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TJhiversity Mcrafflms International
8603064
Thanthianga, Clement
BIOLOGY OF CALLOSOBRUCHUS MACULATUS
The Ohio State University Ph.D. 1985
University Microfilms International300 N. Zeeb Road, Ann Arbor, Ml 48106
BIOLOGY OF CALLOSOBRUCHUS MACULATUS
DISSERTATION
Presented in Partial Fulfillment of the Requirement for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Clement Thanthianga
The Ohio State University
1985
Dissertation Committee Approved by:
Dr. Rodger Mitchell Dr. Roy A. Stein Dr. Valayamghat Rhagavan Dr. Thomas E. Hetherington AAdvis’or Department of Zoology ACKNOWLEDGEMENTS
I would like to thank my adviser, Dr. R. Mitchell, for his guidance and understanding during my study inspite of my many shortcomings. He gives me every opportunity to make me feel at home not only in this University but in the United States of America. He is not my adviser alone but also my benefactor during my years at the Ohio State University. I extend my deep appreciation to my Dissertation Committee members for their understanding throughout this work. I wish to thank Dr. Roy A. Stein, who first welcomed me as a student in Zoology Department, for his advice and valuable suggestions in this reseach. I also extend my acknowledgement to Drs. V. Raghavan and T. E. Hetherington, who read through the draft copies of the dissertation, giving me constructive solutions to many problems. Lastly, I extend my acknowledgement to Dr. Roy A. Tassava whose set an example as a teacher and to many good friends whose names I will not forget.
ii VITA
January 1, 1954 Born - Sialsuk Village, Mizoram, India.
1970-1974 BSc(Hons), North-Eastern Hills University, Shillong, India.
1974-1976 MSc,, North-Eastern Hills University, Shillong,
India 1 1976-1982 Lecturer, Pachhunga Univ. College, Alzawl, Mizoram, India.
iii TABLE OF CONTENTS
PAGE
ACKNOWLEDEMENTS ...... ii
VITA ...... iii
LIST OF TABLES ...... v
LIST OF FIGURES ...... viii
INTRODUCTION ...... 1
LITERATURE REVIEW ...... 3
Biology of C_. maculatus ...... 25
Materials and methods ...... 25
Adult stage ...... 27 1. Emergence and mating ...... 27 2. Intrinsic determinants of fecundity .. 33 3. Oviposition behavior ...... 55 4. A model for host selection ...... 71
Develpment of passive stages ...... 76 1. Development of single larvae in Berkin 76 2. Effects of food on development ...... 80 3. Experimental analysis of competition . 96
The consequences of oviposition decisions .... Ill
SUMMARY ...... 115
LITERATURE CITED ...... 118
iv LIST OF TABLES
TABLE PAGE 1. Influence of mating regimen on fecundity females (n=10) given 50 beans for oviposition...... 35
2. The effect of length of development from egg to adult and competition within the bean on fecundity. The means for each set were tested (t-test) against the early emerging females...... 37
3. Effect of feeding on the fecundity of pairs placed on 50 berkin beans...... 39
4. Pattern of oviposition based on counts from five females taken at each time interval from a cohort of isolated pairs on 50 beans. Negative values can be obtained because the eggs deposited during an interval were estimated by subtracting the average at the start of the period from average at the end of the period...... 43
5. The average (s.d.) fecundity for females (n=10) given different number of beans for oviposition...... 51
6. The eggs deposited by females given 0, 1, and 10 beans and then tansferred to a dish with 50 beans for the rest of her life. The sum is the total eggs laid before and after transfer. Averages (n=10) were compared (t-test) to controls on 50 beans (mean 77.4, s.d. 11.1, n=5)...... 52
v 7. The density of eggs per bean at which the females first begin to add a second egg to beans. The transferred females were placed on 50 beans after being inhibited by having too few oviposition sites (see Fig 8). There were no differences between the means (t-tests)...... 59
8. Outcomes of experiments to determine the discrimination of two sizes of Berkin beans. There are 10 replicates for each experiment. 68 9. Selection of oviposition sites by beetles given choices between 15 rough (chickpea) and 15 smooth (soybean) beans for 24 h. There were no differences (t-test) between the means for 10 replicates...... 69
10. Selection of oviposition sites by beetles (n=10) given choices between Berkin beans and the alternate hosts for 30 h ...... 70
11. Development in days and weight (mg) of C_. maculatus feeding alone in beans. Larvae were weighed (n=10) at the end of the molt and pupae were weighed when first formed... 79
12. Composition of the major hosts of C_. maculatus from Duke (1981). Components that show major differences are given... 83
13. Life tables for C_. maculatus on its principle hosts. With Berkin as controls, confidence limit for percentages were used to test for differences in survival and t-tests for differences in fecundities. High egg mortality was due to high temperatures, up to 30 C, shortly after oviposition... 84
14. Frequencies of various interactions between competing larvae when larvae first come into contact...... 93
15. Average (s.d.) weight (mg) of 20 larvae alone and 10 pairs of competing larvae of C_. maculatus segregated by the position of their burrow...... 94
vi 16. Average (s.d.) weight (mg) of larvae growing singly and in pairs in chickpea and pigeon pea (n=20)...... 95
17. Weights of larvae in beans glued together to simulate conditions of competition within. a single bean. Controls were beans with 1 larva ground and the surface glued...... -98
18. Experiments to alter the transmission of cues between two larvae of C_. maculatus from bean to bean. The experiments were set up while the eggs were fresh and the larvae weighed when they were estimated to be 14 days old...... 108
19. Weights (s.d.) of larvae in beans glued together after larvae had started to feed alone. Weights were taken 12 days after hatching for 20 larvae in beans glued in the first instar and at 13 days for larvae joined in the second instar...... 109
20. Tests to determine the reversibility of the competitive behavior...... 110
vii \ LIST OF FIGURES
FIGURE PAGE
1. Basic life cycle of C_. maculatus with the active non-feeding stage (adult) separated from the passive and feeding stages (egg, larva, and pupa) by the double line. As shown in Fig. 3, passive stages must live in the single bean selected by their mother...... 5
2. Female C_. maculatus ovipositing on a bean (X 20)...... 8
3. Development of C_. maculatus in a bean of the Berkin variety. Larvae are shown at the end of the first (A), second (B) and third (C) larval instars and the pupal stage (D) to show the burrowing pattern of a larva alone in a bean (X 20)...... 19
4. The basic life cycle (Fig. 1) expanded to indicate the interactions proposed or inferred from published reports...... 23
5. The first three days of emergences from an even aged cohort of 500 C_. maculatus. Black horizontal bars indicate dark periods..... 29
6. The daily pattern of emergences (points) and the frequency of mating pairs (bars) found at each 2-h sampling period. The records for the first three days are pooled. Black horizontal dars indicate dark periods..... 32
7. Diel pattern of oviposition by C_. maculatus based on 10 replicates taken every 12 h from a cohort of isolated pairs...... 42
viii 8. Eggs deposited by pairs of C_. maculatus given limited number of oviposition sites and a set of 10 transferred to 50 beans each day. Pairs with no bean (A), 1 bean (B), and 10 beans (C). The outcomes were tested against controls on 50 beans...... 48
9. Daily pattern of oviposition estimated from 10 replicates taken each day from cohorts of C_. maculatus given 0 (A), 1 (B), 10 (C), and 50 (D) beans for oviposition...... 50
10. Discrimination of large size beans by C_. maculatus given 25 large and 25 small Berkin beans during the first 3 days of life. The outcomes expected under a simple encounter model (dashed line) and oviposition based on single comparision (solid line) are indicated...... 64
11. Model for the selection of beans for oviposition. The responses were based on the choices made by females under experimental conditions (see Tables 8-10)...... 73
12. The differential development of two identically aged third instar larvae of C_. maculatus growing in Berkin bean (X 20). 87
13. Experiments to alter the transmission of cues between two larvae in beans glued together. Beans were glued together while the eggs were fresh and the larvae were weighed when they were 14 days old...... 101
14. Experiments to alter the responses of larvae to competition: (A). Beans with one 5 day old larva growing alone glued together and weighed 8 days later. (B). Beans with one egg were glued together. When the larvae were 5 days old the beans were separated and glued to a bean with an 8 day old larva and weighed after 6 days. (C). Competing third instar larvae were transferred to a new bean and held for emergence...... 105
ix INTRODUCTION
Callosobruchus maculatus (Coleoptera: Family Bruchidae) is thought to have evolved in Africa (Decelle 1981) possibly as a pest of cowpeas (Vigna unguiculata) or pigeon peas (Cajanus calan). Now the most widespread and destructive species of Callosobruchus. it commonly attacks dried cowpeas, mung beans (Vigna radiata). and chickpeas (Cicer arletinum). The harvest of these four beans in developing countries totals 39.3 million tons that contain about 24% protein (Duke 1981). This 9.4 million tons of vegetable protein is nearly equivalent to the 9.5 million tons of animal protein reported for those countries (FAO 1983). Because of the immense economic impact of C_. maculatus and the great ease with which it can be maintained in the laboratory, much has been published on its life history, ecology, and physiology. In addition, it has been used in widely cited studies of competition. Utida (1953) began studies of interspecific competition between C_. maculatus and C.chlnensls. Recent studies of intraspecific and interspecific competition using C_. maculatus are the basis for developing theory on which new models for competition and clutch size are based (Bellows 1982a, 1982b, Bellows and Hassell 1984, Charnov and Skinner 1984, Smith and Lessells 1984).
