Progress report for GNE21-247
Project Information
- Determine what host beetle factors influence the preference of C. setosa parasitoids.
- How does the presence of male A. vittatum adults influence parasitism?
- How does the presence of female A. vittatum adults influence parasitism?
- How does host mass, sex, and sequestered defenses influence parasitism?
- Determine what host beetle factors influence the performance of offspring C. setosa parasitoids.
- How does A. vittatum mass relate to offspring parasitoid performance?
- How does A. vittatum sex relate to offspring parasitoid performance?
- How does A. vittatum mass relate to offspring parasitoid performance?
- Determine the effects of parasitism on A. vittatum performance.
- How does parasitism affect A. vittatum survival?
- How does paratisism affect A. vittatum fecundity?
- How does parasitism affect A. vittatum consumption?
- How does parasitism affect A. vittatum pheromone production?
Acalymma vittatum - striped cucumber beetle - is a major agricultural pest of cultivated crops cucurbit crop (Cucurbitiacae) such as cucumbers, squashes, pumpkins, and melon. These insect herbivores are notoriously destructive because they feed belowground on roots as larvae and aboveground on foliage, blossoms, and fruits in their adult stage (Houser and Balduf 1925; Gould 1940; Metcalf et al. 1987; Smyth and Hoffman 2003; Ellerns-Kirk and Fleischer 2006; Haber et al. 2021) and can also vector highly destructive plant pathogens and viruses, such as bacterial wilt (Reviewed in Saalua Rojas et al. 2015). Moreover, pesticides still remain the most effective strategy to manage striped cucumber beetle populations (Reviewed in Saalua Rojas et al. 2015) which adds additional hurdles to organic growers.
Biological control and its enhancement is an alternative solution for both organic and conventional growers. A. vittatum can be heavily parasitized by a parasitoid tachinid fly, Celatoria setosa (Houser and Balduf 1925; Fischer 1981; Toepfer et al. 2009; Smyth and Hoffman 2010; Coco et al. 2020; Haber et al. 2021) there is a gap in the literature investigating the relationship between these two organisms. The purpose of this project has been to investigate the ecological relationship between a major agricultural insect herbivore pest - A. vittatum - and an abundant natural enemy - Celatoria setosa and assess its utility as a biological control agent. Over the last year and a half, the scope of the project has been broadened beyond to effects of parasitism on A. vittatum by C. setosa to also include factors that influence the preferences and performance of C. setosa parasitoids as well such as the release of vittatalactone - male A. vittatum pheromone - and cucurbitacins - sequestered chemical defenses in A. vittatum tissue.
This research is critical for sustainable agriculture in the northeast for two reasons. First, emerging research has shown that vittatalactone - male A. vittatum pheromone - is an important chemical cue in the cucurbit agroecosystem, attracting and influencing the behavior of other relevant cucurbit insect pests (Brzozowski et al. 2022; Weber et al. 2022; and Haber et al. 2023). Investigating how vittatalactone may influence the behavior of an abundant natural enemy, C. setosa, is an important next step. Second, the main crop disease vectored by A. vittatum, bacterial wilt, is geographically specific to the northeastern United States (Reviewed in Saalua Rojas et al. 2015). The spread of bacterial wilt is directly related to
A. vittatum beetle density, consumption, and defecation (Reviewed in Saalua Rojas et al. 2015). C. setosa is a specialist parasitoid that requires its host to continue feeding for its own development (Reviewed in Cuny et al. 2021). Therefore, understanding how parasitism of A. vittatum by C. setosa will help understand the pathogenesis of bacterial wilt.
Research
Insects and organism care
In this study, we used wild-caught A. vittatum collected from two organic farms in the Finger lakes region of New York State including Fellenz Family Farm located in Phelps, NY, and West Haven Farm located in Ithaca, NY from late May of 2022 through August of 2022. All collected beetles were isolated for one to two weeks following collection to rear out potential parasitoids in our sample. Following isolation, male and female beetles were sorted into separate rearing cages. Caged males and females were then used for various lab-based experiments and then moved into fresh cages for future rearing. Caged beetles were supplied biweekly with a fresh C. pepo (var. Saffron Summer Squash) plants grown in Cornell greenhouse facilities, sliced organic cucurbit fruit purchased locally, fresh cut cucurbit flowers if available, cotton soaked in 5% honey water solution, and moistened black gauze (soil mimic).
