Biology Abstracts

There is public concern about the use of chemicals in agriculture causing environmental and health hazards. This has led to an interest in botanical insecticides due to their minimal ecological side effects and cost. Previous studies tested essential oils as  insecticides and didn’t focus on preserving the lives of pests that are also pollinators, namely the cabbage white butterfly (Pieris rapae). We conducted lab and field experiments to determine if solutions of peppermint (Mentha piperita L.), rosemary  (Salvia rosmarinus), and garlic (Allium sativum) are effective oviposition and feeding repellents against P. rapae. We predicted garlic would be the strongest oviposition deterrent. To test this hypothesis decoctions of garlic, peppermint, and rosemary were produced at a 4:1 ratio. Choice and no-choice assays were performed in the lab. Ten females were placed in individual cages with B.oleracea plants treated with the solutions and randomly placed within the cage. The number of eggs was counted after 24 hours. Eggs were allowed to hatch and caterpillars were reared on treated B.oleracea plants. Caterpillars were then moved to petri dishes (ten per dish) with treated leaves to determine larvae survival and performance. In the field, B.oleracea plants were treated with each solution, and late-instar caterpillars were placed on treated plants. The number of eggs laid was counted after 48 hours and caterpillars were monitored to determine survival.The overall highest oviposition deterring effect was recorded with the rosemary treatment (28% of total eggs), followed by peppermint(33%) then garlic(39%). Larvae survival was 50% on garlic-treated plants, with no significant difference between peppermint and rosemary (20%). In addition, the growth rate of larvae on garlic-treated plants was significantly higher, suggesting that rosemary and peppermint are more effective alternatives to chemical pesticides. Our study provides a practical botanical insecticide and remains ethical (link to poster).

Cabbage white butterflies (Pieris rapae) prefer to lay eggs on Brassica plants over other plant species. P. rapae are a major cause of damage to important Brassicaceae vegetable crops. P. rapae is an agricultural pest that harms the plants in the Brassicaceae family, and knowing what prevents the oviposition of P. rapae on the agricultural plants would be significantly useful. Research is very limited when using non-host plants like Lactuca sativa L. to surround Brassicae  plants to deter P. rapae from ovipositing. Oviposition patterns can be affected by non-host plants, outside plants that surround their host plant. When choosing oviposition spots, P. rapae and other butterflies use visual cues like the height of the plant, its color, flatness, and degree of polarization. Our research focuses on how the presence of three non-host plants surrounding a host plant affects oviposition preferences of P. rapae, and how the absence of glucosinolates in non-host plants might affect the oviposition choice of P. rapae. Glucosinolates are compounds that contain sulfur and are found in Brassica plants. We investigated the effects of non-host plants by placing one female butterfly in a habitat that included one Brassica plant that was surrounded by a non-host plant. Each habitat had one Brassica juncea plant surrounded by three Lactuca sativa L. (non-host plants). We hypothesized that surrounding Brassicae plants with a 1:3 ratio with Lactuca sativa L. plants would affect the oviposition choice of P. rapae. Plants in each cage were checked and counted to see the number of eggs laid after the experiment  concluded. We found that only 23% of the butterflies do not lay eggs on their host plant  surrounded by Lactuca sativa L (link to poster).

Conspecific cues, with regards to oviposition, signal that members of the same species have oviposited on the same host plant site. The presence of these cues implies a fitness trade-off: while high oviposition rates may indicate a more desirable oviposition site, it risks greater larval competition among members of the same species. In this study, we investigated whether the presence of either visual or chemical conspecific cues affected oviposition choice of Pieris rapae in a lab environment. Plants treated with conspecific cues were placed in the Edmonds College P. rapae colony to allow for initial egg laying. Removing eggs from the treated plants plants would test for olfactory chemical cues alone, whereas leaving eggs on the plants would represent the retention of both chemical and visual cues. Eggs were removed from the plant in each pair with the most eggs to maintain the strongest chemical cue before being placed into oviposition cages with an adult female P. rapae. We examined oviposition preference between three Brassica oleracea var. palmifolia (kale) plants among the two plant treatments and an untreated plant—one without any previous oviposition—and between two B. oleracea var. viridis (collards) among only the treated plants. The proportion of eggs on each plant was measured after 24 hours. While previous research has found that cabbage white butterflies tend to express moderate conspecific avoidance, our data supports that P. rapae exhibit conspecific attraction. Butterflies laid significantly more eggs on plants with conspecific cues than plants without them. No significant difference was found between plants with both conspecific cues versus with olfactory chemical cues alone. These findings suggest that P. rapae interprets conspecific occupation as an indicator of a viable host plant rather than a warning for offspring competition (link to poster). 

