Running head: THE OVIPOSITION PREFERENCE OF C. MACULATUS 1 The Oviposition Preference of Callosobruchus maculatus and Its Hatch Rates on Mung, Pinto, Kidney, and Adzuki Beans Abbigail Traaseth, BIO 106-77 Piedmont Virginia Community College Introduction Many insects display preference for the placement of their eggs. Different leaves, trees, feces, and even beans can have an impact on the development and success of the insect’s offspring. But can the same be said of Callosobruchus maculatus, commonly known as the bean beetle? C. maculatus is a pest and a nuisance in much of West Africa, where it commonly develops on cowpeas found in storage containers (Sankara, et al., 2009). Hudaib et al. (2010) determined that storage containers are most likely targeted by the beetles due to the dry beans found inside, as opposed to fresh beans in the wilderness; it is thought that this is due to the structural stability of dry beans, and lack of predators indoors. This makes the bean beetle a particular threat to agricultural practices and communities that rely on beans and seeds for food. Most worrisome, the bean beetle is known to thrive on many different varieties of bean; introducing new hosts to the beetle has failed to decrease rates of copulation among similar seed beetles (Stojković et al., 2014). This information supports the notion that the bean beetle is a potential threat worldwide, but as of yet, it has not infested American storage containers. That said, it’s in our best interest to determine the bean and oviposition preferences for C. maculatus to deter the potential future problem. In our experiment, we gave five mating pairs the choice to lay their eggs upon either kidney or mung beans. It’s our hypothesis that there will be no oviposition preference between the beans, and that their hatch rate will be equal to the number of eggs laid on each bean. THE OVIPOSITION PREFERENCE OF C. MACULATUS 2 Methods The experiment compared combinations of two of the following beans for the females to choose from: mung beans, kidney beans, pinto beans, and adzuki beans. Four mating pairs of C. maculatus were placed in a 10cm petri dish with 8g of one bean, and 8g of another bean. After the appropriate beetles were placed among the beans, they were incubated at 27°C for a week. Next, the eggs laid on each bean were counted and tallied, using the Chi-square (χ²) (표−ⅇ)2 method to determine the significance of oviposition, using the formula χ² = ∑ . We then ⅇ compared the data to the critical value provided by the p=0.05 column of the χ² Distribution Table. Finally, the egg-laden beans were left for a month to continue incubation. Following the month, the hatch rate data was collected by counting the hatch holes found in each bean. These data were compared to the previous data, and the significance found by evaluating whether the beans were viable hosts by comparing eggs counted to hatch holes found. Results Table 1 shows the individual team’s results of eggs laid on each bean type. The Chi- square analysis for the collective data produced significant results that suggest oviposition preference was displayed by the female bean beetles. In every case but Team 5’s, where the adzuki and mung comparison proved to be fairly similar, the mung bean was preferred exponentially over any other bean choice. THE OVIPOSITION PREFERENCE OF C. MACULATUS 3 Table 1. Eggs laid on each bean type for each team bean pairing. Total Total Expected Chi-square (χ²) Kidney Pinto Adzuki Mung Observed per Bean Type Value Team 1 31 66 nc nc 97 49 12.51 Team 2 nc 7 54 nc 61 31 35.65 Team 3 65 nc 40 nc 105 53 5.91 Team 4 nc 7 nc 47 54 27 29.63 Team 5 nc nc 68 83 151 76 1.49 Team 6 43 nc nc 101 144 72 23.36 The hatch holes and the hatch rate percentage for each bean per team are shown in tables 2 and 3. Once again, the Chi-square analysis proved these results significant, with a much higher successful hatch rate for eggs laid on mung beans than any other bean host. The experimental bean subjects are shown in figures 1 and 2, which shows Team 6’s petri dish environment, and a mung bean hatch hole. THE OVIPOSITION PREFERENCE OF C. MACULATUS 4 Table 2. Number of hatched eggs counted on each bean type. Kidney Pinto Adzuki Mung Team 1 0 6 nc nc Team 2 nc 3 8 nc Team 3 0 nc 11 nc Team 4 nc 1 nc 33 Team 5 nc nc 15 56 Team 6 0 nc nc 46 Table 