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Table of Contents
- Culture Techniques
- Generation Time
- Identifying the Sexes
- Handling Techniques
- Measuring Beetles
- Mating Beetles
- Isolating Virgins
- Disposal of Cultures
This handbook provides basic information about raising, handling, and maintaining cultures of bean beetles, Callosobruchus maculatus. Our intended audience is the faculty and staff who teach or coordinate undergraduate laboratory courses. The information provided here is based on our own experiences working with this insect in undergraduate laboratory courses, and information available in the research literature. Our references to specific commercial vendors for supplies are intended to assist you in finding the types of supplies we have used, but these references are not an endorsement of these particular vendors. Comments to the authors with corrections or suggestions for additional information to include in future versions of this handbook would be much appreciated.
Bean beetles, Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae), are agricultural pest insects of Africa and Asia that presently range throughout the tropical and subtropical world. This species also is known as the southern cowpea weevil. The larvae of this species feed and develop exclusively on the seed of legumes (Fabaceae) hence the name bean beetle. The adults do not require food or water and spend their limited lifespan (one – two weeks) mating and laying eggs on beans. The systematic placement of bean beetles is as follows: Callosobruchus is one of the genera in the subfamily Bruchinae (seed beetles) that is in the family Chrysomeloidae (Kergoat et al. 2007). This group is part of the order of beetles, Coleoptera (from Greek “sheath-winged” referring the stiff outer, first pair of wings (elytra) that protect the membranous second pair of flight wings). The Coleoptera is largest of the orders that comprise the class Insecta. Insects are the largest and most diverse (750,000 described species) of all the animal classes that are found in all but marine environments. Insects are protostomous animals and are thus more closely related to mollusks and crustacea than to the deuterstomous vertebrate classes. See the Integrated Taxonomic Information System on the subfamily Bruchinae for more information on the systematic placement of Callosobruchus (ITIS 2011). The systematic placement of seed beetles in the family Chrysomelidae (rather than in their own family) is relatively recent so some websites on animal taxonomy may not yet reflect this change. Additional information about the taxonomy of C. maculatus, ecological associations, and information about other species of Callosobruchus may be found at the Encyclopedia of Life website (EOL 2011). Callosobruchus maculatus forms a monophyletic group with C. analis, C. rhodesianus, and C. subinnotatus, all species that use dry beans in the genus Vigna as their natural hosts (Figure 1, Tuda et al. 2006).
Bean beetles exhibit two adult forms (morphs), a sedentary (flightless) form and a dispersal (flying) form. The dispersal morph is induced by high larval density in stored beans or laboratory cultures, and is caused by density dependent micro-habitat temperature increases (Utida 1956, 1972,). Induction of the dispersal morph allows individuals to move to new, higher quality habitats. These two morphs have very different life history characteristics such as longer adult lifespan in the dispersal morph and significantly reduced fecundity compared to the sedentary morph (Utida 1956, 1972). In the sedentary form, the sexes are highly dimorphic and readily distinguished but sex differences are very subtle in the dispersal form. Thus, it is essential to maintain laboratory cultures at low density (one or two larvae per bean) and temperatures no greater than 30°C, if individual beetles need to be unambiguously identified by sex (see Identifying the Sexes).
We are frequently asked, by students and even fellow academics, what is the purpose of bean beetles? A very short adult life span and a larval stage in which most or all life-time feeding occurs is not unusual in insects (for example the Order Ephemeroptera, mayflies), but this life cycle seems strange compared to the dominance of adult stages in familiar birds and mammals. Ultimately, in evolutionary terms, the purpose of bean beetles is the same as in all other living things, reproduce and leave descendants.Ecologically, bean beetles are herbivores that have specialized on seed consumption.They are a part of food webs in that eggs and larvae are prey for parasitoid wasp species (Boeke et al. 2003), and adults may be prey for birds, reptiles and amphibians, so they do have the purpose of providing food for other organisms.
Once inseminated, adult females will lay (oviposit) single fertilized eggs on the external surface of a bean. Individual eggs (0.75mm long) are oval or spindle shaped, clear, shiny and firmly glued to the bean surface (Figure 2a). The larva that hatches from the egg burrows from the egg through the seed coat and into the bean endosperm without moving outside the protection of the egg. Once the larva burrows into the bean, the remaining egg (shell) becomes opaque white (Figure 2a) or mottled as it fills with frass (feces) from the larva. The larva (Figure 2b) burrows and feeds on the bean endosperm and embryo, undergoes a series of molts, and burrows to a position just underneath the seed coat prior to pupation. Although the seed coat of the bean is still intact, a round 1-2mm window is apparent at the location where the beetle is pupating (Figure 2c). Pupation (Figures 2d) is the complete metamorphosis of the larval maggot to a winged adult. The adult that results from pupation chews through the seed coat and emerges from the bean (Figure 2e and 2f). The adults are fully mature 24 to 36 hours after emergence. Males seek females to inseminate (see Mating Beetles) and females store viable sperm in their spermatheca (a structure in the female reproductive tract for storing sperm). Neither male nor female adults require food or water during their short adult lifetime (10-14 days).