1 2
Janzen (1975) and Janzen et a l . (1977) used C_. maculatus to assay for the effects of secondary compounds of legumes suspected to be co-evolved chemical defenses. The variety of responses to food qualities of the beans which range from death to alterations in behavior, indicates an unexpected complexity in defensive mechanisms. Despite the wide interest of applied biologists and ecologists in this insect, most aspects of the basic biology remain open to dispute because of inconsistences in the many isolated accounts. My aim is to resolve these inconsistencies in a comprehensive monograph of the basic biology that can be also used to test the inferences of theoretical ecologists about the mechanisms of competition and evaluate the purported effects of co-evolved traits. Literature Review with Special Reference to Callosobruchus maculatus
More than 80* of the species of the family Bruchidae attack seeds of plants in the family Leguminosae (Johnson 1970, Johnson and Kingsolver 1971). Many species of bruchids are host specific, though a host may be attacked by more than one species of bruchid. The host preferences of certain taxa are rigid enough to be cited as taxonomic characters (Johnson 1980). This co-evolved association of bruchids with the Leguminosae involves the evolution of both behavioral mechanisms and biochemical tolerances to the secondary compounds contained in the seeds by the beetles and defensive traits by the plant (Applebaum 1964, Janzen 1977a, 1977b, Southgate 1979, Johnson 1981a, 1981b). This coevolution is particularly tight because the larvae do not choose their food. Bruchids pass through the characteristic holometabolous life-cycle (Fig 1). Most beetle larvae select food from the area near where they hatch, but many bruchid larvae are sedentary larvae that cannot move from the seed or pod selected by their mother. Bruchids pass through three or four larval instars (four instars in the case of C_. maculatus) , followed by the pupa and the adult stage. Adults feed far less than the larvae and are the only stage able to disperse. Feeding functions
3 Figure 1. Basic life cycle of C_. maculatus with the active non-feeding stage (adult) separated from the passive and feeding stages (egg, larva, and pupa) by the double line. As shown In Fig 3., passive stages must live in the bean selected by their mother. 5
II
ADULT
reserves
PUPA protein fat carbohydrate
survival egg reserves (potential fecundity) larval competition growth
realized fecundity digestive physiology
LARVA
EGG
survival 6
are completed by the larvae of many bruchids so that the adults emerge from the beans with all the food reserves needed for mating, dispersal, and egg production. The life cycle (Fig. 1) can be quite naturally divided into two phases: the active phase, the adult (Fig. 2), and the stages from the egg through the pupa that are either quiescent or feeding stages (Fig. 1). Nearly all research deals with just one of these phases and these separations are used to organise these studies. Figure 2. Female C_. maculatus ovipositing on a bean (X 20). 8
III
*•. •* v’ -‘f' ' ’. 9
The Active Non-feeding Phase
Many bruchids can be maintained in laboratory cultures if given only beans for the larvae to feed upon. The adults do feed on nectar and pollen in nature and when given water and sugar in the laboratory, they feed, lay more eggs, and live longer (Larson and Fisher 1924). Adults of some species are inactive for the several months intervening between the seasons of pod production (Larson and Hinman 1931, Brindley 1933, Parnell 1966, Forister and Johnson 1971, Southgate 1979, 1981, Biemont and Bonet 1981, Hodek, et a l . 1981,). Many of the species that undergo diapause, or are inactive between seasons, are those that oviposit on pods (Whitehead 1930, Larson et al. 1933, Hinman et a l . 1949, Pajni 1981). Bruchids that oviposit on seeds are often opportunistic species passing from generation to generation without an inactive phase (Johnson and Slobodchikoff 1979, Johnson 1981c, 1981d). The species of Callosobruchus are the best known examples of bruchids without a resting phase (Utida 1981). A. Mating. On emergence, the sexually mature females display a calling behavior while they produce a pheromone that excites males (Qi and Burkholder 1982). Females are usually mated within minutes of emergence (Howe and Currie 1964, Booker 1967, Raina 1970, Utida 1972, Mitchell 1975, Utida 1981). Copulation usually lasts about 10
3-8 minutes (Raina 1970, Qi and Burkholder 1982) and one mating may be sufficient (Brauer 1944), although females will mate more than once. Unmated females do not lay eggs (Ouedraogo and Huignard 1981). B. Dispersal of Adults. Casual reports of dispersal (Larson et a l . 1933, Jarry 1981, Pajni 1981, Wright 1983) provide little information about the behavioral basis for dispersal. There are no experiments or observations of Callosobruchus dispersal in nature or in large storage facilities infested with C_. maculatus. Some populations of C_. maculatus have dimorphic adults. The larger flightless form is usually the most abundant morph while the occurrence of the small morph, called the flight, active, or dispersal morph, is generally episodic (Utida 1954, Caswell 1960, Sano 1967, Taylor 1974, Nwanze and Horber 1975a). Although the heritability of the dimorphism has not been determined, it probably has a genetic basis because the small morph is missing in some laboratory cultures. The morphology, physiology, and behavior of the two morphs have been studied (Utida 1954, 1972, Sano 1967, Taylor 1974). The small morph, which Taylor and Agbaje (1974) regard as the field form, have larger fat reserves and live longer, but they lay fewer eggs than the flightless form (Utida 1972, Taylor and Aludo 1974, Nwanze et al. 1976, Ouedraogo and Huignard 1981). The abdominal cavity of both sexes of the dispersal form is filled by the fat body at emergence, whereas mature reproductive organs occupy the abdominal cavity of the large morph (Utida 1972). The two morphs are alternative patterns for allocating larval resources in the differentiation of the adult features (Utida 1981). The cost of developing 11 dispersal capability can be measured as a sacrifice of fecundity, because the flightless form diverts energy from the growth of the flight muscles and fat reserves into a larger reproductive system and eggs. This argument is based on the assumption that the adults differentiate from similar-sized larvae. The fecundity of the large morph, 62.6 eggs per female, is reduced by 42 per cent, to 36.4 eggs, in the small morph (Utida 1972). Utida (1981) postulates that only the dispersal morph can move from area to area. Uncrowded local populations will produce mostly large morphs of high fecundity, but if crowding develops so that nearly all beans are exploited, then, more small morphs will be produced and these will disperse widely (Utida 1972, Messina and Renwick 1985a). Small morphs seem to appear in a population of Callosobruchus as a response to unfavorable local conditions. There are no reports of dispersal by the large morphs in nature though both morphs are found in bean fields (Taylor 1981). C. Fecundity. Total egg production has been quantified for a few bruchids cultured in the laboratory but the daily pattern of oviposition has not been determined. When given an ample supply of beans, but no food or water, C. maculatus lavs 70 to 110 eggs (Wade 1919, Larson and Simmons 1923, Booker 1967, Raina 1970, Mitchell 1975, Nwanze and Horber 1975b, Singh et a l . 1978). Reports of fecundities over 100 eggs result from feeding sugar solutions (Larson and Fisher 1924). All Instances of fecundities below 50 seem to be from females given very few beans for oviposition or under crowded conditions (Alzouma 1981, Wasserman and Futuyma 1981, Bellows 1982b). Realized fecundity is reduced at extremes of temperature, though measurable changes in fecundity occur over the range of 20 to 30° C. Fecundity is quite constant at humidities of 20 to 80% relative humidity, but fecundity 12
rises by 10 to 20% above 809s relative humidity, indicating that the metabolic cost of producing and retaining water must be a substantial part of the activity costs incurred by the adult (Schoof 1941, Howe and Curry 1964). The quality of larval food produces almost two-fold differences in fecundity (Mitchell 1983), presumably by altering reserves that are built up during the larval stage. Maximum egg production is limited by the reserves of critical nutrients built up by the larva with protein reserves likely to be the limiting component. If the energy needed for female activity is obtained from optional feeding on nectar, then all potential resources for eggs can be used in egg production. The potential fecundity can be taken as the highest fecundity obtained by feeding the adult female under the most favorable conditions. The determinants of realized fecundity can be specified as a function of the equivalents of the following components:
(1) Realized fecundity = potential fecundity - (activity - feeding)
The major adult food, nectar, is carbohydrate that can be used to support activity or synthesize fat. If carbohydrate obtained by the female supports her activity, the net gain of carbohydrate will equal the cost of activity, making the second term zero. Realized fecundity will then equal the potential fecundity. Carbohydrate might be used in egg synthesis if there is a surplus of protein, but even if that occurs, protein would still be the ultimate limiting factor. This equation defines concepts the ecologist can use to obtain answers in egg equivalents, but physiological studies with labeled compounds would be needed to obtain caloric values for the equation. 13
D. Ovipositlon Behavior. Nwanze and Horber (1976) suggest that eggs are deposited in batches separated by days of low egg deposition. Others report a single period of egg deposition during the first 2 to 4 days followed by a rapid decline in egg deposition (Brauer 1944, Utida 1972, Nwanze and Horber 1975b, Bellows 1982b). These conflicting views are based on circumstantial evidence and from cultures of females disturbed each day for counting of eggs. The decision of a female C_. maculatus to oviposit is influenced by a variety of cues. Five cues are purported to modify the ovipositlon behavior. Presence of Eggs. Ovipositing C_. maculatus, like many phytophagous Insects, rejects hosts that already bear eggs. Oshima et al. (1973) claim to have isolated a pheromone that inhibits ovipositlon. However, it is now established that females respond to a variety of features of eggs rather than a specific pheromone (Messina and Renwick 1985b). By avoiding beans with eggs on them, females tend to distribute their eggs uniformly over the available beans (Avidov et al. 1965a, Mitchell 1975, Wasserman and Futuyma 1981). Females add a second egg to a bean only after nearly all the available beans have at least one egg on them and this reduces the incidence of larval competition (Avidov, et al. 1965b, Mitchell 1983). Number of Sites. If females reject beans with eggs (Messina and Renwick 1985b), then it seems likely that they might be inhibited from ovipositlon when there are few ovipositlon sites as reported by Bellows (1982b). 14
Bean size. When given a choice, C_. maculatus females select large beans for oviposition (Howe and Currie 1964, Booker 1967, Mitchell 1975, Nwanze and Horber 1975, 1976, Mark 1982). Avidov et al. (1965a) also reported that a related species, C_. chinensls. can distinguish beans by size and seem to use cues related to surface curvature. Reports are confusing because beans with differing surface textures were used to test for size discrimination leaving open the question of whether the ovipositing females were responding to size alone. Surface Texture. Callosobruchus prefers to oviposit on smooth-surfaced beans rather than on rough-surfaced ones (Larson 1927, Howe and Currie 1964, Booker 1967, Raina 1971, Reddy and Singh 1972, Nwanze et al. 1975, Wasserman and Futuyma 1981, Messina 1984). The Increment in egg mortality occurring on rough-surfaced beans (Nwanze and Horber 1976) is thought to be due to poor egg attachment. Eggs must be firmly attached to the bean surface so that the first instar larva can use the egg shell for leverage as it forces its mandibles against the bean surface. Roughness could be a defense evolved by the host, although the preference for smooth beans may be an adaptation of the beetle for rejecting cracked, chipped, and broken beans in which larvae may become exposed as they burrow to dessication or parasites. Cues identifying broken beans may incidentally detect natural roughness and the rejection could be a single response to any surface irregularity. Species of Bean. Bean type affects the developmental time and the fecundity of C_. maculatus (Howe and Currie 1964, Raina 1970, Mark 1981, Mitchell 1983). Apart from size and surface features, various species of beans do not seem to differ in their attractiveness to ovipositing females. Larson (1927) reported that caged females do not discriminate among different hosts on the basis of the 15 host's suitability for larval survival and development. The cues controlling the ovipositlon of C_. maculatus are associated with properties of the bean that do affect the chances of larval survival. Selective ovipositlon increases the chances of eggs hatching and dispersion of eggs reduces the incidence of larval competition. While the ovipositlon responses and the effect of bean traits on survival have been measured for various cues independently, the relative importance of one cue over another, their interactions with each other, and the associated survival functions have never been considered in a coordinated study. 16
Passive and Feeding Stages.
A female placing an egg on a host has responded to the size, surface texture, presence of eggs and, possibly, quality of bean her offspring must use. The subsequent larval and pupal stages cannot leave that resource item (Fig. 3). Individual larvae are, therefore, strictly monophagous throughout their life. Each stage from the egg through the pupa has its prospects determined by the placement of the egg. Egg. The egg, a smooth, somewhat ovoid and ventrally flattened structure, is protected by a tough and elastic chorion. Some species attach the egg to the host with an adhesive secretion. Other species cover the eggs with a layer of a glue-like material. C_. maculatus eggs, about 0.71 mm long and slightly milky in color, are deposited singly and covered with a glue-like secretion extending over the adjacent bean (Raina 1970). The larvae hatch in 4-7 days depending on the temperature (Brauer 1925, Mukerji 1939, Howe and Currie 1964, Osuji 1982). Larva. Bruchids have three or four larval instars and species of Callosobruchus have four instars (Raina 1970). Some bruchid larvae crawl on the pod or Inside the pod before entering the bean. The larvae of bruchids that glue their eggs to beans, such as C_. maculatus, generally 17
cut through the floor of the egg and through the testa of the bean to enter the bean cotyledons on which they feed. The first instar larva has functional legs and an extensive coat of long setae (Johnson 1981a). As mentioned above, the ability of the larva to enter a bean appears to depend on how well the egg is attached to the bean (Nwanze and Horber 1976). Once in the burrow, the spines are probably used to anchor the larva in the burrow as it chews its way into the bean (Howe and Currie 1964). The second instar is usually leg-less and unable to do anything other than feed on the walls of its burrow.x A few species have mobile larvae able to move between the seeds in a pod as they feed (Bridwell 1918, Parnell 1966, Johnson, 1967, Johnson and Kingsolver 1971, Center and Johnson 1973, Center and Johnson 1974, Johnson 1974, Schoonhoven 1976, Southgate 1979). C_. maculatus, however, is a species that is helpless outside of its feeding cavity. A spherical chamber is created as a larva feeds on the chamber walls (Fig. 3) and granular white feces accumulate in the feeding chamber behind the larva. Extremes of temperature and humidity reduce survival but survival is maximal and varies little over temperatures of 20 to 30° C and humidities above 30$ relative humidity (Schoof 1941, Howe and Currie 1964). During the last day or two of the fourth instar, the differentiating head, thorax, and abdomen of the pupa becomes apparent under the larval skin and this stage is called the prepupa (Raina 1970, Osuji 1982). The prepupa chews a circular passage to the testa and clears a thin, circular, translucent window under the testa. For pupation, the prepupa settles with its head facing the window (Howe and Currie 1964, Raina 1970) and the emerging adult pushes open the window to exit. Figure 3. Development of C_. maculatus in a bean of the Berkin variety. Larvae are shown at the end of the first (A), second (B) and third (C) larval Instars and the pupal stage (D) to show the burrowing pattern of a larva alone in a bean (X 20). 19
CO 20
Survival of Callosobruchus larvae has been used as an assay for defensive compounds (Podler and Applebaum 1971, Janzen 1975, Janzen et al. 1977). Certain compounds kill all larvae, while others affect particular stages, for example, the preparation of an exit hole by the fourth instar larva is disrupted by certain foods (Howe and Currie 1964, Applebaum et a l ■ 1968, Podoler and Applebaum 1968, Janzen 1977b, Mitchell 1983). Some species of bruchid will have only one larva mature per bean while multiple emergences from infestations a bean are common for other bruchid species (Pinckney 1937, Janzen 1975, Johnson 1978). When a single exit is the rule, the first larva to enter the bean is thought to seek out and destroy other larvae. The outcome of larval competition by C. maculatus varies and there is, as yet, no % explanation for multiple exits being the rule in some hosts while single exits are the rule in other Jiosts (Janzen 1975, 1977a, Johnson 1967, 1978, Bellows 1982a, 1982b, Osuji 1982). When there is a single emerging adult, it is assumed from casual observations that a dominant larva excludes later invaders (Janzen 1977a). Despite the extensive studies of competing populations, which have even included the development of models for evolutionarily stable systems, no one has tested the assumptions or predictions of the models by opening beans to determine the fates of larvae within a bean. Summary
The wealth of Information on the biology of C_. maculatus is difficult to fit together because of inconsistent outcomes and differences in experimental procedures. The general problems that deserve attention can be defined in terms of the known interactions among different stages. These are added to the outline of the life history (see Fig. 1) to Figure 4 to show all the interactions inferred from the studies reviewed here. This set of relations has never been examined under consistent experimental conditions. Three aspects of C_. maculatus biology require examination: 1) Measures of realized fecundity vary widely. Certainly both feeding and activity can alter the number of eggs deposited. The concept of potential fecundity developed above, serves as the basis for measuring the way activity, egg reserves, and optional feeding by the ovipositing female can alter the number of eggs actually deposited (Fig. 4). Uncontrolled experiments indicating that crowding and availability of oviposition sites reduce realized fecundity need to be verified experimentally. 2) Each of the cues affecting oviposition behavior has been studied individually, using different stocks of beetles under a variety of experimental procedures. The suite of cues needs to be examined under consistent Figure 4. The basic life cycle (Fig. 1) expanded to indicate the interactions proposed or inferred from published reports. 23
optional ADULT feeding rese rv e s I PUPA protein fat carbohydrate / egg reserves/ survival 1 (potential fecundity) activity larval / competition 4 growth mating dispersion realized I fecundity oviposition cues digestive physiology 1. no. of sites 2. bean size 3. egg dispersion (quantity, quality 4. bean species LARVA of food) 5. surface texture
survival 24
conditions to determine how they affect the oviposition behavior of the female and whether the bean traits favored by females affect the potential survival of the feeding stages. 3) The bean is a black box. Anecdotal accounts from unsystematic observations of larvae in beans provide little information about the feeding stages. Thus, the growth, development, and interactions of competing larvae need to be described. I, therefore, present a comprehensive account of these three aspects of the biology of C_. maculatus. These problems are explored under a consistent set of conditions. With these data as a reference, I measure the effects of different foods on the biology of C_. maculatus. using the major hosts of this beetle. The Biology of C_. maculatus
If a comprehensive account is to be a useful reference for comparative studies, it must be verifiable. Thus, a defined stock of beetles and host is required. Mung beans were selected as the host because the genetically defined variety Berkin is readily available. The beetles themselves are a lineage that was originally found on mung beans. The stock of beetles and host association has been continued in the laboratory to avoid the possibility of dealing with concurrent genetic evolution under altered conditions or artifacts due to growing the beetles on an unfamiliar host.
