In the stock culture used in this study, similar unintentional selection for the normal form would probably have existed. However, field populations of C. maculatus would probably be able to maintain a higher capacity to produce active forms than the populations used in the laboratory study, because they spend their lives in 2 contrasting habitats. In the field and storage, natural selection respectively works for and against the active form. This dimorphism, therefore, is delicately balanced by the insect’s life style in 2 contrasting habitats. The active/normal dimorphism (or polymorphism) or its equivalent can be observed in many species of insects. In some species, the different morphs occur in individuals of apparently the same genetic constitution. Here, the gene function is “switched” or latent genes are brought into action under the impact of some change in the environment. The phase polymorphism in locust and armyworm and wing polymorphism in aphid and leafhopper are some examples (reviewed by Harrison, 1980). These polymorphisms are thought to be regulated by density effects and/or external factors (e.g. nutrition and photoperiod). However, the present study shows that not all individuals in the population have the same genetic constitution; there can be genetic variations among individuals in the ability to assume a particular form. The dimorphism of C. macufatus is regulated by the combination of the external factors and the genetic variation among the individuals in the population. This example supports the proposition suggested by some researchers (Denno and Grissell, 1979; Lamb and MacKey, 1979; Harrison, 1980) that there may be a genetic component in many of the cases of phase dimorphism. Acknowledgements-1 wish to express my hearty thanks to Professor Syunro Utida for his valuable suggestions and helpful criticism for this work, to Dr Koichi Fujii for his useful criticism and strong encouragement and to Drs Morris Levy, Bruce Champ and the anonymous reviewer for their careful reading of the earlier manuscript and useful comments.
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Genetic basis of the C. maculutus active form
123
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1986
0022-474X186
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THE GENETIC BASIS OF THE PRODUCTION OF THE ACTIVE FORM OF CALLOSOBRUCHUS MACULATUS (F.) (COLEOPTERA: BRUCHIDAE) Isuzu SANO-FUJII* Entomological Laboratory, College of Agriculture, Kyoto University, Kyoto, Japan (Received 16 January 1986)
Abstract-An experimental study was conducted to determine the hereditary factor in the production of the active form of Callosobruchus muculafus (F.). Through artificial mass selection for or against the active form of C. macularus, the proportion of the active form could be increased or decreased over several generations. A crossing experiment between the selected line created by selection for active forms and the stock line showed that the percentage of the active form among the offspring was 0% in all stock x stock crossings, whereas it was more than 40% in selected x selected crossings. Crosses between stock and selected lines exhibited intermediate values. Whether the parents came from lines which had been subjected to selection or not, played a more significant role than the actual form of the parent in determining the percentage of the active form among the offspring. There was no significant effect of selection on such biological properties as the number of eggs oviposited by a female in her lifetime. However, a modest shift was observed in adult longevities. From the present experiments, it can be concluded that the ability to become an active form in C. maculutus is heritable and that external factors (e.g. larval population density) only stimulate this potential to develop into active forms. The repeated mass selection for active form in the present experiment increased this ability to such a level that some active forms emerged even with no external stimulus.
INTRODUCTION
The southern cowpea weevil, Cuflosobruchus maculutus (F.), exhibits 2 adult forms which are called “flight form” and “flightless form” (Utida, 1954), or “active form” and “normal form” (Caswell, 1960). The forms differ distinctly in morphology (Utida, 1954, 1967, 1972; Southgate et al., 1957; Caswell, 1960), behavior (Taylor and Agbaje, 1974), and physiology (Utida and Takahashi, 1958). Such differences seem to be related to adaptation to divergence in life style. The adult oviposits on the mature crop and the larvae feed in the field or on stored beans in the warehouse. The active form is apparently adapted to life in the field, and the normal form to life in the storage house. This dimorphism may be analogous to phase polymorphism in migratory locusts, or wing polymorphism in aphids or leaf hoppers (e.g. Kennedy, 1956; reviewed by Harrison, 1980). Several environmental factors have been considered responsible for the production of the active form. These include larval density, temperature, water content of the bean, photoperiod, and so on during the preimaginal stages (Utida, 1954, 1965, 1969; Sano, 1967; Sano-Fujii, 1979, 1984). The genetic regulation has not yet been established. Caswell (1960) showed some indirect evidence for genetic regulation of active form production. The proportion of the active form in laboratory cultures decreased with culture age despite the high frequency of the active form in new cultures under identical culture condition. Utida (1954, 1970) has obtained similar results. This paper reports on several experiments designed to provide straightforward evidence for hereditary control of the production of active form. Experiment I concerns the results of artificial selection of active and normal forms. In Experiments II and III, the results of crossing between a stock culture line and the artificially selected active form line are presented. The biological characteristics of the two forms in the two lines are also investigated. MATERIALS