- setosa adult parasitoids were caught primarily at West Haven Farm and housed in a 1oz disposable plastic cup supplied with a small piece of cotton soaked in a 5% honey water solution. We also collected emerged C. setosa pupae biweekly from isolating beetles and their cages and transferred them to moistened black gauze in plastic opaque sealed containers. Containers were monitored daily for eclosion, sexed, and then housed similarly to wild flies.
All organisms were kept in growth chambers at Cornell University set to a 16:8 hr L:D cycle with daytime temperatures set to 25 degrees Celsius, night-time temperatures set to 23 degrees Celsius, and humidity maintained between 30-50%.
Greenhouse and planting
All plants used in organism care and lab-based experiments were grown in greenhouse conditions managed at Cornell University, Ithaca, NY by the authors and greenhouse staff. C. pepo (var. Saffron Summer Squash) seeds obtained from W. Atlee Burpee & Co were planted weekly in 72-cell seed trays using a Lambart 111 premix soil. Seedings were then transplanted between 10-14 days old or after the emergence of a first true leaf into either standard 4-inch garden pots, or 8oz disposal plastic cups, locally purchased. Plants were watered twice daily and composted after fruit set had begun. Greenhouse conditions were set to a 16:8 hr L:D cycle with daytime temperatures set to 25 degrees Celsius, and night-time temperatures set to 23 degrees Celsius with ambient humidity.
Manipulating parasitism in the lab
In certain experiments, we directly manipulated parasitism in the lab by enclosing a single wild-caught C. setosa female with a single A. vittatum adult like previous methods (Fischer 1981). Newly caught wild A. vittatum beetles were first isolated for one to two weeks to rear out potential prior parasitism. One C. setosa female and one adult A. vittatum beetle were then placed into an acrylic insect-rearing cage with a full spectrum light source overhead. The insects would move towards the light source and rest on the top of the cage. Then using a shallow 3oz plastic dish we would quickly enclose the two organisms together at the top of the cage. Once paired in the small enclosure, most parasitoids would respond to the presence of the beetle within a minute and would make an obvious and characteristic oviposition attempt in which the parasitoid can be observed thrusting its ovipositor into its host. Parasitoids that did not respond to their potential host were removed from the cage and a new parasitoid was rotated in. After observing oviposition, we assumed individual beetles to be parasitized.
Experiment 1 - Patterns of parasitoid attraction and performance in field collected organisms
Between May - August of 2022, we conducted a field survey measuring on-farm abundance of A. vittatum across five varieties of cultivated cucurbit crops on an unmanipulated organically managed farm - Fellenz Family Farm. To survey these abundances we flagged 150 individual plants across C. pepo (var. Desert Zucchini), C. pepo (var. Zephyr Summer Squash), C. pepo (var. New England Pie), C. sativus (var. Marketmore), and C. lanatus (var. Crimson Sweet) using stratified sampling technique and measured abundances of A. vittatum per plant. Plants were sampled weekly for ten weeks beginning two weeks after sowing. In addition, we also collected between 1-10 beetles per plant per week, depending on the quantify present, to track and monitor parasitism. Each beetle collected was weighed, sexed, and then isolated in a small 1oz disposable plastic cup supplied with a cube of freshly cut cucurbit fruit. Beetles were then isolated for fourteen days to rear out potential parasitism. Following the isolation period, C. setosa pupae that emerged from host beetles were collected and placed into 2mL centrifuge tubes and then monitored for eclosion. A subset of elytra among parasitized, non-parasitized (surviving), and dead beetles were removed following isolation periods using fine-tipped dissecting forceps and then stored in 2 mL centrifuge tubes aliquoted with 500uL of methanol. Sampled were then stored at -80 degrees for analysis of sequestered dietary cucurbitacins (Ferguson and Metcalf 1985).