Callosobruchus maculatus has a karyotype of ten chromosome pairs (2N=20) (Yadav 1971). Chromosome 10 is a sex chromosome and males are the hetergametic sex (Figure 3). One Mendelian trait has been described for bean beetles, body color which is autosomal and has alleles with incomplete dominance (Eady 1991). Heritable variation in body size is well described (Fox et al. 2004) as are life-history and behavioral traits such as longevity and copulation duration (Brown et al 2009). Some genes have been isolated and characterized, for example, genes with environmentally relevant transcription regulation (Chi et al. 2011). The nucleotide database in the NCBI GenBank has more than 175 entries for C. maculatus. Several transcriptome assemblies are available through GenBank and are available for BLAST search on this website.
Fertilization occurs as females lay eggs and glue them to the surface of a bean seed (Fabaceae). Early embryonic development occurs inside the transparent egg until the first instar larva (maggot) burrows through the seed coat into the seed endosperm directly from the egg. Bean beetles development is long-germ (Patel et al. 1994) similar to Drosophila in which body segment determination occurs by the end of the blastoderm formation. The empty egg shell is typically filled with white frass (fecal waste) as the larva feeds (Figure 2a). There are four larval instars (Figure 4, Devereau et al. 2003) all feeding inside the endosperm of the seed on which the egg was laid. Pupation occurs inside the seed and an adult emerges by chewing and removing a circular piece of the seed coat to form a round exit hole (Figure 2f). Temperature, the species of bean chosen for egg laying, and relative humidity all influence development time and success (see Generation Time).
Starting new cultures requires no more than containers to contain beans and beetles. Virtually any closable containers will work successfully: lidded plastic Petri dishes, screen covered glass jars, snap lid vials, and cotton plugged shell vials are all suitable containers. We prefer to use disposable plastic containers in our teaching laboratories to minimize breakage and to keep cultures relatively small but replicated. Although bean beetles are the easiest of insects to successfully culture, sometimes a culture will fail if adults were very old when introduced to the new culture container or too few adults were introduced for adequate numbers of eggs to be laid. Therefore, we always start stock cultures in pairs (or more if needed) on the same date and we never dispose of old cultures until we see that a new culture has successfully yielded adults. It is always a good practice to check a new culture a few days after it is started to see if numerous eggs have been laid. Plastic Petri dishes 150 x 25 mm (Falcon 351013, Fisher Scientific 08-757-145 or VWR 25379-048) are ideal containers from which students can easily view cultures and remove selected adults. Although the lids fit loosely on the plates, Petri dishes will confine adults and permit adequate ventilation without any modification. Covering the bottom of a Petri dish with a single layer of beans (approximately 50 ml volume) and introducing 10 adults males and 10 adult females is sufficient to produce a dense culture. Cultures established in this manner will typically sustain two or three sequential generations without adding additional beans and without inducing the production of dispersal morph adults. Cultures older than three generations on the same set of beans should be discarded (see Disposal of Cultures).
We also have had good results using plastic snap-lid containers ( 300 ml Corning Snap-Seal Sample Containers, Fisher Scientific 02-540-23) with pin-holes punched in the lid for ventilation. As with the Petri dishes, we use only 50 ml of beans in each 300 ml snap-lid container. The ideal seeds (beans) to use are mung (Vigna radiata = Phaseolus aureus), blackeye peas or cowpeas (Vigna unguiculata) and adzuki (Vigna angularis) (Figure 5). Although bean beetles can be reared successfully on these three species, they differ in nutrient quality (USDA Agricultural Research Service) and secondary compounds (Bisby et al. 1994). We find that raising beetles on blackeye peas is best done in 150 mm petri dishes which minimizes mold growth on the beans. The successful completion of the bean beetle life cycle on most other bean species is minimal (Janzen 1977), but we have successfully cultured bean beetles on pigeon peas (Cajanus cajan) and hyacinth beans (Lablab purpureus). Dark (completely black or brown) varieties of blackeye peas will support normal development of bean beetles and have the advantage of making it easier to see newly laid eggs. These varieties should not be confused with black beans (Phaseolus vulgaris) that are toxic to bean beetles (Janzen et al. 1976). We prefer to use organically grown beans to minimize pesticide problems in our cultures, but it is not essential for successful culturing of beetles. Dry beans and adult beetles in a container that keeps the beetles from escaping are all that you need. Keep the cultures at temperatures between 22° – 30°C (not in direct sunlight and away from radiators).
