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Bruchid methods

 Environmental Conditions for Lab Cultures

A haphazard survey of the literature reveals high variation in temperatures and photoperiods used for Callosobruchus spp. I encountered the following combinations (temperature, hours of light:hours of dark):

27°C, 24:0
27°C, 13:11
25°C, 24:0
30°C, 24:0
30°C, 16:8
25°C, 12:12
30°C, 16:8
30°C, 0:24

Relative humidity (r.h.) also varies among studies. Schoof (1941) observed that "optimum humidity regarding survival is approximately 44 per cent." Development times are reduced at higher values but fungal growth concomitantly rises. A range of 40-70% r.h. is typically recommended.

In my lab, I culture C. maculatus and C. chinensis at 28°C, 16:8, and 40% r.h.

 Manipulating Egg/Larval Densities*

Remove a reasonably large number of adults (250+) from the main culture, soon after they have emerged. Place these individuals in a separate container that has a reasonably thin layer of fresh beans of the variety that you are interested in for your experiment. Males and females will mate (if they haven't already done so) and females should begin to lay eggs within minutes. Approximately 48 hours later, separate the beans from the adults. Waiting this long should allow most of the eggs to harden before you handle them, while preventing larvae from chewing their way into the bean proper. Using a magnifying glass (or equivalent) to help you locate eggs on beans, scrape excess eggs off of beans with a scalpel or other similarly sharp instrument. To minimize the chance that you're dealing with a bad bean, avoid beans with only a single egg on them, even if you are attempting to reduce egg densities to egg per bean. Depending on the requirements of your experiment, you will either want to place individual beans into individual containers (I find that small plastic centrifuge tubes with a hole poked in the lid work well) or you can pool beans with a given egg density into larger (marked!) containers.

* as in Vamosi SM. 2005. Functional Ecology 19: 859-864.

 Collecting Bruchid Egg-Parasitoids**

The eggs of bruchids are commonly elongate in shape and relatively small ca. (0.5-0.6 mm), usually clearly visible on the host plants, especially in high density bruchid populations. They may be easily found, because they are precisely located on the generative parts of plants. Bruchid larvae develop in the seeds of different host plants families: Fabaceae (Papilionoideae, Caesalpinioideae, Mimosoideae) pulse crops - 84%, Arecaceae - 4.5%, Convolvulaceae - 4.5%, Malvaceae - 2%, and 29 other families - 5% (Acanthaceae, Anacardiaceae, Bignoniaceae, Bixaceae, Bombaceae, Boraginaceae, Cistaceae, Cochlospermaceae, Combretaceae, Compositae, Convolvulaceae, Discoceraceae, Ebenaceae, Euphorbiaceae, Lauraceae, Lythraceae, Malpigiaceae, Myrtaceae, Nyctanaceae, Nymphaceae, Ochnaceae, Onagraceae, Pandanaceae, Rhamnaceae, Sterculiaceae, Tiliaceae, Verbenaceae, Vitaceae, Zygophyllaceae). The most common and well known host plants are legumes, and include such well known plants as Phaseolus, Vicia, Astragalus, Acacia, Lathyrus, etc.

Bruchids lay their eggs on the surface of legume pods, covering leaves over the seeds, or flowers. Eggs are usually deposited singly or, rarely, in small groups. They look like bright drops on the surface of the plant pods. Young unparasitized bruchid eggs are pale yellow. When the egg is parasitized, it becomes dark yellow, orange, and then grey. Some bruchid eggs are hidden near the seed boxes or seed pod. For example, Callosobruchus lay their eggs between the covered leaves over the seed boxes of Convolvulus (Convolvulaceae). These eggs are partially flattened and united in small groups. They may be easily found by closely examining the seed boxes of Convolvulaceae.

The fresh eggs of Bruchidae are pale white, and hatchling larvae bore inside the pods of legumes or seed boxes of their host plants. The empty eggs of bruchids are transparent, with small pieces of plant tissues inside, covering the entrance holes of the larvae. The different species of Bruchidae prefer to lay their eggs on different developmental stages of legume pods. The legume pods may be small and green, or more developed and mature, which are usually brown and dried. The parasitized eggs of bruchids change their color from pale white to bright yellow and orange, becoming grey in the last stages. The pupa of egg-parasitoid with its red eyes may be visible through the transparent egg shell.

The emerged egg-parasitoids cut a hole on the upper side of the egg's shell. Usually only one egg-parasitoid can develop inside the single bruchid egg. The rearing of egg-parasitoids can be accomplished in a small glass tube (10-15 mm diameter, 30 mm long). The small sections of legume pods with bruchid eggs on their surface may be placed in these glass tubes until the emergence of egg-parasitoids. The green seeds of legumes can be removed from the pods to reduce moisture accumulation inside the tubes, avoiding damage by fungi. The emerged and dead egg-parasitoids (Uscana sp.) may be saved for future taxonomic study in separate glass or plastic tubes (5 x 15 mm), carefully closed by a cotton plug and kept in dried conditions. There is no need to keep the egg-parasitoids in alcohol. The labels must contain: locality, collector, plant, host, dates of collection, and emergence. Large amounts of field collected pods of Fabaceae can be collected in small tissue bags (10 x 15 cm) for rearing Bruchidae adults and their egg-parasites. A large sample of young, green bean pods is easier and safer to collect in tissue bags rather than glass boxes. Small samples of pods can be selected into separate glass tubes. The adults of Bruchidae can be successfully reared from mature pods of Fabaceae mostly, but also from seeds of other plant families. Identified adults of Bruchidae are valuable for the identification of specific egg-parasitoids.

**based on an article by Victor Fursov for the Chrysomela Newsletter (Vol. 32, 1996), with updated plant family names

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