AND
CONDITIONS
OF
EXPERIMENTS
The strain of C. ,maculutus used in the present experiments has been reared in the Entomological Laboratory, Kyoto University, for more than 20 years and is the same strain as that used in my *Present address: 2-815-I Azuma, Sakura-Mura,
Ibaraki, 305 Japan 115
116
Isuzu
SANO-FUJII
orevious studies (Sano, 1967; Sano-Fujii, 1979, 1984). Stock cultures of the weevils have been maintained at 30°C and 75% r.h., using azuki bean (Vigna angularis, variety Dainagon) with water content of about 16%. The same environmental conditions used for stock cultures were employed throughout the experiments. EXPERIMENT
I
Method
The potential for creating laboratory strains with high and low production of active forms was investigated using artificial selection. Selection for active form. A culture was established with 54 active form females and 37 active form males, and 162 beans. Three beans per female were provided to give high larval densities (1 active form female can oviposit up to 30 eggs in her lifetime, though the actual number is much less at high adult density). The procedure for selection was as follows. Newly emerged active forms (aged O-l day) from the stock culture were introduced into a plastic container (7.5 cm in dia and 3 cm in height) and kept there until they all died. The number of emerged offspring of each form was scored daily and normal forms were discarded. Active forms were immediately transferred into a new plastic container with new beans in proportion to the number of females among them (3 x number of females). A maximum of 70 (usually 50-65) pairs of active forms were placed in the new plastic container. When more active forms than were required for 1 container emerged in one generation, additional plastic containers were set up. They were treated as different subculture(s). This procedure was foilowed for 10 generations. Selection for normal form. A normal form culture was established at the same time as the active form culture was established. Approximately 8 beans per female were supplied to permit larval densities comparable to those in the active form culture (1 normal form female can oviposit up to 80 eggs). The initial culture contained 21 pairs of weevils. The procedure for selection was as follows. Newly emerged normal forms (aged O-l day) were transferred from the stock culture into the plastic container and kept there until they all died. The number of emerged offspring of each form was scored every l-3 days. When 21 pairs of the normal form emerged in 1 day (usually the 2nd or 3rd day after the first adult emergence), they were transferred to a new container with the same number of beans as used initially, and were kept until they all died. The same procedure was repeated each generation. Results
In selection for the active form, the proportion of this type increased gradually, stabilizing at around 55% in the 7th and following generations (Fig. 1). The increase of the proportion of active form was independent of the population density after the first few generations. The sequential establishment of subcultures created 38 lines of the active form. These differed in relative emergence time. This temporal displacement among subcultures continued from generation 5 to generation 9. However, comparison of subcultures of earliest vs latest emergent active forms exhibited no significant differences in the proportion of active form offspring. In all cultures, the earliest emerging individuals were all normal form. The active form began emerging several days later. The proportion of the active form increased with time and the majority of late-emerging adults were active form in all cultures. The results of selection for the normal form are shown in Fig. 2. The proportion of the active form varied during the first severai generations depending upon the popuIation density measured by the number of adults emerged per bean. Thereafter, the proportion decreased and remained low independent of population density. EXPERIMENT
II
Method
Reciprocal crossing experiments using selected and unselected lines were conducted to analyze the effect of parental type on the production of active forms. The main culture of active form selection in Experiment I was used as the “selected line” and the stock culture was used as the
Genetic basis of the C. maculufus active form
-
60 t
117
2400 ‘I.
-
TIII
III 1
3
2
2000
0 J
111
4
5
6
7
8
9
Generation
Fig. I. Result of artificial selection of active form (data pooled from main and all subcultures). Upper; the changes in the proportion of the active form (solid line) and total number of adults emerged (broken line). Lower; changes in mean number of adults emerged per bean.
“unselected line”. Several biological properties of the 2 forms in the selected and unselected lines were also investigated. These included the proportion of reproductive pairs, the longevity of male and female adults, the preoviposition period of female, the number of eggs oviposited by a female in her life and of eggs hatched among them, larval mortality, and the number of emerged offspring. Beans with eggs oviposited by weevils of selected and unselected lines were separated into small vials. Newly emerged virgin females and males were removed from the vials and each pair was introduced into a plastic container with 40 beans for a normal form female, and with 20 beans for an active form female. They were kept in the container until their deaths. Data involving percentages were analyzed after arcsine transformation. The means and 95% confidence intervals were presented after retransformation, instead of means and standard deviations as in other cases. All statistical decisions were made at 5% significance level.