Glucosinolates are a chemical compound vital for the nourishment of Pieris rapae caterpillars in particular. Its presence acts as a chemical signal for larval feeding (de Vos et al., 2008). Thus, we decided to research if altering the amount of glucosinolates available to P. rapae caterpillars would affect their preference, growth, and survival. A significant amount of research has explored the effects of changing chemicals in caterpillar diets in labs when rearing caterpillars and testing their performance during their lifetime, but few have researched the effect of changing the glucosinolate levels in the diets and its effect on the performance of caterpillars over the course of their larval stages (Roeder et al., 2010). We prepared three types of artificial diet based on a recipe for Manduca sexta caterpillars, adapted by Edmonds College Biology department. The colony of P. rapae at Edmonds College is reared on this diet, with collard greens as the source of glucosinolates. The diet we prepared used kale, cabbage, and broccoli as the source of glucosinolates, all of which are brassicaceous plants with statistically different levels of total glucosinolates (Bhandari et al., 2015). All other ingredient concentrations and masses remained the same, though texture varied due to the different water content of each plant. After hatching, neonates were placed on the three types of diet; survival and growth were measured for each. We found that larvae survivorship was positively correlated with glucosinolate content, but biomass growth was negatively correlated with glucosinolate content. Out of the three artificial diet options, kale had the highest glucosinolate content, the highest larvae survivorship, and the lowest biomass growth. Higher glucosinolate levels do not necessarily indicate better performance for P. rapae (link to poster).

P. rapae larvae are one of the leading causes of pest destruction of Brassica vegetable crops, such as kale, cabbage, and broccoli. They cause heavy losses to farmers and consumers alike, so farmers often resort to pesticides and insecticides. However, because of the different ingredients in each type and even brand, the efficiency of the pesticide may differ. Having the most efficient pesticide will save effort and money for farmers, so we tested two commercial pesticides, one neem-based, and one spinosad-based, against control untreated leaves to see which is most effective at killing P. rapae larvae. In our experimental set-up, one collard green leaf was placed inside a 0.5-cm circular disk. As a controlled variable, four collard leaves were not painted with either insecticide. Four collard leaves were painted (front to back) with super soap and four different collard leaves were painted with neem oil. Two caterpillars were placed inside each 0.5-cm circular disk on top of the leaf. The caterpillars were gathered in a laboratory based on two development stages: early larval stage (instar 3) and later larval stage (instar 3-4). This process was done five more times. Our data found that the caterpillars exposed to Neem oil died significantly more than the control, and the Super Soap caterpillars also died significantly more than the control, and significantly more than the Neem oil. Additionally, one time exposure was not significantly different from continual exposure, for any of the pesticides. In conclusion, the Insecticidal Super Soap was the most effective at killing the caterpillars and minimizing consumption of the Brassicae leaf (link to poster).

Pieris rapae females examine a variety of cues when hunting for a host plant. While many studies have focused on single cues or multiple cues, none have overlapped multiple cues and insecticides. We focus on determining the effects of a long distance and a short cue on oviposition preference of P. rapae. The long distance cue is plant size and the short distance cue is Neem Oil insecticide. We hypothesized that butterflies will prefer feeding on large collards (Brassica oleracea var. Viridis) over small collards and Neem Oil pesticide will deter egg laying no matter what plant sizes are, based on preliminary research. Oviposition preference was measured by taking the average proportion of eggs laid among all treatments. Plant sizes were conducted with small 1-2 week-old collards and large 4-5 week-old collards. Neem Oil’s effect was conducted with either two-choice small collard or two-choice large collard to keep size variable controlled. Four-choice assays with small control, large control, Neem-Oil-treated small, and Neem-oil treated large collards were conducted to determine the impact of both short distance and long distance cues on oviposition preference. In plant-size assays, a large proportion of eggs, 0.706, was laid in large collard, while only 0.294 was laid in small collard. Between the control collards and Neem-Oil-treated collards, the Neem-Oil-treated plants had approximately three times the proportion of eggs laid than control plants regardless of their size. In four choice assays, large and small Neem-Oil-treated collards have around the same proportion of eggs laid. The large control collard received the highest proportion of egg, 0.372. Overall, our experiment showed that the large plant is more dominant in oviposition than the small plants and Neem Oil attracts P. rapae to lay more eggs instead of acting as a deterrent as advertised (link to poster).