The elapsed time from newly laid eggs to the emergence of adult beetles varies between bean beetle strains and environmental conditions. Previous studies indicate that temperature and relative humidity (Howe and Currie 1964, Schoof 1941) are the most important variables influencing generation times (egg to adult) when beetles are raised on preferred host beans. Within a limited range, increasing temperature will decrease the generation time. In our laboratory, we have observed generation times as short as 3-4 weeks in a 30°C incubator (12:12 day:night light cycle) and ambient humidity (averaging 30% RH and ranging from 20%-40% RH). Cultures raised in a 25°C incubator (12:12 day:night light cycle) and ambient humidity (averaging 50% RH and ranging from 40%-60% RH) had generation times of 4-5 weeks. Cultures maintained on a laboratory bench at room temperature (22°C) with indirect outdoor window lighting and ambient humidity (averaging 50% RH and ranging from 40%-60% RH) had a generation time of a full 7 weeks. Reliably obtaining newly emerged adults for a specific date (a scheduled laboratory class meeting) requires that you grow cultures for a few months under your laboratory conditions so you can predict emergence times. In low humidity locations, or during the winter in North America, when RH is typically low, the emergence rates of beetles may be improved by increasing the RH of an incubator or culture container. Simply placing a tray of water in a temperature controlled incubator may be all that is necessary to bring RH to the 40%-60% range, and improve emergence success rates.
Generation time also depends on the host species of bean you choose to use. We have found longer generation times in adzuki beans compared to either mung or black-eyed peas. At 30°C, it takes seven weeks for emergence from adzuki beans compared to 3-4 weeks from mung beans. Bean beetles grown on pigeon peas and hyacinth beans have generation times similar to those on adzuki beans.
Identifying the Sexes
Male and female bean beetles (of the sedentary morph) are easily distinguished from one another by general appearance. The most distinguishing characteristic is the coloration on the plate covering the end of the abdomen. In the female, the plate is enlarged and is darkly colored on both sides (Figure 6). In the male, the plate is smaller and lacks stripes. Generally, females are larger in size than males, but there is much variation. In some strains, females are black in coloration and males are brown (Figure 6), but in others both sexes are brown.
Although sedentary morph bean beetles are capable of flying, they rarely do. As a result, they are easy to handle. Beetles can be moved either using Drosophila sorting brushes (Carolina Biological 17-3094; Ward’s Natural Science 15 V 3846) or soft forceps (Bioquip TM featherweight forceps 4748 or 4750; Ward’s Natural Science 14 V 0520).
When removing beetles from stock cultures, individual Petri dishes, or well plates, tap the containers lightly on the lab bench before removing the lid to prevent beetles from crawling out immediately. If the lid is left off for more than a minute or so, beetles will escape from the culture dishes.
Especially when kept individually, bean beetles will often “play dead.” Don’t be fooled! A gentle prod with forceps or brush will cause them to move.
Body Mass – To weigh individual beetles, a 0.1 mg analytical balance, at a minimum, is necessary (for example, Ohaus Analytical Balance Model PA64, Carolina Biological 70-2498, Fisher Scientific S97282) . Individual beetles can be placed in the bottom of a 35mm Petri dish (for example, Falcon 351008, Fisher Scientific 08-757-100A or 60mm dish, Carolina Biological 741246) to be weighed.
Linear Measures – Linear measures of body size, such as the length of the elytra (the hard wing covers), may be readily collected on dead adults. Such measurements may be facilitated by using an inexpensive microscope video camera, such as the Moticam 352 (Carolina Biological 591282) attached to the eyepiece of a dissection microscope. This video camera connects directly to a computer (Mac or Windows PC) via the USB port and measurements are made by using image analysis software (included with the camera) to evaluate the length of a line drawn on a body part in a captured image or in a live video image. Dead animals may be sorted by sex and glued to file cards for measurement under a dissection microscope. A free image analysis program (NIH Image J) may be used to make measurements on any digital image, including those captured with a Motic camera.
Both virgin and non-virgin beetles will mate readily. However, virgin males may not produce fully formed spermatophores until 24 hours after emergence. In addition, females may not mate for several hours after a previous mating.
To mate beetles, place beetles into a 35mm Petri dish (for example, Falcon 351008, Fisher Scientific 08-757-100A or 60mm dish, Carolina Biological 741246). Males will chase females until they are able to mount and copulate with females (Figure 7). Copulation generally begins within 10-15 minutes, but sometimes may not begin for 30 minutes to an hour.