Materials and Methods
The cultures and experiments were maintained in the laboratory at 22-24 C using the Berkin variety of mung beans obtained from the Johnson Seed Co., Enid, Oklahoma. Berkin is grown from foundation stock maintained as a genetically uniform line by the Oklahoma Foundation Seed Stocks, Inc. (Matlock and Oswalt, 1963). The C_. maculatus culture was established in 1979 from naturally infested mung beans obtained from near Tirunelveli in south India. The beetles were presumably adapted to, mung beans in the field and have been maintained on mung beans since their isolation about 30 generations before these experiments
25 26
were started. Stock cultures were maintained In ventilated plastic boxes, 31 x 17 cm. Each generation a large number of fresh beans were put into a clean box and newly hatched beetles were allowed to oviposit for no more than 24 h. Beetles were usually removed before beans began to receive a second egg. Thus, stock cultures produced adults from beans carrying single uniformly aged eggs. The lineage was monomorphic for the flightless morph. Unless otherwise noted, virgin females, less than 4 h old, isolated as they emerged from beans or obtained from isolated beans, were used. The standard experiment used a pair of virgin beetles placed with 50 beans in a petri dish, 8.5 cm in diameter and left undisturbed until they were dead or moribund, by day 9 or 10.
t Adult Stage
The success in the adult stage depends on the females being mated promptly and on her ability to locate oviposition sites. Having located potential oviposition sites, the female determines the fate of her eggs as she chooses whether to place her eggs on that host or not.
1. Emergence and Mating
Emergence and incidence of mating were recorded for a set of 500 beans carrying one egg every 2 h beginning before beetles were expected to emerge. Twelve hours after observations began, the first emergences occurred and observation were continued for 72 h. The number of mating pairs was recorded at the time of sampling and all emerged beetles were removed at that time. The frequency of inseminated females was determined by isolating each female in a vial with about 20 fresh beans. Only inseminated females lay eggs. The first emergences occurred between 0600 and 0800 h and about 65% of all emergences occurred between 0800 and 1200 h. Only 1% of beetles emerged between 1800 and 0600 h the next day (Fig. 5). There were no sex differences in emergence time.
27 Figure 5. The first three days of adult emergence from an even aged cohort of 500 C_. maculatus. Black bars indicate dark periods. Number Emerged 30 40 50 20 10 ■ ■ - I I 16
20 4
1 i i i i i i 8
a 2 Day 12
16 i
20 4
o o Females of No. □ 8
o o Males of No. a 3 Day 12
16
20 Mating lasts for only a few minutes and 26 mating pairs were observed at the times of sampling. Most of the matings (92 per cent) were recorded between 0800 and 1200 h and no pairs were found before 0800 h or after 1400 h (Fig. 6). Seventy eight per cent of the isolated females (n=170) deposited batches of viable eggs; hence, they must have emerged and mated during the 2-h sampling period. Frequency of mating was likely to be underestimated because the samples included 26 females whose mating was disrupted at the time of sampling. The beetles began to emerge between 0600 and 0800 and emergences declined throughout the afternoon with very few night emergences. Mating occurred shortly after emergence with 80* of the females mated within 2 h of emergence. Figure 6. The daily pattern of emergences (points) and the frequency of mating pairs (bars) found at each 2 h sampling period. The records for the first 3 days are pooled. Black bars indicate dark periods. mating emergence 8 12 Time of Day 33 2. Intrinsic Determinants of Fecundity A. Efficiency of mating. While females are known to mate soon after emergence and rarely mate thereafter (Qi and Burkholder 1982), neither the adequacy of a single mating nor the effect of the presence of a male has been determined. Four treatments tested the effect of mating and male presence on female fecundity. i. Virgin females kept alone. ii. Virgins paired together for 6 h and observed to copulate. Females were kept alone. iii. Virgins pairs left together. iv. Virgin females less than 2 h old and 3 day old males that had mated two or three times with other females left together. Unmated females laid no eggs (Table 1) while all mated females laid at least a few eggs. Females with males in the culture dish laid at least 65 eggs, average 80.0 eggs. Age of male had no effect on fecundity (t18=l.354, P=< 0.20). Females isolated after mating deposited 2-99 eggs. One female laid 3 infertile eggs, another laid 2 eggs that developed, and the remaining eight laid from 39 to 99 eggs, average 77.0 eggs. Some sort of a insemination threshold seems to exist because females either lay a full clutch or very few eggs. Possibly females laying only 2 or 3 eggs received very little sperm, while all the other females received a full supply of sperm. When the females laying 34 two or three eggs were excluded, the fecundity of isolated females did not differ from the control (t16=0.672, P>0.5). A single mating was enough for a female to produce a full set of eggs in most cases. The lower fecundity and greater variability among females isolated after mating was due to occasional females producing an incomplete clutch. Fecundity must be measured from pairs kept together to avoid the bias from females that are inadequately mated. Table 1. Influence of mating regimen on fecundity of females (n=10) given 50 beans for oviposition. Experiments N of females Average laying (s.d.) Range < 10 eggs Unmated 0.0 0 10 Mated and isolated 62.1 (35.8) 2-99 2 Virgin pairs 80.0 (9.3) 65-92 0 Mated with old males 85.2 (7.7) 72-95 0 36 B. Conditions of larval growth. Development of the adult could vary in two ways. Development from the egg to adult required 23 to 38 days and adults developed from larvae that had either fed alone in a bean or else had competed with other larvae. If competition occurred, only one adult emerged from a bean, usually a day or two after adults from larvae that did not compete. To investigate these factors, females were isolated and paired as they emerged from beans with one, two, or three larvae in them. Females that emerged earliest laid an average of 73.2 eggs and females with the longest developmental time laid significantly more eggs, 93.8 (Table 2). Females from competing larvae had consistently higher averages but they did not differ from the females from single larvae (ti7=0.745, P>0.05). A second replicate for variation in fecundity with day of emergence did not show a clear pattern when pooled for comparisons with the first set of experiments. Although fecundity tended to increase with time of development and the extent of competition, the increase was quite small. 37 Table 2. The effect of length of development from egg to adult and competition within the bean on fecundity. The means for each set were tested (t-test) against the early emerging females. ______Days of Development 31-33 35-39 41-46 One larva (Rep. 1) 73.2 92.4* 93.8* s.d. 9.9 14.3 12.8 n 9 10 10 Two larvae 78.0 92.4* 96.4* < s.d. 16.9 17.6 9.3 n 10 10 7 Three larvae 97.0 99.4 s.d. 10.2 9.3 n 9 20 One larva (Rep.2) 83.0 86.6 80.6 s.d. 18.3 13.2 12.1 n 20 30 16 * PC0.05 38 C. Feeding by the female. These experiments follow those of Larson and Fisher (1924) with one exception. Fed females also were given a separate container of water. It seemed possible that beetles living solely on metabolic water might require additional water to absorb nutrients from concentrated sugar solutions. Virgin pairs were placed in dishes with 100 beans and given nothing (control), water alone, sugar water plus water, and honey plus water. The sugar solution was 40$ sucrose by weight and the honey, which was principally d-fructose and d-glucose, was diluted to 40$ sugar by weight. Fluids were put in a small glass tube with a cotton wick and replaced every 2 days to keep them fresh. Four females given honey and three females given sugar water drowned and were rejected from the analysis. The average fecundity of control females, 69.2 eggs, did not differ from those females given water alone (Table 3). Females given sugar water and honey had higher fecundities and the fecundity of females given honey was significantly higher (t31=2.047, P< .05) than those with sugar. All eggs were about 95$ viable. Controls and females getting water lived about 9 days, while one fed females lived 47 days. The longevity of the two sets of fed females was similar. Females fed carbohydrates lived longer and laid as many eggs as reported by Larson and Fisher (1924). The maximum fecundity, attained with honey, was a 45$ increase over the controls. Hence, it appeared that the unfed females use, on the average, 55$ of their potential egg reserves for activity when they did not have food or water. 39 Table 3. Effect of feeding on the fecundity of pairs placed on 50 Berkin beans. Controls Water Water Water and and sugar honey n 20 20 17 16 * * * * Mean fecundity 69.2 75.8 102.1 125.0 s.d 19.9 33.7 29.6 34.6 * * * * Longevity (days) 9.0 9.3 16.7 22.5 s.d 1.6 3.5 8.9 12.3 Egg viability 0.96 0.95 0.97 0.95 Male frequency 0.53 0.49 0.52 0.53 ** P < 0 .01 t-test 40 D. Oviposition rate. The initial oviposition peak obtained by Bellows (1982b) when he transferred females to new beans every day, differs from the reports of a sequence of peaks (Booker 1967, Nwanze and Horber 1975a). To avoid the effects of disturbance, a large number of dishes were set up simultaneously and egg production was counted from a set of females removed at each interval. A set was used to determine the diel periodicity of oviposition from samples taken every 12 h and two replicates, one with pairs and one of females isolated after mating. These treatments were sampled every 24 h. The oviposition during an interval was estimated by subtracting the average number for samples at the start of the interval from the average for the samples taken at the end of the interval. Females laid an average of 8.5 eggs per day but each experiment revealed three cycles of egg deposition (Table 4, Fig. 7). Samples taken at the end of a day were not significantly different from the samples taken at the start of the next day; hence, very few, if any, eggs were deposited at night. Oviposition in C_. maculatus was not a continuous process. Three batches of eggs were produced with a lapse of a day or two before another set was produced. The three cycles of egg production are unaffected by the absence of males and represent an endogenous cycle of egg production. The single peak reported by Bellows (1982b) may be due to either genetic differences in stocks or the result of changing females from dish to dish each day. Figure 7. Diel pattern of oviposition by C_. maculatus based on 10 replicates taken every 12 h from a cohort of isolated pairs. Estimated eggs/day 25 i 0 2 10 ■ - Days Eggs deposited* deposited* Eggs Eggs deposited deposited Eggs t night. at in the day. the in 10 42 43 Table 4. Pattern of oviposition based on counts from five females taken at each time interval from a cohort of isolated pairs on 50 beans. Negative values can be obtained because the eggs deposited during an interval were estimated by subtracting the average at the start of the period from average at the end of the period. ______Number of eggs deposited______Pairs______Isolated Set 1______Set 2 Set 3 day Day Night 24 hr 1 6.0 0.4 6.4 15.4 12.2 2 11.8 0.0 11.8 12.4 20.5 3 12.2 6.6 18.8 9.6 -2.5 4 11.8 6.2 18.0 0.8 21.0 5 6.4 -2.6 3.8 17.4 2.6 6 17.2 5.8 23.0 7.4 7.2 7 3.2 -5.0 -1.8 -6.2 15.8 8 4.0 -1.6 2.4 14.6 7.5 9 11.0 1.0 12.0 8.1 -7.0 10 3.6 -6.4 -2.8 -2.1 3.5 Sum 91.6 77.4 80.2 s.d 6.3 11.1 4.2 44 E. Presence of oviposition sites. Fecundities of less than 50 seem to be obtained from crowded conditions (Alzouma 1981, Wasserman and Futuyma 1981, Bellows 1982b). Thus, beetles that prefer to disperse their eggs (Avidpv et al. 1965b, Mitchell 1975) should be inhibited by a shortage of oviposition sites. The effect of reducing the number of beans available for oviposition was determined for pairs placed in dishes with 0, 1, 10, 25, 50, and 100 beans. The number of eggs laid by females with 50 and 100 beans, 92 eggs and 82 respectively, was similar (Table 5). Only 3 of the 50 females laid an egg or two on the surface of the dish rather than on beans. Some females with 25 beans had fecundities as high as the females with 50 to 100 beans, but the average, 68.1 eggs, was lower than females with 50 or more beans (t18=3.23, P<0.01) and 0.6% of the eggs were deposited on dishes. The inhibition of oviposition was apparent and Increased as fewer than 25 beans were available. With fewer beans the number of eggs placed on dishes increased. If the number of beans available to a female was about equal to or greater than half her realized fecundity, there was no apparent inhibition of oviposition . With a number of beans less than a quarter of her realized fecundity, a female laid fewer eggs and the inhibition Increased as the number of available beans was reduced. Is the Inhibition of oviposition reversible? Pairs were put in a dish with 0, 1,10 or 50 (control) beans. Every day 10 pairs of beetles were transferred from each of the experimental treatments to a dish with 50 fresh beans to determine how many eggs a female would deposit after transfer to control conditions (Table 6). The last transfer was on day 10; all the females were dead or moribund by the end of day 11. 