Experiment 2 - How parasitism affects A. vittatum survival and performance
To determine how parasitism by C. setosa affects its host, we manipulated parasitism of wild-caught female adult beetles under laboratory conditions and then monitored survival, leaf consumption, and change in mass over two weeks [the average development time of C. setosa larvae (Fischer 1981)].
In this experiment, we used beetles primarily collected from West Haven Farms. Collected beetles were isolated for one to two weeks prior to rear prior parasitism. Following isolation, female beetles were starved for four hours and then had their mass recorded before being assigned to either a parasitism treatment or a non-parasitized (control). Individuals assigned to the parasitism treatment were then parasitized directly in the lab using wild-caught C. setosa females. Individuals assigned to the non-parasitized treatment were treated similarly except wild-caught parasitoids were swapped for either lab-reared male parasitoids or lab-reared non-gravid female parasitoids [eclosed in isolation], depending on availability. Parasitized and non-parasitized beetles were then placed in 16oz plastic cups containing a single C. pepo (var. Saffron Summer Squash) leaf taken from a plant at the two-true leave stage using a sterilized razor. Stems of cut leaves were then mounted in 1oz plastic cups and filled with water to prevent wilting. We then monitored the survival of each beetle and tracked their performance measured by mm2 leaf area consumed and their respective change in mass (mg) over fourteen days and measurements were taken every forty-eight to seventy-two hours. We measured leaf area by tracing damaged leaves and integration removed areas using ImageJ.
Over the course of six weeks, we tracked the survival and performance of 105 A. vittatum adults including 44 parasitized beetles and 61 control or a non-parasitized beetles. About 8% of the females used (N = 9) had been parasitized prior in the field, either yielding a parasitoid offspring in the non-parasitism (control) treatment or yielding a parasitoid offspring a week earlier than the average development time (Fischer 1981). These individuals were omitted from our analysis.
Experiment 3 - Investigating the effects of parasitism on pheromone release
To determine how parasitism affects pheromone release we collected headspace volatiles of parasitized and non-parasitized male A. vittatum beetles during active feeding for seventy-two hours.
In this experiment, we used beetles primarily collected from West Haven Farms. Collected beetles were isolated for one to two weeks prior to rear prior parasitism. Following isolation, male beetles were starved for four hours and then had their mass recorded before being assigned to either a parasitism treatment or a non-parasitized (control). Individuals assigned to the parasitism treatment were then parasitized directly in the lab using wild-caught C. setosa females. Individuals assigned to the non-parasitized treatment were treated similarly except wild-caught parasitoids were swapped for either lab-reared male parasitoids or lab-reared non-gravid female parasitoids [eclosed in isolation], depending on availability. Parasitized and non-parasitized male beetles were then isolated alone in small 1oz disposable plastic cups supplied with a cube of freshly cut squash and left alone for seventy-two hours.
After this time, male beetles were then assigned to groups of six, marked with dots of colored acrylic paint, and placed into 1-gallon glass jars with a single C. pepo (var. Saffron Summer Squash) plant at the two-true leave stage. Pushed air was filtered through activated charcoal and humidified through distilled water was pushed into glass containers at 0.5mL/min. Volatiles were then trapped on Orbo ® charcoal filters and eluted with 1000uL of MeOAc spiked with a known standard into 2mL screw cap Agilent glass vials. Volatile samples were then stored at 0 degrees Celsius until further analyzed.
Experiment 4 - How the presence of parasitoids affects A. vittatum survival and performance
In addition to investigating the direct effects of parasitism, we also sought to determine how the presence of parasitoids may also affect A. vittatum survival and performance. In this experiment we compared leaf consumption measured in mm2 and the change in mass measured in mg between beetles in the presence or absence of parasitoids.
In this experiment, we used beetles primarily collected from West Haven Farms. Collected beetles were isolated for one to two weeks prior to rear prior parasitism. Following isolation, female beetles were starved for four hours and then had their mass recorded before being assigned to either a presence of parasitoid treatment or a absence of parasitoid (control) treatment. Beetles were then placed in 16oz plastic cups containing a single C. pepo (var. Saffron Summer Squash) leaf taken from a plant at the two-true leave stage using a sterilized razor. Stems of cut leaves were mounted in 1oz plastic cups and filled with water to prevent wilting. Cups were then loaded with either a C. setosa parasitoid or no parasitoid respectively and left for twenty-four hours. After this time, we recorded the final weights of our beetles and then placed them in 1oz plastic cups for parasitism monitoring. Living flies were returned to their rearing conditions and leaves were imaged for analysis of consumption.