Results The percentages of active form offspring in the crossing experiments are shown in Table 1. When the 16 combinations were classified into 4 major groups, the order of the percentages of active form emergents was as follows; crosses within selected line > crosses between selected and unselected
I
11”
T’ 1
2
” 3
4
5
6
”
1’ 7
6
9
10
’ 11
12
Generation
Fig. 2. Results of artificial selection of normal form. For explanation, see Fig. 1.
Isuzu
118
Table
I.
The
percentages
SANO-FUJII
of active
form
emerged
Form Stock
w
line
ForIll
AS
0.0%
0.0%
(OsM.0)
(0.0-0.0)
Selected
~_
line SAC?
0.0%
I I .4% (0.0-45.8)
0.9%
form
from
stock
and SA: active
line,
2.2%
5.7%
(0.&9.1)
(0.5-16.3)
form
from
form
selected
(2
7)
(6) 47. I %
I .9-59.6)
(0-C
100.01
(5) 55.2% (35.4-74.
(10)
confidence
A: active
16.4% (I.741
40.0%
(O&-12.4)
(10)
(6)
(5)
(14)
(9) Top line: mean; Second line: 95%
(12)
(0.0-0.0)
(0.1-5.5)
SNZ
I .O% (0.0 -7.4)
(0.7-5.9)
0.0%
1.7%
male
SA$
3.0%
(0.0-0.3)
..
line
SN:’
(5)
(9)
of
Selected A:
and line
line
Ni
(281
_
Stock
in the offspring
and line of female
limit;
from
Bottom
stock line.
73.2% I)
(63.1 +Q.?)
(81 line: numbers SN:
normal
WI
of replicates. form
from
N: normal
selected
Ime.
line.
lines (both reciprocals) > crosses within unselected line. The reciprocal selected x unselected crosses were not significantly different (by Student-Newman-Keuls (SNK) multiple comparison test [Sokal and Rohlf, 19691) from each other, suggesting that any genetic factor(s) involved in the production of the active form were not sex-linked. Four values within each major group of crosses were not significantly different from each other, except in the crosses within the selected line where a SNK multiple comparison test showed that SN$! x SNg was significantly different from the rest and SAQ x SAd was again significantly different from the rest. Overall, there were significantly more active form offspring in the progeny of selected line females than in stock line females. There was no significant difference between forms within each line (multiple comparison by S-method, Scheffe, 1959). The same conclusions also apply to males. These data analyses can be summarized as follows: The selected line produced significantly more active forms than the unselected line and the potential to produce the active form was similar between normal and active forms within the selected line. When the percentages of active form offspring were calculated for each sex within each crossing combination (not shown in Table 1), there were no significant differences between sexes in all 16 combinations. The results of the biological properties investigated are shown in Table 2. Not all data are available for all replicates due to such events as the accidental escape of one sex of the overlooking of the exact date of death or initiation of oviposition, So sample sizes are provided when necessary in Table 2. Over all crosses, there was a significantly higher percentage of non-reproductive individuals in active form females than in normal form females (column 1). However, there was no significant difference between selected and unselected females of the same form (multiple comparison by S-method). Over all crosses, there was no significant difference between females with active and females with normal male mates. Most non-reproductive pairs in all combinations were those which produced no eggs at all. This suggests that reproductive success seems to depend primarily on the female. Column 2 shows female adult longevity (based on reproductive pairs only). The selected active form female (SA?) has a significantly longer adult life than the 3 other types of female. Although the effect of the form of the male mate was a significant factor in a 2 way analysis of variance test, there was no male significantly different from the others according to multiple comparisons by the S-method. Non-reproductive females usually lived longer than reproductive females in each female form and line (but the differences were not statistically significant). The forms and lines of the female mate had no significant effect on male adult longevity (column 3). Normal form males had shorter lives than active form males within each line. When all forms of both lines are compared, the following order is established at the 5% significance level; normal form male of unselected line (Nd) < active form male of the unselected line (Ad) = normal form
*Based on adults ti’ercentage and IMean, standard §Mean and 95%
Form and line of female
0.0 0.0 9.1 25.0 50.0 38.5 54.6 31.3 1.7 25.0 10.0 38.5 57.1 22.2 60.0 10.7
(28) (IO) (II) (12) (10) (26) (1 I) (16) (13) (8) (20) (13) (14) (9) (IO) (28)
%Nonreproductive pairs?
in reproductive pairs only. sample size (in parentheses). deviation and sample size (in parentheses). confidence interval of mean.