45 Females with 10 beans were inhibited even on the first day, but they recovered full fecundity if transferred to 50 beans by day 9 (Fig. 8C). The total fecundity of females with one bean was reduced slightly by day 5 and irreversibly reduced by day 8 (Fig. 8B). Females with no beans suffered an irreversible inhibition after only 2 days of inhibition (Fig. 8A). Only one of the 2,398 eggs in controls was deposited on the dish. Females with 10 beans deposited 2% of their eggs on the dish but none of these were deposited until after day 5. After 3 days those with one bean began to deposit eggs on the dish and these represented 10.135 of the eggs. When egg sites were limited, beetles withheld eggs for the first 7 days. At 7 days females have about 3 days of life remaining and the oviposition rate and deposition of eggs on inappropriate flat surfaces increased sharply. Inhibitions affecting both the numbers and site of egg deposition were lost. Curiously the cyclic pattern of egg deposition persisted in inhibited females (Fig. 9). The first peak in the controls lasted about 3 days and the subsequent peaks lasted 2 days each. The first peak for females with 1 and 10 beans lasted only 1 day before egg production dropped. The second peak of females with both 1 and 10 beans lasted 3 days, 1 day longer than the controls. The third peak lasted 3 days for females with 1 bean, while it was only a small 1 day peak for females with 10 beans. The beetles with no beans did not show clear peaks. The persistence of cycles supports the idea of there being an endogenous rhythm in the ripening of eggs. Beetles with limited oviposition sites did withhold ,eggs but females deposited the equivalent of the withheld eggs later if they found more beans. The inhibition may be 46 permanent. Females with 0 or 1 bean suffered an Irreversible inhibition within 2 days. On day 7, with about 3 days of life remaining, inhibited females ceased to withhold eggs and lost their inhibitions against laying eggs on flat surfaces. Figure 8. Eggs deposited by pairs of C_. maculatus given limited numbers of oviposition sites with sets of 10 transferred to 50 beans each day. Pairs with no beans (A), 1 bean (B), and 10 beans (C). The outcomes were tested against controls on 50 beans. Day of Transfer 10 4 6 9 3 5 8 7 2 1 0 50 □ gs eoie after deposited Eggs gs n mt dish. empty onEggs rnfr o 0 beans. 50 to transfer Control 100 Fecundity □ rnfr o 0 beans. 50 to transfer after deposited Eggs deposited Eggs hl wt 1 bean. 1 with while □ ControlControl gs deposited Eggs while with 10 beans. 10 with while gs eoie after deposited Eggs rnfrt 5 beans. 50 to transfer 03 <£• 49 Figure 9. Daily pattern of oviposition estimated from 10 replicates taken each day from a cohort of C_. maculatus given 0 (.A), 1 (B) , 10 (C), and 50 (D) beans for oviposition. Estimate of eggs/day Ol Ol o Ol __1_ to to co CO ■&. Ol 01 O) 0) ■Nj 00 o CO CO o a o> W to to CO co ■&. -(*> Ol Ol J 0> 0) -v| 00 00 w CO CO J on o o Table 5. The average (s.d.) fecundity for females (n=10) given different numbers of beans for oviposition. Beans Fecundity % eggs on dish 0 11.0 (9.2)a 100 1 23.2 (15.9)a 32.7 10 50.3 (13.7)b 5.6 25 68.1 (22.9)c 0.6 50 92.0 (6.6)d 0.4 100 81.9 (22.1)d 0.1 Letters indicate similar means (t-test, P>0.05) 52 Table 6. The eggs deposited by females given 0, 1, and 10 beans and then transferred to a dish with 50 beans for the rest of her life. The sum is the total eggs laid before and after transfer. Averages (n=10) were compared (t-test) to controls on 50 beans (mean 77.4, s.d. 11.1, n=5) . 10 beans 1 bean 0 bean Day Before Sum Before Sum Before Sum transfer transfer transfer 1 11.0 80.8 2.5 73.5 0 69.5 2 11.4 84.8 3.7 73.1 0 70.0 sk 3 14.2 77.5 6.7 77.6 0.7 64.4 4 24.8 76.7 10.6 68.8 1.0 58.1** 5 31.9 80. 2 11.4 71.3 4.5 55.7** 6 34 . 2 80. 1 11.3 60.7* 2.9 46.5*** 7 45.3 78. 2 19.0 71.4 7.0 55.3** 8 46.3 75.5 24 .4 53 . 8* 7.4 41.5*** 9 48.4 68.4 24.8 57. 2* 11.6 38.6*** 10 52.6 56.7 19.1 47.9** 11.0 31.5*** * P <0.05 ** P <0.01 *** P <0.001 Summary Using a single stock of C_. maculatus, the reported range of fecundities, 34 to 128, was easily obtained by feeding and altering the availability of beans for oviposition. The increase of fecundity to 125 when females were fed carbohydrates verifies the findings of Larson and Fisher (1924). Based on the consistency of the outcomes, the potential fecundity of the species appears to be about 125 eggs. Fecundities of 60 to 90 eggs, obtained if females were not fed, corresponded with published accounts. It appears that the cost of the metabolic activities of unfed females producing 75 eggs was equivalent to about 40* of the potential fecundity (50 eggs). Developmental time of the larva and competition with one or two other larvae had small but consistent effects on the fecundity. Fecundities of less than 60 eggs were obtained by restricting the number of potential oviposition sites to less than half the realized fecundity. All the published reports of lower fecundities seem to be from females that were crowded, or deprived of oviposition sites, or both. If deprived of beans for oviposition, females withhold eggs and, as they get older, begin to lay eggs on inappropriate surfaces. Both the inhibition and inappropriate oviposition behavior increased as fewer beans were available. Except for females with 0 or 1 bean, the 54 inhibition was reversed if females were given ample beans. The determinants of realized fecundity (F) can be defined as (2) F = (Potential fecundity -[activity-feeding]) f(bean number). The function of bean number is complex. It is about 0.15 when females have no beans and becomes 1.0 when bean number exceeds 0.5 realized fecundity. Its effect is reversed if a female finds more beans. At present, values.can be substituted as follows: (3) F = ( 125 -[ 50 - feeding]) f(bean number). with f(number of beans) = 0.15 to 1.0 for the original site but increasing if a female dispersed from an area of low resource availability to a place where more beans were present. There was a rhythm to oviposition with eggs deposited in the day time in three peaks of oviposition during the 10-day life span of the female. These peaks seemed to be the result of waves of egg production that even persisted in the inhibited females. While stocks used over the last 70 years had very different histories, and had fed on many different beans, and might have had genetic differences in fecundity, the range of fecundities was the same as obtained here. The fecundity of C_. maculatus is easily modified by a range of extrinsic and intrinsic factors. 55 3. Oviposition Behavior Ovipositing females respond to a variety of cues, such as presence of eggs, size of bean, texture of bean, and species of bean (Nwanze, et al. 1975, Nwanze and Horber 1976, Wasserman 1981, Messina 1984, Messina and Renwick 1985b). While the evidence for selective oviposition based on these cues is convincing, the relative importance of the cues has not been determined under conditions that allow females to make unrestricted choices among beans. Size, texture, and species of bean are simple cues acting to release or inhibit oviposition. Responses to eggs seems more complicated because it varies with the relative number of eggs on a bean (Messina and Renwick 1985b). In addition females forced to oviposit on beans carrying many eggs or no beans displayed a feedback that reduced realized fecundity (see Table 6). The following experiments were designed to determine preferences and the strength of preferences of females when they could choose between beans. A model for size selection (Mitchell 1975) exists and a model for oviposition choices will be developed to incorporate all the known cues from this set of experiments. Eggs. Previous experiments established that females deposited their eggs one by one on beans and added a second egg to a bean only after nearly all the available beans carried an egg. Because an individual female cannot be 56 continuously monitored for several days, the nature of her choices must be inferred from the pattern of egg dispersion of females presented with various numbers of beans for various periods of time. To avoid artifacts, all data for distributions of eggs must be obtained from undisturbed females. Females used in the fecundity experiments (see Tables 1-6) were not disturbed and the egg distributions were recorded at the end of each experiment. Egg dispersion by 637 females that were used in the fecundity experiments were gathered together. These cover oviposition periods of 1 day to the lifetime (10 days) for females with 10, 25, 50, and 100 beans. The sequence and nature of oviposition decisions was reconstructed from these data. If females were indifferent to egg cues, then random placement of eggs would be expected under the standard experimental conditions. Females traversed circular petri dishes in which beans were scattered haphazardly in less than 10 s; hence, they were unlikely to be restricted in any way. Any non-randomness in encounters would produce clumping. In the 144 dishes with 10 beans, 97& of the distributions were significantly (P<.01) less variable than expected under a Poisson. Three egg distributions fit the Poisson and one was in the direction of being clumped. Of the females ovipositing on 50 or 100 beans, only one set fit the Poisson. All the others showed a strong tendency (P>.01) toward a less variable dispersion of eggs. That dispersion was possible only if females consistently rejected beans carrying eggs. Because the variance in eggs per bean was so much less than expected under the Poisson, the distributions were compared to a uniform distribution. Under a uniform distribution all beans carry 0 or 1 egg per bean until the 57 mean reaches 1.0 egg per bean; hence, the frequency of attacked beans equals the mean. With 1.0 to 2.0 eggs per bean, the frequency of beans with two eggs is the mean minus 1.0. The remaining beans carry one egg. This defines the uniform distribution. A total of 86 records covered a female's dispersion of eggs on 100 beans. In 43 cases the egg dispersion fit a uniform distribution and the coefficient of variation was below 10% in all but three of the remaining 46 cases. Females must be avoiding any bean with an egg on it as long as beans without eggs were regularly encountered. The avoidance of beans with eggs was even more clear when females had fewer beans. While females given 10 beans had their oviposition rate inhibited (see Table 6), they still responded almost perfectly to egg cues. Of females depositing up to 20 eggs on 10 beans, 82% (31/38) deposited their eggs uniformly. The records for all females with 10 beans showed and increase in the coefficient of variation relative to eggs per bean (t95=10.23, P<.001). The increasing coefficient of variation indicated either a relaxation of the avoidance of beans as there were more eggs per bean or else a decline in the ability of females to evaluate the number of eggs per bean. The slope reached the point at which the variance equalled the mean, i_.e_. , fit a Poisson, when there were 11.8 eggs per bean. The three females that dispersed their eggs randomly deposited 59, 63, and 76 eggs on 10 beans. Females must have counted or discriminated egg numbers up to 5 to achieve the observed uniform distribution. The samples in which one bean carried a second egg can be taken as measures of the conditions under which females ceased to avoid beans with eggs on them and began to add a second egg. Samples with a single bean carrying a second egg averaged of about 0.8 eggs per bean when females 58 had 50 or 100 beans available. Evidently females did not add a second egg to a bean as long as at least one bean out of every five they explored was free of eggs. With 10 beans, females never added a second egg until all beans carried a single egg. The "transferred" females (Table 7) were taken from the experiment in which females were given either 0, 1, or 10 beans and a set transferred to 50 beans each day (see Table 6). Although the oviposition rate was inhibited by being on few beans, their subsequent responses to egg cues did not differ from uninhibited females. Clearly, the cues inhibiting oviposition rate had no effect on the rejection of beans with eggs on them. Obviously, females must, explore many beans to produce and maintain the observed dispersion of eggs over a wide range of egg densities. If the 20% of the beans with no eggs represent zero encounters, the number of encounters needed to discover 0.8 beans under the Poisson is 1.6 encounters per bean. Any non-randomness in encounters would result in some beans being discovered more often than the expected frequency of 1/(number of beans) under the Poisson and attaining a uniform distribution would require more exploration than predicted from the Poisson. The rejection of beans carrying eggs is strong enough to be a standard for measuring the relative strength of responses to other cues. If given choices, females use the least preferred bean before adding eggs to the preferred bean, then the associated cues can be taken to be stronger than egg avoidance cues. If females add a second egg to the preferred bean before using the less preferred, the bean cues are stronger than the egg avoidance cues. 59 Tabie 7. The density of eggs per bean at which the females first begin to add a second egg to beans. Transferred females were those placed on 50 beans after being inhibited by having too few oviposition sites (see Table 6). There were no differences among the means (t-tests). Sample Females Eggs/bean mean s.d. On 50 beans 9 0.88 0.14 On 100 beans 17 0.85 0.14 Transferred to 50 beans 30 0.75 0.26 60 Eggs versus bean size. Will females ignore large size differences when given the choice of either avoiding beans with eggs or ovipositing on egg-free small beans? Two size classes of Berkin beans were selected to give females clear choices as to size: large beans of about 5.9 mm long, averaged 78.9 mg (57 to 105 mg). Small beans averaged 35.7 mg (26 to 46 mg) and were 4.2 mm long. Pairs were given 50 beans, half large and half small, and distribution of eggs was recorded for each of the first 3 days. The preference for large beans was clear (Fig. 10) and, with one exception, females avoided placing a second egg on large beans until the third day when an average of 43.0 eggs had been laid. The threshold for adding a second egg, about 0.8 eggs per bean, was the same as reported elsewhere (see Table 7). Bean size was an important cue, but it was secondary to the avoidance of beans with eggs on them. How is bean size discriminated? Three processes could result in more eggs being deposited on large beans: a threshold response set at some absolute size, responses based on comparisons of bean size, and encounter rates determined by bean size. Models for the general expectations for each of these explanations are considered. They are then used to interpret the results from the above experiments that used two discrete size classes of Berkin beans. i. Threshold responses. If oviposition occurs whenever a bean is larger than an innately determined threshold, then eggs should be randomly dispersed over beans above some critical size as long as the female has a choice. When a female cannot find beans larger than the critical size, the innate threshold might be broken and eggs dispersed at random over the available beans. The 61 size of beans with eggs should not be correlated with number of eggs deposited. ii. Discrimination of size based on comparisons. As females move from bean to bean, bean size is compared and that determines the oviposition response. This model has been formalized (Mitchell 1975) to give the expected outcomes if females compare the bean she is examining with the bean encountered previously. If the second bean is found to be larger she will oviposit on that bean, and beans that are smaller than the earlier bean will be rejected. The comparison model has a peculiar outcome when there are two discrete size classes of beans. Four comparisons are possible: from large to small class beans, from small to large class beans, and movements between beans of the same size class. The oviposition decisions following each encounter and their frequencies are as follows: from L+ to L- pass .125 from L- to L+ oviposit . 