Analysis of volatile emissions of parasitized and non-parasitized beetles
All headspace volatile samples were quantified using GC/MS technique at Cornell AgriTech, Geneva, NY, USA between June, and September of 2022.
Specific GC/MS run methods and parameters to be completed for final report.
Analysis of sequestered cucurbitacins in parasitized and non-parasitized beetles
All cucurbitacins samples from beetle elytra were quantified using HPLC-Q-TOF technique at Cornell AgriTech, Geneva, NY, USA in January of 2023. Samples were thawed and then homogenized in solution using [insert specific bead beater and run parameters]. Homogenized solutions were then double centrifuged [insert specific centrifuge parameters], and then 200uL of each supernatant was transferred to 250uL glass insects set in 2mL screw cap Agilent glass vials.
Specific HPLC-Q-TOF run methods and parameters to be completed for final report.
Statistics and Data Analysis
All statistical analyses were conducted using R and RStudio (Ver. 4.2.1; R Core Team, 2022). All figures were generated in RStudio using the ggplot2 package. The script used to clean, organize, and analyze the data presented is publicly available for final report.
[Below is a detailed results and discussion section taken from two manuscripts in preparation. Not all results have been included but will be for the final report. Please do not copy or distribute this information].
Patterns of parasitoid attraction and performance in field-collected organisms
Male A. vittatum beetle abundance attracts parasitoids, not females.
Preliminary results from our field data suggest that male striped cucumber beetles attract C. setosa parasitoids whereas female beetles do not. We used a negative binomial analysis to model the number of parasitized male and female beetles (y-axis) with increasing abundances of total male and female beetles on a macroscale (x-axis). From our models, we find that as the number of male beetles increases, the number of males parasitized also increases (Figure 1a - top - blue [N = 90, AIC = 442.5, P < 0.001]) whereas the number of female beetles is independent of the number of females parasitized (Figure 1b - bottom - red [N = 90, AIC = 452.19, P = 0.57]).
![Figure 1 - Plot displays two negative binomial regression models measuring the relationship between the total number of beetles counted (x-axis) and the total number of those beetles parasitized (y-axis) for male A. vittatum beetles (top 1a) and female A. vittatum beetles (bottom, 1b). The top model (blue) shows that as the number of male beetles increases, the number of males parasitized also increases [N = 90, AIC = 442.5, P < 0.001]) whereas the bottom model (red) shows that the number of female beetles counted is independent of the number of females parasitized (Figure 1b [N = 90, AIC = 452.19, P = 0.57]). All statistical analyses were conducted using R and RStudio (Ver. 4.2.1; R Core Team, 2022). All figures were generated in RStudio using the ggplot2 package.](https://projects.sare.org/wp-content/uploads/fig1-11.jpg)
Characteristics of field-collected A. vittatum beetles
Between May 30, 2022, and August 15, 2022, we processed 1,659 field-collected A. vittatum beetles. Within this sample, 942 were identified as male, 438 were identified as female, and 279 were unable to be identified either from desiccation before processing, the emergence of parasitoid offspring before processing, or severe damage. Within the subset of identified beetles (N = 1,380), 1,030 beetles (74.64%) survived isolation and yielded no parasitoid offspring, 159 beetles (11.52%) yielded parasitoid offspring, and 191 beetles (13.84%) died without yielding parasitoid offspring. Using a Pearson's Chi-squared Test we found that we caught about twice as many male A. vittatum beetles as females, (N = 942 and N= 438) respectively, [N = 1380, X2 = 184.07, df = 1, P < 0.001], and that there was no difference in parasitism rates between male and female beetles when adjusted for our observed sex ratio, N = 110 and N= 49 respectively, [N = 159, X2 = 0.06529, df = 1, P = 0.7983]. We did however find that more males died during isolation than female beetles when adjusted for our observed sex ratio, N = 151 and N= 40 respectively, [N = 191, X2 = 10.257, df = 1, P < 0.01] (Table 1). Using a two-tailed Wilcoxon Rank Sum Test we found that male beetles were on average 3.80 mg smaller than females [N = 1373, W = 335596, P < 0.001], and using a Kruskal-Wallis Rank Sum Test and a nonparametric pairwise-comparison we found that both male and female beetles that yield parasitoid offspring were larger than those that did not yield parasitoid offspring, [N = 938, X2 = 34.957, df = 2, P < 0.001] and [N = 438, X2 = 8.7005, df = 2, P = 0.013], for males and females respectively.