3. Hatch rate percentage per team’s bean pairings. Kidney Pinto Adzuki Mung Team 1 0% 9% nc nc Team 2 nc 43% 15% nc Team 3 0% nc 28% nc Team 4 nc 14% nc 70% Team 5 nc nc 22% 68% Team 6 0% nc nc 46% THE OVIPOSITION PREFERENCE OF C. MACULATUS 5 Fig. 1 (left). Team 6 petri dish environment of 8g kidney beans and 8g mung beans, photographed during the second experimental observation. Fig. 2 (right). Detailed photo of one mung bean hatch hole made by an emerging C. maculatus. We see in table 3 that the mung beans were the only bean host that had close to or greater than 50% hatch rate, compared with its rivals, which were all below 50%. With this information, we reject both null hypotheses: we see that there is an oviposition preference, and the success rate of hatching is dependent on bean. THE OVIPOSITION PREFERENCE OF C. MACULATUS 6 Discussion Our initial experiment was aimed to explore the possibility of the female bean beetle’s oviposition preference, and whether her choice was indicative of the future hatching success of her offspring. As we discovered, her preference for the mung bean was significant, and for good reason; the offspring had the highest chance of surviving when laid on mung beans. The least successful host was the kidney bean, which had zero hatch holes across all team pairs; followed by the pinto bean; and finally, the adzuki bean, whose structure was most similar to the mung bean in size and shape. Though we don’t yet understand the biological reasons for the success rates found on mung beans (nutrition? Penetrability of shell?), our original bean beetle mating pairs were laid on and hatched from mung beans, so it follows that when given a choice between a familiar or an unknown host, they would prefer what they know. Unfortunately, there were no experimental parameters put in place to measure how the beetles recognized the mung beans as familiar, but we assume that the smell, taste, and sight were all recognizable from their original petri dish environment, to their experimental environment. While there is enough data to feel confident that a preference has been indicated, the results may be skewed to represent higher hatch rates than came from our original four mating pairs. This is due to the timing of our original observations, which were carried out a month before the hatch data was collected; this gap would offer sufficient time for a second or third generation of bean beetles to contribute to the eggs laid and hatched on our host bean population. For example, the total original egg count on Team 6’s 8g of kidney beans was 43; the following month, the second counting yielded roughly 20 eggs per bean, resulting in ~200+ eggs. This increase is monumental, yet none of these eggs hatched from the kidney beans. The subsequent choice to lay eggs on kidney beans for the future generations could have been due to the limited THE OVIPOSITION PREFERENCE OF C. MACULATUS 7 choice available to them, as all of the newly hatched beetles came from mung beans, reducing the available mung bean hosts from 8g to something unobserved, but almost definitely smaller. Given little to no choice, the female has proved to be willing to risk laying her eggs on questionable beans, but even so, her offspring will not be viable. Overall, this is a good sign for kidney bean farmers. In the meantime, barring any unforeseeable advancements of the C. maculatus’ proliferating abilities, kidney beans will not contribute to the spread of bean beetles in any part of the world, nor will they be targeted by these particular storage container pests. THE OVIPOSITION PREFERENCE OF C. MACULATUS 8 References Hudaib, T., Hayes, W., Brown, S., & Eady, P. E. (2010). Effect of seed moisture content andd- limonene on oviposition decisions of the seed beetle Callosobruchus maculatus. Entomologia Experimentalis et Applicata, 137(2), 120-125. doi:10.1111/j.1570-7458.2010.01044.x Sankara, F., Dabiré, L. B., Ilboudo, Z., Dugravot, S., Cortesero, A. M., & Sanon, A. (2014). Influence of host origin on host choice of the parasitoid Dinarmus basalis: Does upbringing influence choices later in life?. Journal of Insect Science, 141-11. Stojković, B., Savković, U., Đorđević, M., & Tucić, N. (2014). Host-shift effects on mating behavior and incipient pre-mating isolation in seed beetle. Behavioral Ecology, 25(3), 553-564. .