125 from L to S pass .250 from S to L oviposit .250 from S+ to S- pass .125 from s - to S+ oviposit . 125 The "+" and indicate beans above and below the average for their size class. Because all movements from large to small class beans result in oviposition and none of the reverse movements do, more eggs will be placed on large class beans. Within one size class movements will be balanced with equal numbers of rejections and ovipositions. Females presented with two discrete size classes of beans are predicted to deposit 7595 of their eggs on beans of the large size class. In addition, the largest beans of each size class should be used first; hence, as more eggs are 62 deposited, the average weight of beans with an egg should decline with mean eggs per bean. That association differs from the threshold model. The single comparison given here is the simplest form that a comparison model could take. If more comparisons are made, then the discrimination will be greater. iii. Encounters related to the dimensions of the beans. The dimensions of beans bias the encounters of a beetle moving at random. Larger beans will be encountered more frequently than smaller beans just because of their size (Janardan et al. 1979). If females simply oviposit on egg-free beans as they are encountered, the bias in favor of large beans will be proportional to the differences in bean size. Predictions from these models were tested by obtaining the individual bean weights from the experiments given in Table 8. The expectations for the performance of females choosing between two size classes of beans were as follows: i. Threshold. While females can choose, all beans above a critical size should be equally likely to receive eggs. Number of eggs deposited and the weight of beans chosen should not be correlated. ii. Comparisons. As long as choices can be made, 75* of the eggs should be on large size class beans. The average weight of the beans carrying eggs should decline as more beans are occupied and be consistent at across all egg densities. iii. Size-related encounters. The large bean class was 40* larger (5.9 mm vs 4.2 mm); hence 60* of the ovipositions should be on large beans. The encounter explanation is obviously wrong because far more than 75* of the eggs occurred on large beans until well after the 25 large-class beans were used (Fig. 10). 63 Figure 10. Discrimination of large size beans by C_. maculatus given 25 large and 25 small Berkin beans for the first 3 days of life. The outcomes expected under a simple encounter model (dashed line) and oviposition based on single size comparison (solid line) are indicated. f Per Cent eggs on larger beans 100 50 75 oa eg laid eggs Total 25 □ □» □ r a 2 hrs 72 • 4 hrs 48 □ 4 hrs 24 50 64 65 Discrimination was much greater than the 75SS expected from the comparison model. An excess of eggs were on the large beans as long as females were able to make choices (up to 25 beans). Mean weight of attacked beans declined as more eggs were deposited (tig=5.64, P<.001), suggesting large beans were chosen first. Because bean weight declined with number of beans carrying eggs, the threshold model can be excluded. However, the discrimination by beetles is much better than the single comparison model predicted. The model was not restrictive enough to account for the performance of the female; hence, it seemed that the beetles either made more than one comparison or combined comparisons with a sense of absolute size. Some evidence existed for a weak influence of absolute size. Females with only small size class beans showed a small (6.1%), but significant inhibition of oviposition up to day 4 (Table 8) that disappeared later. Small size may have affected a female's early choices even though it did not alter lifetime fecundity. The perception of size seems to be a complex phenomena involving comparisons that are made independently of the density of eggs. The exceptionally high level of discrimination suggests an ability to determine absolute, as well as relative, size. Eggs versus surface texture. Females were given similar-sized beans differing in their surface texture. Soybeans (Glycine spjaj were used for smooth beans because of their size. Soybeans are not a suitable host for development. The rough surfaced beans were chickpea (Cicer arietinum), a common host. Beans were sorted by weight to the nearest 0.001 g and five sets of beans were presented to pairs left on 30 beans for 24 h. 66 Rough beans were rejected (Table 9) with 86% of the eggs placed on smooth beans. Females had some perception of their universe before making the first oviposition decision. With the two beans in equal number, half of the females would be expected to deposit at least one egg on a rough bean before discovering that there were choices. Of the females laying 15 or fewer eggs, 82% did not deposit an egg on a rough bean. Differences in bean size did not influence the rejections based on surface roughness. The six females laying more than 15 eggs appeared to put second eggs on smooth beans as often as they put eggs on unoccupied rough beans; hence, roughness was a stronger deterrent to oviposition than size differences in many cases. Seventeen females had to choose between putting a second egg on smooth beans or placing a single egg on a rough bean and 35% put more second eggs on smooth beans rather than add single eggs on rough beans. When given either rough or smooth beans alone, females deposited equal numbers of eggs. Although the rejection of rough beans was strong when choices were open, the rejection did not inhibit oviposition when females had no choice (Table 9). Response to species of beans. The stock of C_. maculatus used in these experiments was from a population adapted to mung bean and black gram (Viqna mungo) and a preference for these hosts would be likely. Experiments tested preferences for the three other major hosts: pigeon pea, chickpea, or cowpea relative to Berkin. Pairs were left for 30 h in mixtures of 25 beans of each species. Ovipositing females preferred Berkin over pigeon pea and chickpea despite the very large size of these beans. Chickpea may have been rejected for its rough surface but pigeon pea was much smoother than Berkin and twice the weight of Berkin; yet, it was rejected as strongly as 67 chickpea (Table 10). Nearly half the females started to add eggs to less preferred hosts before all the preferred hosts carried eggs. Cowpea and Berkin were equally attractive (t18=l.69) with 6 females placing more eggs on cowpeas. Cowpea had a sculptured surface, weighed twice that of Berkin, but the two were equally attractive. The ambiguous outcome may have reflected complex responses to a variety of conflicting cues. The host preferences required recognition of host specific cues independent of size and texture. The nature of these cues was not examined, but the beetle had a basis for recognizing mung beans independent of physical cues. Choices between species were not as clear as the choices made in tests for discrimination of surface texture (see Table 9) and size (see Table 8). Species recognition was based on an independent set of cues. 68 Table 8. Outcomes of experiments to determine the discrimination of two sizes of Berkin beans. There are 10 replicates for each experiment. Time Beans Mean no Replicates with h of eggs __2 eggs/bean 24 25 Large 12.0 1 25 Small 2.7 0 48 25 Large 19.7 0 25 Small 12.3 0 72 25 Large 24.5 3 25 small 18.5 0 96 50 Large 56.7 (4.76) 96 50 Small 50.7 (4.14) 240 50 Large 77.5 (10.06) 240 50 Small 75.6 (13.57) 69 Table 9. Selection of oviposition sites by beetles (n=10) given choices between 15 rough (chickpea) and 15 smooth (soybean) beans for 24 h. There were no differences (t-test) between the means. Beans Mean no Females laying of eggs 2 eggs on smooth 15 smooth 170+5 mg 14.2 3 15 rough 120+5 mg 1.6 0 15 rough 170+5 mg 1.3 0 15 smooth 120+5 mg 12.8 3 15 smooth 145-y.O mg. 11.4 0 15 rough 145+10 mg. 3.3 0 30 smooth 130-180 mg 14.4 (s.d. 6.10) 30 rough 130-180 mg 10.1 (s.d. 8.74) 70 Table 10. Selection of oviposition sites by beetles (n=10) given choices between Berkin beans and the alternate hosts for 30 h. Beans (weight mg) Surface Mean no q.f eggs 25 Chickpea (447 mg) Very rough 2.7 25 Berkin (72 mg) Smooth 17.2 25 Pigeon pea (148 mg) Smooth 2.9 25 Berkin (72 mg) Smooth 15.7 25 Cowpea (235 mg) Finely sculptured 10.0 25 Berkin (72 mg) Smooth 6.4 A Model for Host Selection A gravid female encountering a bean will walk over and around the bean and then either oviposit on the bean or move on. C_. maculatus responded independently to all the oviposition cues suggested in the literatures. In addition, these experiments can be fit into a six-step model explaining the basis for the choices made in response to surface texture, host species, and eggs per bean. The model defines the information a female must process before an oviposition decision is made (Fig. 11). Choices are arranged according to the strength of the response to each cue. The strongest rejection is for rough-surfaced beans. They are rejected in choice situations even if they are larger and carry fewer eggs (see Table 9). Thus, roughness takes precedence over other cues, but the rejection of rough beans is conditional. Females given both rough and smooth beans reject rough beans at first. To exclude the occurrence of occasionally depositing their first egg on a rough bean, they must explore oviposition sites and use this information when the first oviposition choice is made. The female will leave a rough bean to search out a new bean if she has encountered smooth beans earlier. If she has encountered only rough beans in her explorations, then she continues to evaluate the bean (decision 1A). Figure 11. Model for the selection of beans for oviposition. The responses were based on the choices made by females under experimental conditions (see Tables 8 - 10). 73 EXAMINE ANOTHER BEAN YES Is this Are there bean rough? YES saooth beans Of the \ beans, is this Do all the preferred NO preferred beans species? carry eggs? YES YES Does N . this bean carry ^ fewer eggs than ave NO eggs/bean? JES. Is the size above average for beans carrying NO under ave egg no? . YES OVIPOSIT 74 Females reaching decision point 2 evaluate host species by a set of unidentified cues independent of size and surface texture. The choice experiments (see Table 10) indicate the rank of preferences for the four common hosts to be Berkin = cowpea >> pigeon pea = chickpea. If experience indicates that she is sitting on the most preferred of the available beans, she goes to decision point 3; otherwise, further evaluation occurs at decision point 2A. Females consider whether the preferred beans she has explored already carry eggs. If that is the case, she continues to evaluate a bean of the less preferred species. Additional information gained from explorations are used to evaluate the number of eggs per bean and the relative size of the bean among beans of that species carrying eggs. This may appear, at first glance, to be beyond the capacities of a beetle but the data are clear. Females with only 10 beans never added a second egg until all 10 beans carried a single egg. They must be able to count 0 or 1 and record their frequencies if they end up putting each of 10 eggs on a separate bean before ever adding a second egg to a bean. The evidence suggests the ability to accurately record numbers up to 6 when dealing with 10 beans. Even when females had 50 to 100 beans, they did not add a second egg until at least 80% of the beans carried 1 egg (see Table 7). In addition, eggs were added in a way that followed a ranking of beans by weight. At decision point 3 females reject beans carrying more than the average number of eggs/bean. The final decision to oviposit is made at the fourth step when the perceptions from exploration were used to answer the question: Is this bean larger than most beans of this species that carry eggs? As a result of making that decision there is a negative correlation between the mean 75 weight of beans carrying eggs and the density of eggs per bean (see Fig. 10, Table 8). This elaborate oviposition strategy can be presumed to be the product of natural selection favoring females selecting host most favorable for the survival of eggs, larvae, the fecundity of her daughters, or some combination of the three. These matters are taken up in the next section with experiments measuring the gain in survival or fecundity or both, associated with each of the six decision points. Development of Passive Stages The consequences of selective oviposition, survival of eggs and larvae, and fecundity of daughters, must have driven the evolution of the oviposition behavior (see Fig. 11). Life history studies of survival at each stage and host-influenced fecundity will measure the selective pressures currently operating to maintain the oviposition behavior. Berkin is the appropriate host as the reference for comparative studies of life history traits because it is preferred by ovipositing females and is the species of host attacked in the field by the lineage of beetles used here. The location, activity, and weights (wet and dry) of larvae were recorded from beans opened daily and used to reconstruct the sequence of events and growth rates during larval development. 