Host mass relates to parasitism by C. setosa in both male a female beetles, but parasitoid offspring are less likely to develop.
Given that we found a relationship between beetle mass and parasitism we modeled the probability of parasitism (Figure 2a) and the probability of parasitoid offspring eclosion (Figure 2b) as a function of host beetle mass using logistic regressions. From our models, we find that as host beetle mass slightly increases the probability of parasitism also increases [N = 1141, SE = 0.02843, AIC = 888.31, P < 0.001], whereas the probability of parasitoid offspring eclosion decreases as host beetle mass increases [N = 119, SE = 0.06992, AIC = 147.8, P < 0.01]. We did not find any relationship between beetle mass, sex, and the sex of the parasitoid offspring.
![Figure 2 - Plots display logistic regression models of the probability of paratitism (2a - top) and the probability of parasitoid offspring eclosion (2b - bottom) as a function of host A. vittatum beetle mass (mg). The top model indicates that within a sample of beetles, the probability of an individual beetle yielding an parasitoid offspring increases slightly with per unit increases in host beetle mass [N = 1141, SE = 0.02843, AIC = 888.31, P < 0.001]. The bottom model indicates that the probability of parasitoid offspring eclosing is inversely proportional to the mass of the host beetle it emerged from ie. decreasing with per increases in host beetle mass [N = 119, SE = 0.06992, AIC = 147.8, P < 0.01]. All statistical analyses were conducted using R and RStudio (Ver. 4.2.1; R Core Team, 2022). All figures were generated in RStudio using the ggplot2 package.](https://projects.sare.org/wp-content/uploads/fig2-8.jpg)
Experiment 2 - How parasitism affects A. vittatum survival and performance
The effects of parasitism on survival, death, and fecundity
About 75% (N =30) of beetles parasitized in the lab died and about 25% (N = 11) survived, whereas about 15% (N = 8) of beetles not parasitized (control) died and about 85% (N = 47) survived. Using a Pearson's Chi-squared Test we found that C. setosa is effective in killing its host [N = 96, X2 = 112.46, df = 1, P < 0.001] as expected. Of the parasitized beetles that died about 63% (N = 19) of beetles died and yielded a parasitoid offspring whereas 36% (N=11) died without yielding a parasitoid offspring. On average, parasitoids emerged from female host beetles between 8-13 days with a median emergence on day 10. Unexpectedly, about 25% (N = 14) of beetles from our control treatment oviposited eggs into their experimental cups. Some beetles from the parasitism treatment also released eggs but at much lower frequencies. Using a Pearson's Chi-squared Test we found that parasitized females (yielded offspring) oviposited eggs less than control females [N = 30, X2 = 4.6473, df = 1, P < 0.05] whereas females that survived parasitism oviposited at a similar frequency to controls [N = 11, X2 = 0.0191, df = 1, P = 0.8899].