Form and line of male (27) (9) (9) (7) (5) (13) (5) (8) (12) (6) (16) (7) (6) (7) (4) (22)
(28) (IO) (IO) (9) (5) (16) (5) (II) (12) (6) (18) (8) (6) (6) (4) (25) 78.9 f 21.1 (27) 54.3 f 19.4 (IO) 71.2521.6 (9) 70.0 + 3 I .8 (9) 42.6 + 22.0 (5) 31.8+ 13.3 (13) 27.0 + 13.8 (5) 37.1 f 13.8 (9) 81.9 f 21.6 (12) 70.8 f 16.7 (6) 69.8 _+ 18.9 (16) 66.3 + 23.4 (8) 53.8 f 21.7 (6) 47.4 f 10.5 (5) 39.0 f 7. I (2) 44.9 f 13.1 (23)
(27) (9) (9) (9) (5) (15) (4) (7) (12) (5) (16) (8) (6) (7) (4) (24)
0.0 + 0.0 0.5 f 0.8 0.0 f 0.0 l.2k2.1 I.6 + 0.5 l.9+ I.5 I .4 f I .7 2.7 *_I.7 0.1 f 0.3 0.2 + 0.4 0.1 + 0.2 0.0 f 0.0 0.8 f 0.4 I.3 f 0.5 I.3 * I.9 1.7 f I.1
II
7.9 + I .7 12.0 + 4.0 9.7 + 2.0 14.1 + 5.1 7.8 + 0.8 9.4 + 3.6 ll.6k2.7 15.1 + 3.6 7.9 + 1.0 13.5 + 5.1 9.8 fr 3.4 13.9 + 5.2 7.5 + I .8 13.0+ 1.5 IO.5 rfI 2.1 15.1 + 3.7
of crosses in Experiment
8.6 + I .7 1.7 + 1.9 7.1 f 2.0 9.7 + 2.8 II.8 k4.3 10.3 + 3.4 7.3 _+ I.0 10.3 f 3.9 8.3 f 1.7 8.4 1: I.1 9.0 + 3.1 9.3 f 2.4 ll.8f5.2 11.7k3.8 I I .O ?r 3.2 14.5 + 5.0
combinations Total no. of eggs oviposited per femalef
in various
Male adult longevity (days)?
observed
Female adult longevity (days)*t
nronerties Female preoviposition period (days)*t
Table 2. Biolonical
78.9 88.9 84. I 84.3 64.8 75.8 64. I 16.7 84.8 87.5 75.0 78.3 81.0 82.1 45.9 66.0
(75.6-82.0) (83693.2) (8 I .5-86.5) (73.s92.6) (52.2-76.4) (64.6-85.5) (48.9-78. I) (69.9-82.9) (81.1-88.2) (79.2-93.9) (66.8-82.3) (66.9-87.9) (69.3-90.5) (59.8-96.6) (t&100) (60.2-71.5)
% Egg hatchability
12.4 13.6 9.6 16.4 6.8 9.2 13. I 24.4 13.0 19.1 18.2 17.8 21.0 21.6 36.8 30.9
(9.2-l 5.9) (9.tH9.1) (2620.3) (11.8-21.6) (0.3-20.7) (2.6-19.3) (0.&50.5) (17.C32.6) (6920.6) (11.628.0) (9.628.8) (3.140.9) (12.9-30.4) (5957.6) (f&100) (25.7-36.4)
%Larval mortality§
Isuzu SANO-FUIII
120
male of selected line (SN$) < active form male of selected line @AS). The longevity of the male was not affected by whether it was paired with reproductive or non-reproductive females. Normal form females of both lines had, in most cases, no preoviposition period, whereas active form females of both lines had preoviposition periods of l-2 days (column 4). Multiple by the S-method showed the following order in preoviposition period; comparisons SN9 = NO < SA9 < A$?. The male mate was a significant factor in the preoviposition period according to a 2-way analysis of variance. There was a tendency for preoviposition period of females to be longer when selected active form males (SAJ) were the mates, except in selected normal form females (SN?). There was no clear relationship between preoviposition period and female adult longevity. The three characteristics, the number of eggs oviposited per female in her lifetime, the numbers of eggs hatched, and the numbers of emerged offspring showed a similar tendency. The numbers of eggs oviposited are shown in column 5. Normal form females (NO and SNQ produced significantly more eggs, more hatched eggs and more emerged offspring than active form females (A’$ and SA?). However, there was no significant difference between females of the same form. As with the previous properties, the numbers of eggs oviposited and hatched were independent of the form or line of the male mate. However, there was a significant effect of the male mate on the number of emerged offspring. The number of offspring was significantly higher when an unselected normal form male (NS) was the mate. The percent egg hatchability of active form females of the unselected line was significantly lower than those of the 3 other types of females (column 6). There was significantly higher larval mortality in selected active form females than in the other types (column 7), resulting in the production of more offspring by normal form females than by active form females in both lines. There was no significant effect of male parent on hatchability or on larval mortality. No clear trends were detected in the relationship between female longevity and number of eggs oviposited per female, or between preoviposition period and number of eggs oviposited per female.