1. Development of Single Larvae in Berkin. The life cycle (Table 11, see Fig. 3) from egg to egg was completed in about 29 to 32 days at temperatures of 22-24° C. Eggs with a fresh weight of 0.0235 mg were milky colored. Embryonic segmentation was apparent by day 4 and the differentiation of the head by the next day gave the embryo a larva-like appearance. The head capsule acquired a deep-black pigment by day 6 and larvae hatched a day or two later at a weight of 0.0133 mg (67.5% water). First instar larvae have a large black head capsule and numerous long 76 77 spine-like setae on a body tapering towards the posterior end. They chewed perpendicularly through the floor of the egg into the bean and penetrated the testa in 2 days to enter the cotyledons on which they fed for the rest of their life. Shortly after the body was entirely inside the bean, the larva turned almost 90 degrees to tunnel parallel to the surface as reported by Raina (1970). After feeding for 5 days, the first instar larva had increased in weight by 8 times to 0.107 mg (70.9% water) and it then molted to the second instar. This instar lacked legs and had a head capsule reduced to reddish black plates supporting the mandibles and providing apodemes for muscle origins. As it fed, the abdomen distended dorsally giving the body a strong convex shape (see Fig. 3). The larva continued burrowing parallel to the bean surface for 2 or more days, then turned to cut a path towards the center of the bean. During the 3 days of the second instar, the weight increased by nearly 400% to 0.211 mg (68.9% water). The second through fourth instars were leg-less, thin-skinned white larvae with a characteristic pattern of cuticular folds and a few fine setae. Instars were difficult to distinguish by appearance, but the instar could always be verified by a count of the cast head capsules packed together with frass and the granular, slightly yellowish fecal material in the tunnel behind the burrowing larva. As the third instar larva continued to chew toward the center of the bean, its weight increased 5-fold to 1.190 mg (68.2% water) in 3 days. It then molted to the fourth instar which fed vigorously for 5 to 6 days and consumed much of the remaining cotyledons to open out a large chamber. Feeding slowed down during the next day or 78 two when the larva took on a pupa-like shape, the prepupa. The prepupa chewed a passage to the surface of the bean where it prepared an exit hole by clearing the cotyledons away from a circular area under the testa. This circular window was partially perforated. The prepupa then lined the feeding chamber with fecal matter, frass, and cast skins as described by Johnson (1977) for Ctenocolum janzeni. The terminal fourth instar larva or prepupa, had increased its weight 10 times to 8.76 mg (52.6% water). On completing the pupal chamber and exit, the prepupa oriented itself to face the exit and pupated. Pigmentation began to develop in the mandibles and the compound eyes by day 4 and was complete within 6 days. On day 7, the adult structures were apparent and adults began to emerge the next day. 79 Table 11. Development in days and weight (mg) of C_. maculatus feeding alone in beans. Larvae were weighed (n=10) at the end of the molt and pupae were weighed when first formed. Instar Berkin Cowpea Chickpea Pigeon Pea First duration 5 5 5 7 weight 0.106 0.102 0.099 0.071** (s.d.) (.017) (.018) (.021) (.013) Second duration 3 3 3 4 weight 0.211 0.171 0.157* 0.083*** (s.d.) (.056) (.071) (.056) (.033) Third duration 3 3 3 4-5 weight 1.190 0.990 0.909* 0.272*** (s.d.) (.188) (.418) (.349) (.053) Fourth duration 5 5 5-6 6-7 weight 8.579 7.626 7.856 8.238 (s.d.) (2.89) (1.18) (1.28) (1.02) Pupa duration 7-8 8-9 9-10 8-10 weight 8.272 7.367 7.463 7.507 (s.d.) (1.74) (1.68) (1.53) (1.18) t-tests against Berkin * P<0.05 *** P<.001 80 2. The effects of food on development. The beans attacked by the beetles differ greatly in their food value for humans and it would be expected that beetles may also be affected, especially because each larva feeds on one bean. The major nutrients of the hosts of C_. maculatus as listed by Duke (1981) are similar (Table 12). Of the 20 amino acids, glutamine is missing in mung beans and the other hosts. Tryptophan is missing from all but mung beans. Levels of methionine are low enough in the other three hosts to cause nutritional deficiencies in humans. All four beans have trypsin and chymotrypsin inhibitors and cowpeas have cyanogen. Substantial differences exist in the mix of fatty acids in these four beans and the concentrations of the water-soluble vitamins. Combine differences among beans in nutrients with the larvae feeding on a single bean and it is not surprising that the performance of beetles differs from host to host. Comparative development. The development of a cohort of eggs laid within 6 h of each other was followed of all four hosts. All eggs hatched within 7 days, but egg mortality was exceptionally high. This was probably caused by a brief rise in laboratory temperatures early in the experiment. The level of mortality is above the usual levels but such temperature fluctuations occur in nature and the differences between hosts can be used to indicate relative mortality differences associated with hosts. Egg survival (11%, n=196) was highest on Berkin (P<0.01) than on the other three hosts. One of 151 larvae died in Berkin. The mortality of first instar larvae in the other hosts was limited to the first 3 days of life and was similar in all hosts. About a third of the first instar deaths occurred on hatching, another third died while chewing testa, and the remainder died within a day of 81 feeding on the cotyledons. The deaths were either larvae that were weak at hatching or due to nutritive qualities of the testa. While surface texture may have affected egg and larval survival (Nwanze and Horber 1976), it did not account for all of the early mortality. Deaths of larvae and eggs on pigeon pea, with its smooth highly polished surface, were as great as those for the rough-surfaced chickpeas and cowpeas which have a finely sculptured surface. In both Berkin and cowpeas, the first instar larvae burrowed perpendicular in the bean, then turned to burrow almost parallel to the bean surface for 2 or 3 days and again turned toward the center. Larvae in chickpea and pigeon pea did not follow this path; instead, they burrowed directly toward the center. Pirst-instar larvae performed similarly in all but pigeon peas in which development and growth was much slower. Only 5 of the 15 larval weights were significantly different from larvae in Berkin (see Table 11), but all the averages were less than Berkin indicating a small but consistent deficiency in growth in the other three hosts. Aside from the first week, burrowing and development time was similar for larvae in all but pigeon pea. Weights were lower for larvae in both chickpea and pigeon pea and differences increased with age. Pupal weights were similar as a consequence of increased growth during the fourth instar compensating for the earlier differences in larval weights. The prepupa was disoriented in both chickpeas and pigeon peas. Some prepupae in chickpea cut exit paths at an angle to the bean surface and either opened the exit or cut incomplete windows. These prepupae still pupated normally but 82 mortality of 42% occurred because of pupae falling out of open pupal chambers or adults emerging to find themselves trapped behind an incomplete exit. The faulty exits could be due to the irregular surface of chickpeas, but the angled exit paths were clear evidence of a disorientation unrelated to surface features of the bean. The exits were cut perfectly in pigeon peas, but 15% of the prepupae oriented themselves to face the rear wall of the pupal chamber. Pupae developed and adults emerged only to die because they faced the wrong way. This was never observed in other beans. Despite the adverse affects apparent from the growth of larvae and the disoriented behavior of prepupae, adults were unaffected. Judging from fecundity and oviposition behavior, the nutritional reserves from all hosts were adequate for normal fecundity (Table 13) and there was no disorientation in the oviposition behavior. Females that could not exit from the pupal chamber in chickpeas and pigeon peas were dissected out Of the beans just after adults had emerged from normal pupae. Females from disoriented prepupae were paired with normal males grown in Berkin beans and given 50 beans for oviposition. Four of the 10 females from chickpeas deposited a few infertile or oddly shaped eggs over a normal life span. The average fecundity of the six remaining females, 32.1 (s.d. 25.3) eggs, was half that of adults from normal pupae, but the dispersion of eggs was uniform. Females from prepupae that pupated backwards in pigeon peas had a normal fecundity and also dispersed their eggs uniformly over a normal life span. While food quality affected the behavior of prepupae and the fecundity of adults from disoriented prepupae from chickpeas, it had no effect on the oviposition behavior of females. 83 Table 12. Composition of the major hosts of C. maculatus from Duke (1981). Components that show major differences are given. Mung Cowpea Chickpea Pigeon pea Nutrients dry wt) Carbohydrate 69 66 70 73 Protein 26 27 22 22 Fat 1 2 4 1 Amino acids (mg/g N) Cysteine 0.5 0 .0 0 Methionine 2.0 1.2 1.2 1.4 Phenyalanine 5.5 6.0 10.0 6.6 Vitamins (mg/lOOg) Thiamine 0.59 1 .29 0.53 0.80 Riboflavin 0.29 0.39 0.18 0.16 Niacin 2.80 4.82 2.00 3.22 Ascorbic acid 4.00 87.00 0.00 0.00 Cyanogen present 84 Table 13. Life tables for C_. maculatus on its principle hosts. With Berkin as controls, confidence limits for percentages were used to test for differences in survival and t-tests for differences in fecundities. High egg mortality was due to high temperatures, up to 30 C, shortly after oviposition. Stage Berkin Cowpea Chickpea Pigeon Pea n 196 306 269 266 Egg lx 1.000 1.000 1.000 1.000 (qx ) (0.771) (0.553**) (0.617**) (0.650**) I lx 0.771 0.553. 0.617 0.650 (qx ) (0.993) (0.888**) (0.914**) (0.868**) II-IV lx 0.766 0.491 0.558 0.564 (qx ) (1.00) (1.00) (0.993) (1.00) Pupa lx 0.766 0.491 0.554 0.564 (qx ) (1.00) (1.00) (0.247**) (0.734**) Adult lx 0.766 0.491** 0.137** 0.414** Mean developmental time (days) 34.5 * 36.0 38.0* 38.5* Fecundity 70.5 68.1 69.0 72.4 R0 27.00 16.72 4.73 15.00 * PC0.05 ** P<0.01 85 3. Interactions of competing larvae. Competition was followed for a cohort of eggs deposited within 4 h. Eggs hatched on the same day and the process of competition was reconstructed from daily records of larval weights and the burrowing of two larvae sharing a bean and compared to single larvae in a bean. The details of competition in Berkin beans were the basis for comparisons of competition in alternative hosts. On hatching, all larvae burrowed perpendicularly into the bean and then turned to burrow under the testa. All larvae molted to the second instar about day 4 and the burrows were far apart because females deposited second eggs far from the first. On day 6 all single larvae and one of the competing larvae turned in their superficial burrow to chew toward the center of the bean. By day 8 the larvae sharing a bean differed in their appearance, position of burrow, and weight. One larva remained in a shallow burrow under the surface of the bean and appeared flaccid, feeble, and largely unresponsive to disturbance. The larva burrowing toward the center of the bean was pljimp, firm in body tone, and responded to disturbance. That larva was indistinguishable from larvae growing alone. The large larva chewed on the walls of a central cavity and seemed indifferent to the larva remaining in a superficial burrow (Pig. 12). Some burrows of active larvae became so large that some intersected the burrows of the small larvae by day 10 and, as feeding continued, more burrows intersected until day 16 when all burrows had intersected. Burrows intersected as a result of normal feeding by the large larva, not burrowing directed toward the small larva. When burrows first joined, larvae were often mutually aggressive (Table 14). They pushed each other back and forth, extended their heads toward each other, and snapped Figure 12. The differential development of two identically aged third instar larvae of C_. maculatus growing in Berkin bean (X 20). 88 their mandibles frequently. Even after being exposed, larvae continued their aggressive behavior for as long as 2.5 h. As a rule aggression ended in a few minutes when the small larva from the superficial burrow ceased to push and snap their mandibles. It turned away and curled up while its whole body twitched or shivered every 2 s. These interactions were scored one of three ways: mutual aggression, small larva turned away, and small larva moribund or dead. The frequencies of these interactions (Table 14) revealed a clear behavioral sequence starting with aggression on days 11-12 and ending with a loser giving up by days 13-15. Larvae alone and the larva of pairs that fed centrally grew to 6 mg, whereas the larva remaining in a superficial burrow averaged only 2.5 mg. This weight differential was well established by day 8, 2 days before any larvae were in contact. Communication between these larvae must occur with one larva responding by staying in a superficial burrow and reducing its feeding by about 40%. That larva was destined to lose; yet, it still responded aggressively to the large larva. Berkin beans are so small that either mutual death owing to lack of food for both larvae or competitive exclusion was inevitable. Multiple emergences are regularly reported for larger beans (Janzen 1977a, Bellows 1982b, Osuji 1982). Competing larvae in cowpeas burrowed the same way as in Berkin, though winners turned toward the center a day or two later than usual in Berkin. By day 8, the superficial larvae weighed less than single larvae while the average weight of the dominant larvae was greater than those larvae growing alone in a bean (Table 16). Except for the time difference, the behavior of the competing larvae in cowpeas 89 was similar to those in Berkin. As feeding continued, the burrows began to intersect on day 11 and all the burrows were in contact by day 16. The central larva always won. There are reports of two larvae emerging from cowpeas (Janzen 1977a, Bellows 1982b, Osuji 1982),but only one larva emerged in my experiments. The reports of multiple exits are from beans with more than two larvae in them and for situations in which the second larva could have entered long after the first. The chances of the second being inhibited and maturing after the first may be much higher when there are large differences in the times of entering. When two larvae competed in the largest of the hosts, chickpeas and pigeon peas, they were indifferent to one another and burrowed directly toward the center of the bean. Without behavioral differences between the larvae, they could not be identified for weighing. An interaction 1 was suspected because the weights of competing larvae were more variable than those of single larvae and the deviations in weights (Table 16) followed the same pattern as found in normally competing larvae (see Table 15). A simple rank test was devised to determine if the larvae were drawn at random from a single population. The weights of all competing larvae were ranked and competing pairs scored for weights above or below the median. A random combination would give equal numbers of competing pairs with weights below or above the median versus pairs in which one larvae had an above median weight and the other a below median weight. Competing pairs with larvae from two sides of the median greater than expected under a binomial in both chickpeas (chi-square2=15.0, PC.005) and pigeon peas (chi-square3=8.0, PC.05). An interaction exists between larvae, even though they behave similarly in these two 90 hosts. Weights taken on the day 8 demonstrated some inhibition in chickpea but not in pigeon pea and the competing larvae continued to be indifferent in their burrowing nature. These results in chickpeas and pigeon peas indicate that though the competing larvae burrowed similarly, there was an interaction between the two larvae resulting in differential growth before they encountered each other. These interactions determined the outcome of competition. Determination of the winner. The identity of competing larvae was established by exposing Berkin beans to beetles just long enough to obtain 1 egg per bean. A pencil mark was placed around that egg and beans exposed at intervals of 1, 24, 48 and 72 h to obtain a second egg on the bean. Beans were examined every day and the path of each larva followed. The winner could be identified as soon as it turned to burrow toward the center while the other larva remained in a superficial burrow. Knowing the source of the winner allowed a test of the assumption that the first larva into a bean destroyed other larvae entering the bean (Pinckney, 1937, Dickason, 1960, Janzen 1975). Larvae coming from eggs deposited within 1 h of each other hatched on the same day and the larvae from the older egg won 5 5 . 