Pieris rapae butterflies use a variety of short and long distance visual cues in order to choose host plants for oviposition. Previous research has shown the effects of leaf color, leaf density, and size of host plants on P. rapae oviposition. They found that all of these factors contribute towards a butterfly's behavior. Other studies have also confirmed that P. rapae butterflies are able to distinguish between different colors of the same plant species. To expand on these visual cues, we wanted to determine if P. rapae oviposition is influenced by artificially changing the color of napa cabbage leaves. In order to examine how P. rapae’s oviposition behavior is affected by a change in visual color cues, we dyed napa cabbage leaves using four different colors– yellow, red, green, and blue. The leaves were dyed using a ratio of 20 drops of dye to every 3 oz of water and then left for 24 hours prior to the experiment. We separated our research into two parts 1) a no choice experiment to determine if the food dye solvent showed any adverse reactions on P. rapae, and 2) a multi-choice test in which the females were given the choice to oviposit on cabbage leaves dyed different colors. Our preliminary work showed that solvent in the dye had no effect on the butterflies or their oviposition. We found that females laid the greatest number of eggs on the cabbage dyed blue (134 eggs) and cabbage dyed green (89 eggs) while laying less eggs on leaves dyed red (72 eggs) or yellow (21 eggs)– for this reason we decided to eliminate yellow from part two of the experiment. For the second part of our research, we gave P. rapae females a choice between green, blue, red, or no dye leaves. This experiment confirmed that when given the choice P. rapae females prefer blue colored leaves and green colored leaves for oviposition. This behavior implicates that P. rapae butterflies can distinguish different colors and even different shades of the same color and use this information to select host plants (link to poster).

Competition between pollinators play an essential role in structuring communities, which results in the normal function of ecosystems. Honeybees (Apis spp.) are competitors with Pieris rapae. Honeybees are commonly generalist foragers that monopolize floral resources. Therefore,  it can be hypothesized that the presence of this competitor may decrease flower acceptance, despite the butterflies’ flower color preference. This was found in our previous research to be green (Boieriu et al., unpublished). Ring feeders in the colors red, pink, green, purple, and blue were used to test the effects on landing behavior of P. rapae butterfly in the presence of a competitor. The butterflies were placed on each flower and timed for the amount of time spent on the flower in the presence or absence of a honeybee. The color yellow was not used in this experiment since P. rapae have been reared on yellow artificial flowers. The color orange is not used as well as in our research before, none of the butterflies landed on orange. Males and females were not observed separately as we previously found that they do not have significantly different preferences. Our findings showed that out of the entire sample size, 45% had preferred to land on the green flower. The amount of time spent longest was on green, regardless of the competitor, compared to the other flower colors. As for all the colors, even though it was not a significant difference, the acceptance time of the flower was slightly higher when there was no competitor. Thus, we concluded that there was no overall effect of a competitor on flower visiting behavior. Butterflies and bees are currently threatened by habitat loss due to development. This may lead to loss of flower color diversity and increase competition between pollinators as they commonly have the same flower preference (link to poster).

Nectar is a natural sugar-rich energy source from plants found in the nectaries at the base of the flower that is essential for sustaining butterflies through the taxing process of oviposition. Pieris rapae (cabbage white butterflies), are a pest species which lays single, energy intensive eggs, unlike their clutch-laying relatives. Thus, P. rapae has a higher nectar demand, and may use flowers as a visual indicator of host plant viability. We tested whether P. rapae prefers to oviposit on a plant with a nectar reward or without. In our first trial, we performed a 24-hour, two choice-test on potted Brassica oleracea between a plant that contained an artificial flower with a 0.156 percent nectar content and a plant that did not contain a nectar source. Our second trial was an observational study of oviposition-related behaviors that examined the first-landing choice and abdomen-curling of the cabbage white butterflies in 5 minute increments when given a choice between a Collard leaf with flowers and without. There was not a significant difference in the first trial between the mean number of eggs laid on the plants on either plant. However, in the second trial, we found that the butterflies chose the plant with the brassica flower at a significantly higher rate for its initial landing and abdomen-curling behaviors. In conclusion, true flowers appear to have a more significant impact on oviposition and serve as an important cue in signifying host suitability for oviposition. This data can be applied to the theory that it may be beneficial to agricultural workers to limit the number of Brassica flowers that grow in their crops as to limit economic losses due to herbivore damage caused by oviposition (link to poster).

Transforming DNA plasmid pGLO and resulting conclusions (link to poster).