EXPERIMENT
III
Method In Experiment II, 20 beans for an active form female and 40 beans for a normal female were provided for oviposition. With this experimental arrangement, there were some beans on which more than one egg was oviposited. High larval density facilitates the production of active form offspring. The following experiment was designed to eliminate this effect. Pairs of newly emerged virgins (NO x No’, A9 x A&, SNP x SNd, or SA? x SA6) were prepared in the same way as in Experiment II and liberated into a container with 150 beans. After the pair died and eggs hatched, beans with only 1 egg deposited were selected and kept until the offspring emerged. The number of emerged offspring of each form was scored daily. The numbers of replicates for each cross are shown in Table 3. Results
The results are summarized in Table 3. Active forms were found only in the offspring from the selected parents. There was no significant difference in the percentage of active form offspring between the 2 forms of the selected line parents. Table 3. The uercentaaes of active form emerned from beans with onlv one eaa
Crossing
Number of replicates
N?xN$
4
AP x Ad
5
SN9 x SNd
12
SAF x SAd
13
Mean number of beans used per replicate
Mean number of adults emerged per replicate
48.0 (Ck109.6) 42.8 (20.265.3) 51.1 (35.6-66.6) 54.8 (36.6-73.0)
45.5 (Q-102.9) 38.4 (15.7-61.1) 46.3 (31.1-61.4) 48.2 (30.4-66.0)
Mean percentage of aclive form (E) (E) 24.2% (15.3-34.5) 33.0% (23.U3.9)
The numbers in parentheses show 95% confidence limit. The symbols are the same as in Table
I
Genetic basis of the C. macularusactive form
121
DISCUSSION Experiment I demonstrates that the proportion of active forms in the laboratory Culfosobruchus can be increased or decreased by artificial selection, implying that there is an hereditary factor involved in the production of active form. The result seen in the proportions of active form offspring in the crossing experiments (II and III) confirms that the ability to produce active forms is heritable. Most importantly, the selected line (either active or normal form) could produce more active forms than the unselected line (i.e. stock culture). Normal form individuals from the selected lines produced a higher proportion of active form offspring than did crosses between active forms from stock cultures, and the potential they had to produce active forms was similar to that of active forms of the same line, i.e. selected active forms. It may be hypothesized that the external factors known to produce active forms release an internal genetic potential to develop into this form. Only those individuals who have this genetic potential would develop into active forms under such external stimuli. Those individuals with this internal ability would not emerge as active forms without such external stimuli. In the present experiments, larval population density was presumably the only environmental stress parameter. High larval population density generates a variety of environmental changes including sudden increase of temperature and/or water content in the heap of beans (Sano, 1967; Sano-Fujii, 1984). For the system to stimulate active form production, there must exist individuals which generate the environmental changes and serve as a source of stimulus. These individuals are generally those that develop faster or are oviposited earlier. During the artificial selection for the active form, some normal forms always emerged earlier than the active forms in each generation. This consistent phenomenon observed in every generation makes the above hypothesized mechanism plausible. The difference in emergence time between active and normal forms is, as stated above, also in part due to the slower development of active forms than normal forms, as clearly shown in Fig. 3. The results in Experiment III suggest that the internal ability to be the active form can be increased by selection to such an extent that active form development can take place with no apparent external stimulus. However, by comparing the mean percentages of active form offspring in Table 3 (with 1 egg on each bean) and those with density effects (Table 1), we notice that the latter values are twice as high (24.2% vs 40.0% and 33.0% vs 73.2%). It is evident that external stimulus (larval population density) still plays an important role in the production of active form. It remains to be seen whether a line consisting only of the active form (without any external stimulus) can be established by further artificial selection. However, in the present experiment, the percentage of the active form remained essentially the same (ca. 60%) after the 7th generation in spite of further selection. When the biological properties of the 2 forms in the selected and stock lines are compared, normal forms from the stock line (N) show most typical traits of the normal form, e.g. high fecundity, almost no preoviposition period, low percentage of non-reproductive females, and short adult life. All of these properties appear optimal for populations adapted to life in stored beans. The normal form of the selected line (SN) shows similar tendencies. Active forms of both lines, on the other hand, show low fecundity, long preoviposition period, high percentage of nonreproductive females, long adult life, and low viability (low hatchability in active forms from the stock line, and high larval mortality in active form from the selected line). Many of these properties
macularus population
Days after oviposit ion
Fig. 3. Cumulative percentage of the emergence of normal (solid line) and active (broken line) forms from beans with only 1 egg oviposited by the females of selected line. Left; from a replicate in the Sn9 x SNd cross in Table 3. Right; from a replicate in the SA? x SAg cross in Table 3. The numbers at the end of the lines show the total numbers of adults emerged.