9 % of the contests (n=68, chi-square^i.06) , no more than expected at random. The outcome was not unexpected because the development of eggs varies by much more than 1 h. Very little overlap exists in the hatching time of larvae from eggs differing by 24 h, yet larvae from older eggs won only 61.835 of the contests, not a departure from 503j (n=68, chi-square1=3. 76) . Larvae from eggs differing by 48 and 72 h never overlapped in their hatching and the older larva won 7935 (n=34, chi-square^li. 7, P< . 001) and 1635 (n=70, chi-square1=32 . 9 , PC. 001) of the contests respectively. Although older larvae had an 91 advantage, it was not the preemption older larvae were presumed to enjoy. Larvae must be responding to signals passing from larva to larva through the bean which determine the winner while the burrows were separated by a few mm of cotyledon tissue (see Fig. 12). Factors other than age are involved. Males are in excess among adults from beans carrying a single egg, ranging from 0.5322 (chi-square1=5.32, PC.05, n=1295) to 0.584 (chi-squarei=5.78, PC.05, n=200), but the frequency of males, 0.488 (n=3102) and 0.500 (n=200), did not differ from 0.5 in beetles emerging from beans with two larvae in them. Summary. Surface texture appears to affect egg survival by interfering with attachment of the egg. Deaths of early first instar larva have a different pattern than in eggs, hence, they may reflect the combined effect of being unable to obtain leverage for chewing from a weakly attached egg as well as nutritive qualities of the testa. Once larvae begin to feed on the cotyledons, there is no mortality and the realized fecundity based on the reserves built up in the larval stage is the same for all hosts. While food quality does not affect survival or fecundity, it does have specific effects on the behavior of the prepupa. Prepupae in chickpea often cut faulty exits in which case pupae either died from exposure or the adults died behind an incomplete exit. A few larvae pupate backwards in pigeon peas and the emerging adult dies because it cannot escape. Except for aborted or stunted beans, the principle host species are large enough to support a larva. Preferences for large beans may, along with the uniform egg distribution reduces the incidence of larval competition and restricts competition to the largest available bean. When competition exists, larvae interact before coming into 92 contact and age is not the sole determinant of the winner. These mortality factors are combined in the net reproductive rate t(RQ ), a measure of potential contributions to the next generation. The host specific RQ for single larvae measures the effect of host quality. When egg density is high enough for competition to occur, then, a single larva survives from each competing pair and R0 falls in direct proportion to the frequency of competing larvae. These estimates of mortality are the basis for comparing larval success with the oviposition choices made by the females. 93 Table 14. Frequencies of various interactions between competing larvae when larvae first come into contact. Days In Mutual Smaller Smaller _QD_ contact aggression turned away dead 1-10 0.04 0.00 0.00 0.04 (6 6 ) 11-12 0.62 0.29 0.08 0. 25 (24) 13-15 0.75 0.04 0.39 0.31 (48) 16-19 1.00 0.00 0.00 1.00 (20) 94 Table 15. Average (s.d.) weight (mg) of 20 larvae alone and 10 pairs of competing larvae segregated by the position of their burrow. Age in 1 larva/bean 2 larvae/bean______days (control) central superficial Berkin 8 0.44 (0.01) 0.39 (0.17) 0.19***(0.10) 12 3.13 (0.08) 2.54 (0.06) 1.37***(0.04) 14 5.98 (0.04) 6.02 (0.08) 2.46* **(0.06) Cowpea 8 0.38 (0.02) 0.41*(0.03) 0.31***(0.09) 12 1.73 (0.29) 1.75 (0.42) 0.89* **(0.47) 14 4.08 (1.86) 3.35 (1.52) 1.32* **(0.59) * PC0.05 *** P<0.001 level 95 Table 16. Average (s.d.) weight (mg) of 20 larvae growing singly and 10 pairs of larvae competing in chickpea and pigeon pea. Competing larvae were segregated by weight to test for a pattern of deviations matching those in Table 15. Age in 1 larva/bean 2 larvae/bean days (control) heaviest lightest Chickpea 8 0.39 (0.03) 0.39 (0.03) 0.34**(0.05) 12 1.30 (0.17) 1.69*(0.36) 1.14 (0.18) 14 3.46 (1.52) 3.94 (1.36) 2.02*(0 .66) Pigeon pea 8 0.15 (0.02) 0.20* *(0.01) 0.15 (0.04) 12 0.35 (0.19) 0.40 (0.08) 0.25 (0.11) 14 0.90 (0.24) 1.06 (0.34) 0.60*(0.24) 16 3.34 (0.99) 2.99 (0.86) 1.80* *(0.50) * PC0.05 ** PC0.01 level Table 18.*41 Experimental Analysis of Competition. Novel competitive environments were produced by gluing beans containing larvae of known age together. The procedures for joining beans together consisted of grinding a flat surface and gluing the surfaces of two beans together. A set of controls tested the effect of glue, grinding, or both on a larva in a bean. A second set of controls compared larval responses in beans glued together to those of larvae in a single bean. In addition to gluing, larvae were transferred from bean to bean to determine how that affected their responses. Glue and grinding. Beans with a single egg of uniform age on them were subjected to four treatments and the larvae weighed when they were 15 days old. The treatments and average weights (s.d.) for 10 replicates were i) Controls: Not ground or glued: 8.58 mg (2.67) ii) Glue applied to bean testa: 8.36 mg (2.94) iii) Glue applied to ground surface: 7.27 mg (2.53) iv) Ground surface on beans with eggs and applied glue to surface when larvae was 1 day old: 7.85 mg (2.63) 97 Average weights did not depart from the controls (t-test); hence, neither the glue nor the grinding had measurable effect on the larvae, either alone or in combination. Because the weights for all treatments were less than the controls, there may have been a reduction of growth that was too small to be statistically significant. The treatments had no measurable effect on the time of hatching. Developmental time was the same for all sets of larvae. Beans glued together. At the same time as the single beans were set up, the following combinations of beans were assembled: i) Beans with fresh eggs ground and glued together. ii) Beans with fresh eggs ground and held until larvae were active and then glued together. iii) Beans held until after hatching and then ground and glued together. The larvae in beans glued together did not exhibit the differential burrowing behavior; both turned to burrow toward the center after about 7 days. The average weights of the larvae in the treatments deviated from the controls (P<0.05) because one larva grew normally (mean weights of the larger larva equalled controls) while the other larva was significantly smaller. This was the pattern of differential growth predicted if larvae in beans glued together responded as if they were in a single bean; hence, beans glued together can be used to manipulate competitive environments. 98 Table 17. Weights of larvae in beans glued together to simulate conditions of competition within a single bean. The controls were beans with one larva that were ground and the surface glued. Treatment mg weight (s.d.) All larvae Smallest of pairs i . Glued as eggs 5 .47 3.03 (3.39) (1.97) ii. Ground and later 6.14* 4.01 glued as larvae (3.16) (1.71) iii. Ground and glued 5.74 3.64 as larvae (2.73) (1.36) Controls 8.58 (2.67) * P<0.05 ** P<0.01 *** PC0.001 99 Analysis of the Competitive Responses. The technique of gluing beans together opened unlimited opportunities for manipulation. Any desired combination of larvae could be produced at will and the competitors later separated. Such manipulations were used to address three major questions about competitive behavior: i . What are the cues exchanged between larvae? ii. When do larvae respond to competitive cues? iii. Can a larva destined to either lose or win have its behavior altered? Identification of the Cues. What kind of signals can travel through a few mm of the dry cotyledon tissue? Vision can be excluded because cotyledons are opaque. The cues that might pass from larva to larva through bean cotyledons are i) odors or pheromones produced by the competing larvae. ii) vibrations produced by the mandibles chewing on the bean cotyledons. iii) movement of beans produced by larval activity. Four treatments were devised to interfere with diffusion of chemicals and transmission of vibrations (Fig. 13, Table 18). A) 40 beans were packed in a tightly plugged 15 x 45 mm vial. Odors could accumulate but vibrations were not likely to be transmitted from bean to bean. B) The point contact between the testa of intact beans glued together gave little area for the transfer of chemicals and vibrations. C) The area of contact was broad and the layer of glue was the only barrier to chemicals in pairs of beans with ground surfaces glued 100 Figure 13. Experiments to alter the transmission of cues between two larvae in beans glued together. Beans were glued together while the eggs were fresh and the larvae were weighed when they were 14 days old (see Table 18 for the complete data on these experiments). 101 14 days Eggs Deposited 5.68 mg Controls 0 days 2.75 mg Ground and 5.17 mg glued to foil. 2.69 mg Ground and 5.43 mg glued. 4.28 mg Glued by testa. 5.42 mg 102 together. D) The area of contact was the same for beans with the surfaces glued to either side of aluminum foil but the foil was a barrier to chemicals but not odors. The transmission of vibrations was predicted to increase in efficiency from treatment A through C. Beans with single eggs on them were set aside for controls. Weights were taken 14 days after hatching and larvae from experimental pairs tested for differences in weight expected under competition; a reduction in average larval weight that was the result of one larva in each pair weighing the same as the controls and one weighing less than the controls. All larvae from pairs of beans glued together fit the prediction that the lower average weight of competitors was due to one larva of each pair growing the at the same rate as the controls and one growing less than the controls. The response increased as the contacts for the transmission of vibrations became more effective and the difference was significant for the beans glued to foil. The final possibility, one larva rocking beans about so as to inhibit the other larva, was tested. Beans with two eggs differing 1 h in age were glued to 19-mm round glass cover slips so they could not move. Five beans were opened daily to follow the burrowing behavior of competing larvae and weights were taken on day 11. Burrowing behavior in immobilized beans was normal (see Fig. 12) with the differential growth of competing larvae fully expressed. The mean weights of controls (2.26 mg, s.d. 0.56, n=15) and the centrally located competitor (1.99 mg, s.d. 0.43, n=15) were similar. The superficial competitors weighed much less than the controls, 0.96 mg (s.d. 0.21, n=15, t28=8.46, P<0.001). Differential burrowing and growth were fully expressed in beans immobilized on glass cover slips. 103 The experiments exclude bean motion and odors as possible cues, leaving only vibrations. Vibrations are inevitably produced by larvae as they chew and the few fine body setae would be very sensitive vibration receptors, hence, the means of producing and sensing the supposed cue are present. When are larvae responsive? In nature, competition always involves a newly hatched larvae entering a bean occupied by an older larva . Behavioral responses were apparent by day 5 when the winner changed its burrowing and earlier experiments showed that even 72 h difference in age was not enough to make the older larva a sure winner. The capacity to be inhibited by another larva persists at least 72 h. Beans from a cohort of larvae from eggs laid within a 6 h period were glued together (Fig. 14) daily from the time larvae were 1 day old until they were 10 days old. If glued together while in the first instar (days 1-4), the characteristic competitive response was seen. When second instar (days 5-8) larvae confronted each other, they inhibited each other to the extent that the largest competitor was still significantly smaller than the controls. Two second instar larvae would never encounter each other in nature and the mutual inhibition they showed was never seen in natural competition. The abnormal outcome demonstrated that the larvae continued to be receptive and, moreover, that there must be an exchange of signals between larvae. The effect is even more drastic for a third instar larva (days 9-10) confronted by a larva of its age (Table 19). Within 4 days of confrontation, one larva was dead in 3035 of the pairs and 7535 of the surviving larva weighed less than the controls. The extreme responses in the third instar could be due to larvae reducing their feeding but 104 Figure 14. Experiments to alter the responses of larvae to competition: (A) Beans with one 5-day-old larva growing alone glued together and weighed 8 days later. (B) Beans with one egg glued together. When larvae were 5 days old the beans were separated and glued to a bean with an 8 day old larva and weighed after 6 days. (C) Competing third instar larvae were transferred to a new bean and held for emergence (see Table 19). 