To determine if a male transferred a spermatophore successfully during copulation, weigh the male before and copulation. Males may lose as much as 5% or more of their body mass due to spermatophore transfer. However, spermatophore size will decrease with subsequent mating by a particular male.
To isolate virgin beetles, place a mated female into a 35mm Petri dish (for example, Falcon 351008, Fisher Scientific 08-757-100A or 60mm dish, Carolina Biological 741246) with a single layer of beans. The vast majority of females in a stock culture will have mated and are capable of laying fertile eggs. After 12-24 hours, females will begin to lay eggs on beans. Withan excess of beans, females will lay only a single egg on each bean. Finding eggs on beans can be facilitated by using a dissection microscope (at 10x total magnification) or a large magnifying glass (2.5x magnifier Fisher Scientific 14-648-19 or VWR 62379-535). Remove the beans with single eggs (Figure 8) and place each bean in an individual 35mm Petri dish or the well of a 6 or 12-well flat bottom tissue culture well plate (Falcon tissue culture plates, Fisher Scientific). Replenish the beans as they are removed. A single female can produce more than 100 eggs in her lifetime. In general, the sex ratio is 1:1.
Disposal of Cultures
Bean beetles are a potential agricultural pest insect that is not distributed throughout the United States and Canada. This species is absent in much of North America because it is intolerant to freezing temperatures and suitable host plant species are not among our native (non-agricultural) flora. None-the-less, it is prudent and appropriate to dispose of living adults, and beans that have had contact with living adults, in a manner that will prevent their release to the natural environment. Placing a live culture (or any beans exposed to adults beetles) in a freezer (0°C) for a minimum of 72 hours prior to disposal will ensure that beetles at every life cycle stage are dead. Then, dispose the frozen culture in the same manner as food waste.
Boeke, S.J., A.A.C. Sinzogan, R.P. de Almeida, P.W.M. de Boer, G. Jeong, D.K. Kossou, and J.J.A. van Loon. 2003. Side-effects of cowpea treatment with botanical insecticides on two parasitoids of Callosobruchus maculatus. Entomologia Experimentalis et Applicata 108:43–51.
Brown, E.A, L. Gay, R. Vasudev, T. Tregenza, P.E. Eady, and D.J. Hosken. 2009. Negative phenotypic and genetic associations between copulation duration and longevity in male seed beetles. Heredity (2009) 103, 340–345.
Chi, Y.H., J.-E. Ahn, D.-J. Yun, S.Y. Lee, T.-X. Liu, and K. Zhu-Salzman. 2011. Changes in oxygen and carbon dioxide environment alter gene expression of cowpea bruchids. Journal of Insect Physiology 57:220-230.
Devereau, A.D., I. Gudrups, J.H. Appleby, and P.F. Credland. 2003. Automatic, rapid screening of seed resistance in cowpea, Vigna unguiculata (L.) Walpers, to the seed beetle Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) using acoustic monitoring. Journal of Stored Products Research 39:117-129.
EOL. 2011. Callosobruchus maculatus (Fabricius, 1775)”. Encyclopedia of Life. http://www.eol.org/pages/1172676 Accessed 20 March 2011.
Howe, R.W. and J.E. Currie. 1964. Some laboratory observations on the rates of development, mortality and oviposition of several species of Bruchidae breeding in stored pulses. Bulletin of Entomological Research. 55:437-477.
ITIS. 2011. Bruchinae (Latreille, 1802). Integrated Taxonomic Information System. http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=678800 ITIS Report accessed 20 March 2011.
Kergoat, G.J., J.-F. Silvain , A. Delobel , M. Tuda, K.-W. Anton. 2007. Defining the limits of taxonomic conservatism in host–plant use for phytophagous insects: Molecular systematics and evolution of host–plant associations in the seed-beetle genus Bruchus Linnaeus (Coleoptera: Chrysomelidae: Bruchinae). Molecular Phylogenetics and Evolution 43 (2007):251–269.
Tuda, M., J. Rönn, S. Buranapanichpan, N. Wasano, and G. Arnqvist. 2006, Evolutionary diversification of the bean beetle genus Callosobruchus (Coleoptera: Bruchidae): traits associated with stored-product pest status. Molecular Ecology 15:3541–3551.
USDA Agricultural Research Service, Nutrient Data Laboratory. USDA National Nutrient Database for Standard Reference.http://www.nal.usda.gov/fnic/foodcomp/search/ Accessed 25 June 2009.
Utida, S. 1956. “Phase” dimorphism observed in the laboratory population of the cowpea weevil, Callosobruchus quadrimaculatus . 2 nd Report. Differential effects of temperature, humidity and population density upon some ecological characters of the two phases. Researches on Population Ecology 3:93-104 (in Japanese, abstract in English).