Oviposition, or the laying of eggs, is broken down into pre-oviposition and post-oviposition. In this experiment we observed pre-oviposition factors using the model species Callosobrachus maculatus, commonly known as the bean beetle. Small and large lima beans were used to test whether or not bean beetles had a preferred site size for oviposition. We hypothesized that if a preference was shown, the majority of eggs will be oviposited on the larger lima beans when compared to small lima beans. To do this we placed 15 small and 15 large lima beans in 3 petri dishes, then added 3 female and 2 male bean beetles in each and waited 2 weeks to allow fertilization and oviposition. Because our subject was exposed to both sizes of beans throughout the experiment, we interpreted our results by observing the number of eggs laid on each individual bean. From our observations we calculated the mean, standard deviation, standard error of mean, and for statistical purposes, ran a t-test. We found our results to be statistically significant, therefore we rejected the null hypothesis. From this we can concur that C. maculatus prefers smaller sized beans for oviposition.
Introduction
Oviposition, a commonly used term to describe “laying of eggs”, is broken down into two stages, pre-oviposition and post-oviposition. Pre-oviposition comprises of “all the behaviors and factors involved in the selection of, or attraction to, an oviposition site and oviposition itself” (Downes and Lancaster 2013). Individuals locate suitable sites for their offspring by seeking visual and chemical cues throughout the environment; even the behavior through which these individuals lay their eggs depends on environmental factors. Post-oviposition occurs after the eggs have left the female and involves several different “strategies for ensuring that development of the embryo can proceed” (Downes and Lancaster 2013). Some examples include camouflaging the egg to resemble its site, “devices for attaching eggs to substrates”, and nutrient and gas exchange within the egg (Downes and Lancaster 2013). The site of oviposition is determined by behavioral and environmental cues that increase the probability of the offspring’s survival and the parent’s fitness. This is why the significance of where the organism selects to oviposit his or her offspring is crucial for their offspring’s survival, and his or her fitness.
Laying eggs is crucial part of the life cycle for oviparous animals. “An important factor affecting the life-history of an organism is parental investment in reproduction: reproductive decisions are almost invariably costly” (Kotiaho and Paukku 2008). Our experiment is focused on pre-oviposition by measuring whether or not bean beetles prefer small or large lima beans to oviposit on. Oviposition preference is dependent on size, small or large, of a lima bean. If bean beetles prefer large lima beans, will we see any eggs deposited on small lima beans? Or, if there is no preference, will we see near-equal numbers for both categories?
For this experiment our model organism is Callosobrachus maculatus, commonly known as the bean beetle. C. maculatus is frequently found in reproductive experiments because they are easy to maintain, adults do not require food or water and the tropical/subtropical regions they are naturally found in can be easily duplicated, and they spend their entire lifespan (around 1-2 weeks) mating and laying eggs (Beck and Blumer 2011). The reason why this organism was chosen for our experiment is because the larvae of this species exclusively feeds and develops on the seed of legumes (Beck and Blumer 2011). We believe that if a preference is shown, a greater number of eggs will be oviposited on the larger lima beans when compared to small lima beans.
Methods
To begin we gathered 3 individual petri dishes with lids and put 15 large and 15 small lima beans in each dish. All lima beans used in this experiment were gathered from the same source. Next, we obtained the beetles from a laboratory container which supplied the class for this experiment. 3 randomly chosen female and 2 randomly chosen male bean beetles were placed in each petri dish. This was done by using an aspirator to suck each beetle into a collecting vial, then releasing the beetle into its prearranged petri dish. Finally, the petri dishes were placed in a room-temperature location for 2 weeks to allow enough time for fertilization and oviposition.
After 2 weeks, each individual bean was observed under a microscope and examined for eggs. To calculate our results we used the final measurements. Our ending measurements were obtained by observing, through the microscope, which beans had eggs laid on them. We recorded our observations by numbering how many eggs were found on each bean. After observing all 90 lima beans, we returned the beans back to their original groups to determine group averages. After, all 45 small and 45 large beans were placed together in separate categories. From this we were able to use our findings to calculate mean, standard deviation, and the standard error of the mean. After analyzation, we ran a t-test to determine whether size of the lima bean had any influence on the oviposition of C. maculatus.