105 6.68 mg 5 days Controls (13 days) 2.29 mg (13 days) 4.58 mg (13 days) B. 3.57 mg 5 days Controls (11 days) 2.50 mg 20% die (11 days) 8 days 70% die 7.12 mg (14 days) 106 not their metabolic demands. Larvae of all ages were responsive and the experimental environments most unlike those in nature, produced the most abnormal responses to competition. Can the responses of the competing larvae be altered? The winner and loser determined in competition were identified and competing larvae that had their fate determined were dissected from their burrows. Each larva was transferred to a cavity cut into a fresh bean (Fig. 14) which was sealed with parafilm R and the larvae left to develop undisturbed. Thirty of 36 larvae transferred to new beans survived and emerged as adults from the new bean. Inhibited larvae, even those at late 3rd instar stage, emerged as adults. Both inhibited and dominant larvae required more time to develop (46 versus 33 days) and laid fewer eggs (55 versus 80), suggesting that all larvae suffered from the transfer. Obvious, the inhibition is reversible and an inhibited larva could mature if the dominant larva died. A second experiment was set up to determine if a winner could have its behavior reversed. Beans carrying single eggs were glued together and held until the larvae were 5 days old. Two treatments applied: one set of glued beans were separated and an unoccupied bean glued to it. The second set of glued beans were split and attached to a bean with a 9-day-old larva in it. A set of the original glued beans were controls. Six days later, when the larvae were 11 days old, the larvae from controls were separated into the largest and smallest representing the winner and the loser respectively (Table 20) and compared to larvae from the experiments. Larvae in beans split and attached to an unoccupied bean were in a non-competitive environment and they came to 107 weigh as much as winners from the controls. The larvae in beans attached to a bean with a much older larva faced a very large competitor and their weight fell to that of the loser control. Both the winner and the loser can reverse their responses, even after their fate had been established. The events in those combinations of 5 day-old larvae confronted by 9 day old larvae were quite remarkable. Within 6 days 70% of the 9 day old larvae had died. Whether the older larva died or not, the experimental larva was inhibited. Summary. The events of competition are almost certainly mediated by the exchange of vibrations between larvae. There must be a mutual evaluation of signals indicating more than just age differentials. The nature of these traits is not known. Under natural competition, the larvae respond to each other and one is inhibited while the other behaves in the same way as a larva alone. Larvae continue to be sensitive to cues indicating competition even after their fate has been fixed. Both winners and losers could be forced to reverse their responses and older larvae destined to win often died when presented with an extremely abnormal combination of competitive cues. The potential advantage associated with the competitive behavior can be demonstrated in beans glued together. Losers reverse their behavior and can start to grow and mature whenever the winner either dies or ceases to feed at the time of its pupation. Such opportunities almost never occur in the hosts considered above. Table 18. Experiments to alter the bean to bean transmission of cues between larvae of C_. maculatus. Beans with fresh eggs were glued together and the larvae weighed when 14 days old. Treatment Transmission Transmission Weight (mg) of vibrations of odors (s.d.) Control, single beans 5.68 (n=5) (0.79) 40 beans tightly packed Beans touching Diffusion among 5.50 in vial (n=10) each other. beans. (0.93) 2 intact beans glued From testa Diffusion 4.76 together (n=10) through glue. at a point. (0.83) 2 beans ground and glued Through glue at Diffusion at 4.06 together (n=10) broad contact glued contact. (1.65) 2 beans glued to opposite Broad cotyledon No diffusion 3.96* surfaces of aluminum foil contact through (1.42) barrier (n=10) foil and glue. 109 Table 19. Weights In mg (s.d.) of C_. maculatus larvae in beans glued together after larvae had started to feed alone. Weights were taken 12 days after hatching for 20 larvae in beans glued in the first instar and at 13 days for larvae joined in the second instar. Treatment Age of larva All larvae Heaviest Lightest3 at treatment ___ 1st instar Controls 2.90 (1.20) 2 days old 1.90**(0.61) 2.37 1.43 3 days old 1.96**(0.88) 2.57 1.35 4 days old 2.05*(1.00) 2.69 1.42 2nd instar Controls 7.28 (2.14) 5 days old 3.44**(1.43) 4.58 2.29 6 days old 3.17**(1.35) 4.28 2.06 7 days old 4.04**(1.75) 5.52 2.57 8 days old 5.35 (2.45) 7.30 3.39 3rd instar 9 days old 5.68 (2.13) 6.68 3.87 Controls 10.39 (1.89) 10 days old 6.87 9.06 4.13 * PC0.05 ** P<0.01 a All weigh less than the lightest control larva. 110 Table 20. Tests to determine the reversibility of the competitive behavior in C_. maculatus. n Mean weight in mg (s.d.) Beans with eggs glued together Control winners 18 4.17 (1.27) Control losers 18 2.04 (0.64) Separated at day 5 and glued to unoccupied bean. Winning environment 16 3.55 (1.19) Separated at day 5 and glued to bean with 9 day old larva. Losing environment 15 2.50 (0.91) The Consequences of Oviposition Decisions Do the oviposition decisions of females (Fig. 11) maximize RQ for the set of eggs she lays? The life table (Table 13) and observations of competition provide the basis for measuring the consequences of each oviposition decision. If these decisions increase RQ , then it is this differential larval success that maintains the observed oviposition behavior. To examine the effect of oviposition choices on survival, I first reviewed the components of RQ given in Table 13. Surface texture was a pivotal cue in oviposition, but additional studies are needed to determine if the surface structure of the testa per se interferes with egg, attachment. Without knowledge of how surface structure acts, beans can be subjectively ordered by surface texture and associated with host specific egg survival (see Table 13) compared to the Berkin standard: Surface texture: chickpea >> cowpea > Berkin ~ pigeon pea Egg survival: 0.80 0.72 1.00 0.84 The roughness of chickpea is coarse and irregular, whereas, the cowpea surface is finely sculptured. Egg survival is low on both rough beans but egg mortality must involve more than surface because there is high mortality of the smooth polished surface of pigeon peas. Egg mortality on various 111 112 surfaces is not yet understood. If roughness acts as suggested, then, there must be some additional defensive mechanism, possibly chemical, associated with the surface of pigeon peas. Whatever the explanation, it is clear that avoiding rough beans increases the prospects for egg survival. Both the surface texture of the testa and its nutritive qualities may affect success of first instar larvae. If larvae must struggle to chew through the testa because they cannot get enough leverage from a poorly anchored egg shell, they may die before reaching the cotyledon. Such deaths cannot be distinguished from larvae dying due to chemical features of the testa. If first instar mortality and egg mortality are both associated with surface factors, then the ranking of egg survival should match the ranking by survival of the first instar larva: Egg survival: Berkin > chickpea < cowpea < pigeon pea 1.00 0.80 0.72 0.84 I instar survival: 1.00 0.92 0.89 0.87 Larval survival is highest in Berkin and deaths of first instar larvae appear to be unrelated to egg mortality. Thus, the nutritive quality of the test may affect larvae chewing through the testa. Larvae reaching cotyledons vary in their growth rates but all survive. Mortality among pupae in chickpea and adults in pigeon pea occurred because disoriented prepupae failed to cut appropriate exits (chickpea) or pupated facing away from exits (pigeon pea). Food quality must have modified normal behavior. Combining first instar survival with pupal survival gives the effect of food quality. Survival relative to that in Berkin is compared 113 to an independent ranking by the oviposition preferences given in Table 10: Oviposition preference: cowpea = Berkin >> pigeon pea = chickpea II through IV larval survival: 0.89 1.00 0.68 0.22 The interaction of the three life table components just considered determines the overall prospects for an egg in the survivorship (lx ) of the adult, which is used, with fecundity, to obtain RQ . All the consequences of host preferences are combined in RQ : Host preferences: cowpea = Berkin >> pigeon pea = chickpea Rq : 0.62 1.00 0.55 0.15 Survival in the four hosts follows expectations based on the evolved host preferences. Chickpeas and pigeon peas are poor hosts in nearly all respects. Cowpea is a poor host because of high egg mortality. Competing larvae suffer a mortality of 50% and females search extensively for egg-free beans before putting second egg on a bean. Second eggs added within a few hours of the first have nearly a 50& chance of success, but prospects fall sharply as the age difference in eggs increases. The mortality of 50% or more suffered by larvae entering competition is a strong selective pressure maintaining the behavior that generates a uniform dispersion of eggs. Selection of hosts by size cannot be shown to have any effect on the survival or growth of single larvae. When larvae are in competition, larger beans may provide space for inhibited larva to escape and mature. Such escapes did not occur when two larvae of similar age were present, but multiple emergences do occur when densities 114 exceed two larvae per bean in cowpea (Bellows 1982b). Each of the decisions made by ovipositing females can be evaluated with direct measures of egg and larval survival. All oviposition decisions contribute positively egg success. SUMMARY A comprehensive analysis of the behavior and development of Callosobruchus maculatus, a pest of the major grain legumes in the tropics, provides detailed information on the complete life cycle from egg to egg. Adults emerge from beans, mate, females commit each egg to a bean when they oviposit and these oviposition choices fix the resources on which the larva must develop. The prospects for maturation are determined when females place eggs. Below, I summarize the events of the life cycle and the factors determining the success of the beetle. 1. Beetles emerge between 0800 and 1300 h and mate soon after emergence. About 80% of females are mated within 2 h. Multiple matings occur, though 85% of the females mated once produce the normal number of fertile eggs (60-90). 2. Unfed females deposit about 75 eggs, but if females feed on carbohydrates, the realized fecundity increases by about 40% to 125 eggs. This appears to be the potential fecundity attainable when all the protein reserves carried from the larval stage can be put into eggs. 3. Eggs are released in three cycles during the 10-day life span of a female and cycles are expressed even by females inhibited by lack of oviposition sites. These cycles appear to be waves of eggs maturing in the ovary. 115 116 Females denied oviposition sites withhold eggs. If beans become available the rate of egg deposition rises and normal fecundity may be realized. 4. Females discriminate among beans by surface texture, species, relative size and egg load. Rough-surfaced beans are strongly rejected. Bean species are discriminated independently and the largest available beans of a species are chosen first. Beans with more than the average number of eggs per bean are rejected, consequently, eggs are uniformly distributed over a set of beans. 5. Egg mortality is high on rough-surfaced beans, perhaps due to the difficulty of attaching eggs to these beans. The reason for high mortality on certain smooth-surfaced species is unknown. Deaths during the first larval instar are due to differences in the testa of various hosts. There are no deaths among larvae feeding on the cotyledons and, except for small differences in developmental times, the cotyledons of the four common hosts provide adequate nutrition for larval growth and fecundity of the female. 6. Chickpea and pigeon peas probably have chemicals that specifically affect the orientation of the prepupa when it prepares the pupal chamber. Adults from disoriented pupae die because they are unable to exit from the pupal chambers. If extracted from the cavity, such adults mate and oviposit normally. 7. A six compartment decision model accounting for the oviposition decisions of a female, gives the order of preference among the four principal hosts as; mung bean > cowpea > chickpea > pigeon pea. This order corresponds to the ranking of net reproductive rate for eggs maturing in each of these hosts. 117 8. If females have access to a bean population numbering about 20% greater than their fecundity, they will eliminate larval competition becuase they do not add a second egg to any bean. 9. If crowding forces females to place a second egg on some beans, and two larvae enter a bean, they respond to each other. A potential winner and loser are decided while larvae lie in separate burrows. The feeding of losers is inhibited. If larvae contact each other, the inhibited larva loses. Inhibited larva can survive if the burrows do not intersect or the dominant larva dies. 10. Larval responses are mediated through an exchange of signals between larvae with the cues almost certainly being the vibrations from larval chewing. Larvae remain responsive throughout life and the responses to competitive cues can be reversed at any time. 11. The life history of C_. maculatus can be divided into three sub-systems. The first is an algebraic expression specifying the realized fecundity of a female as the outcome of her activity, feeding, and abundance of oviposition sites. Second, the oviposition responses determined by the surface texture, species, size, and egg load of beans can be fit into a single model for oviposition decisions. Lastly, the life table for eggs at each kind of oviposition site defines the success of eggs and the positive correlation of net reproductive rates with cues inducing oviposition verifies contemporary selective pressures favoring the observed oviposition behavior. LITERATURE CITED Alzouma, I. 1981. 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