122
Isuzu SANO-FIJJII
are related to the high capacity for flight and are definitely adaptive for migration and life in the field. It was expected that significant differences in the biological properties might appear between the same form in the selected and stock lines. However, the forms and the properties associated with them in stock culture are apparently tightly linked genetically and this association did not break down dramatically during mass selection. Nevertheless, there are some shifts in selected lines. For example, the male and female adult longevities of the active form from the selected lines are significantly longer than the three others. The longevities of the selected normal form are similar to those of stock active forms and different from those of stock normal form (although not significantly so in females). The results of Caswell(l960) and Utida (1970) which showed that the proportion of the active form in their laboratory populations decreased over a number of generations, could be interpreted from Experiment I as being due to an unintentional selection for normal forms under their experimental conditions (perhaps in the method of husbandry). In the stock culture used in this study, similar unintentional selection for the normal form would probably have existed. However, field populations of C. maculatus would probably be able to maintain a higher capacity to produce active forms than the populations used in the laboratory study, because they spend their lives in 2 contrasting habitats. In the field and storage, natural selection respectively works for and against the active form. This dimorphism, therefore, is delicately balanced by the insect’s life style in 2 contrasting habitats. The active/normal dimorphism (or polymorphism) or its equivalent can be observed in many species of insects. In some species, the different morphs occur in individuals of apparently the same genetic constitution. Here, the gene function is “switched” or latent genes are brought into action under the impact of some change in the environment. The phase polymorphism in locust and armyworm and wing polymorphism in aphid and leafhopper are some examples (reviewed by Harrison, 1980). These polymorphisms are thought to be regulated by density effects and/or external factors (e.g. nutrition and photoperiod). However, the present study shows that not all individuals in the population have the same genetic constitution; there can be genetic variations among individuals in the ability to assume a particular form. The dimorphism of C. macufatus is regulated by the combination of the external factors and the genetic variation among the individuals in the population. This example supports the proposition suggested by some researchers (Denno and Grissell, 1979; Lamb and MacKey, 1979; Harrison, 1980) that there may be a genetic component in many of the cases of phase dimorphism. Acknowledgements-1 wish to express my hearty thanks to Professor Syunro Utida for his valuable suggestions and helpful criticism for this work, to Dr Koichi Fujii for his useful criticism and strong encouragement and to Drs Morris Levy, Bruce Champ and the anonymous reviewer for their careful reading of the earlier manuscript and useful comments.
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Genetic basis of the C. maculutus active form
123
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Utida S. (1970) Secular change of percent emergence of the flight form in the population of the southern cowpea weevil, Callosobruchus maculates. Jap. J. appl. Ent. Zool. 14, 71-78.
Utida S. (1972) Density dependent polymorphism in the adult of CaNosobruchtLF muculutus (Coleoptera, Bruchidae). J. stored Prod. Res. 8, 11l-125. Utida S. and Takahashi F. (1958) “Phase” dimorphism observed in the laboratory population of the cowpea weevil, Cullosobruchus quadrimaculufus III. Chemical differences of body constituents between two phases. Jup. J. uppl. Enf. ZOOI. 2, 33-37.