Results
Our results were obtained from observing the amount of eggs laid on each individual lima bean within their groups. For group 1 we found 11 eggs oviposited on the small beans, 5 were on the large. In group 2 there were 29 on small, 13 on large. In group 3, 48 on small, 0 on large. In total, 88 eggs were laid on 45 small beans, whereas only 18 eggs were laid on the 45 large beans. This information was used to calculate group averages, visualized in graph 1. Data is expressed using a clustered column histogram; the small lima beans are represented in blue, the large in red.
Graph 1. Average number of eggs laid by Graph 2. Average number of eggs laid by C.
C. maculatus per bean for all 3 groups. maculatus per bean. Averages were calculated
Each group contains 2 categories comprised by taking total number of eggs observed divided
of 15 small and 15 large lima beans. by 45, each cat. sample size. SEM error bars are
Averages of each category were found by shown: small is +/- .2998, large is +/- .1163.
dividing number of eggs observed by 15.
Next all 45 small and 45 large beans were placed together into 2 sizable groups. We used our findings to calculate mean, standard deviation, and the standard error of mean, as seen in table 1 on the next page. The mean amount of eggs left on small lima beans is 1.95556, with a standard deviation of +/-2.010829. For large lima beans, the mean is 0.4 with a standard deviation of +/- 0.780442. Means can be observed in graph 2. After conducting a t-test, we found that the averages of our two sizes were significantly different (t = 4.83779, df = 57, p = 1.0367E-05). Because our two-tailed P value equals 0.0000103, we reject the null hypothesis because this result is extremely statistically significant.
Small Bean Large Bean
Mean 1.95556 0.4
SD +/- 2.010829 +/- 0.780442
SEM 0.299756 0.116341
N 45 45
Table 1. Results of experiment. 2 categorical groups,
small and large lima beans, are compared. Mean and
SD measurements are in “eggs per bean”. Sample size
is reflected by N.
Discussion
In this experiment, small and large lima beans were used to test whether or not bean beetles had a preferred site size for oviposition. Our hypothesis proposed that if a preference was shown, a greater number of eggs would be oviposited on the larger lima beans compared to small lima beans. We predicted that they would prefer the larger bean because it has a greater surface area, therefore finding a spot on the bean to oviposit would be easier to do. On the contrary, our results show that we reject the null hypothesis due to the fact that our p-value was so extremely small. Because of this, we can conclude that C. maculatus prefer smaller sized lima beans for oviposition. The preference for a smaller lima bean could be due to a chemical cue preferred by, or undesirable to, bean beetles. Another reason could be that the nutrient to surface area ratio is greater, or because larger beans may be more appealing to predators. According to a similar experiment conducted by Jason Cope and Charles Fox, bean beetle eggs were distributed so that resources were maximized per individual offspring (2002). They found that females preferred a larger mass compared to surface area due to the quantity of resources available inside the seed. Although our experiments measured different variables, in both findings we can identify that a larger surface area is not ideal for bean beetle site preference for oviposition.
In an experiment conducted by Grace Pitman, Tyler Flockhart, and Ryan Norris, they measured which size and what density of a milkweed patch was preferred by the monarch butterfly for oviposition (2018). Their results showed that a small, low-density patch had the highest egg density. This was because larger patches showed an increase in predator abundance (Flockhart et al. 2018). When determining sites for oviposition, the one that increases probability of offspring survival is more desirable. Therefore, choosing a small, low-density site for oviposition increased the probability that the offspring would survive and reproduce. This relates to our experiment because we tested to find the preferred site that would increase survival in bean beetle offspring and increase fitness for its parent.
This experiment could be helpful towards future studies determining site preferences for other insects. Although, there are several ways to adjust our experiment for future studies. For example, acquiring a larger sample size and doing additional trials would reduce random error. Another way to improve our experiment would be to record the mass and surface area of each individual bean.