The effects of parasitism on leaf consumption and change in mass over time
Using a Kruskal-Wallis Rank Sum Test and a nonparametric pairwise comparison we found that parasitized female adult beetles removed less leaf area measured in mm2 compared to non-parasitized control females [N = 96, X2 = 44.205, df = 4, P < 0.001] (Figure 3). For this analysis, we used relative leaf area consumed calculated by summing the total leaf area removed and dividing by the number of days they survived to account for parasitized beetles dying sooner than non-parasitized control females. In addition, we found that females that survived parasitism consumed the same amount of leaf area as controls. We also found that parasitized females that died without yielding parasitoid offspring consumed more leaf area than control females that died, however, both consumed little plant material at all. Most subgroups of parasitized and non-parasitized (control) beetles lost weight over time. Using a Kruskal-Wallis Rank Sum Test and a nonparametric pairwise-comparison we found that parasitized female adult beetles lost marginally more weight compared to controls (P = 0.0770) however females that survived parasitism lost on average about 25% mass over time a two week time period, significantly more than control females (P < 0.05). Moreover, different subgroups of beetles consumed more or less than others over time (Figure 4). For example, females that survived parasitism consumed more leaf area than control females during the first few days post-parasitism whereas parasitized beetles fed similarly to control beetles.
![Figure 3 - Boxplot displays the variation in relative leaf area consumed measured in mm2 across subgroups of parasitized and non-parasitized (control) A. vittatum adult females. Relative leaf area removed was calculated by dividing the total leaf area removed by the number of days the individual survived. Relative leaf area removed across subgroups was then compared using a Kruskal-Wallis Rank Sum Test and a nonparametric pairwise-comparison [N = 96, X2 = 44.205, df = 4, P < 0.001]. Letters denote significance between subgroups. All statistical analyses were conducted using R and RStudio (Ver. 4.2.1; R Core Team, 2022). All figures were generated in RStudio using the ggplot2 package.](https://projects.sare.org/wp-content/uploads/fig3-12.jpg)
Experiment 3 - Investigating the effects of parasitism on pheromone release
Parasitism impacts the likelihood of calling but not signal strength
In total, we quantified the emissions of 29 groups of male adult A. vittatum (N = 174). We were able to detect vittatalactone in 21 out of 23 (91.3%) non-parasitized (control) groups but only in 4 out of 6 or (66.0%) of parasitized (experimental) groups. Using a Pearson's Chi-squared Test we found that our group parasitized males were less likely to release detectable concentrations of vittatalactone [N = 29, X2 = 4.4722, df = 1, P < 0.05]. Groups of non-parasitized males released a median of 59.14 mg/mL of vittatalactone whereas groups of parasitized males released a median of 31.46 mg/mL of vittatalactone. Using a one-tailed Wilcoxon Rank Sum Test we found no difference in vittatalactone emissions [N = 29, W = 86, P = 0.1866].
![Figure 5 - Plot displays the concentrations of male A. vittatum pheromone (vittatalactone) measured in mg/mL between groups of non-parasitized (control) beetles (N = 23) and groups of parasitized beetles (N = 6). Groups of non-parasitized males released an median of 59.14 mg/mL of vittatalactone whereas groups of parasitized males released an median of median of 31.46 mg/mL of vittatalactone. Using a one-tailed Wilcoxon Rank Sum Test we found that these groups did not release different amounts of vittatalactone [N = 29, W = 86, P = 0.1866]. All statistical analyses were conducted using R and RStudio (Ver. 4.2.1; R Core Team, 2022). All figures were generated in RStudio using the ggplot2 package.](https://projects.sare.org/wp-content/uploads/fig5-4.jpg)
Experiment 4 - How the presence of parasitoids affects A. vittatum survival and performance
Beetles in the presence of parasitoids consume less leaf area and gain less weight
Using a two-tailed Wilcoxon Rank Sum Test we found that adult female A. vittatum beetles in the presence of C. setosa parasitoids consumed about 54% less leaf area than beetles in the absence of parasitoids [N = 58, W = 243, P = 0.011] (Figure 6). In addition, we also found that beetles in the presence of parasitoids gained about 57% less weight after the 24-hour feeding period using a two-tailed Wilcoxon Rank Sum Test [N = 58, W = 243, P = 0.015]. Surprisingly, no beetles yielded a parasitoid offspring after monitoring all individuals for two weeks.
![Figure 6 - Boxplot displays the leaf area consumed measured in mm2 between female adult A. vittatum beetles in the presence or absence of C. setosa parasitoids. Using a two-tailed Wilcoxon Rank Sum Test we found that adult female A. vittatum beetles in the presence of C. setosa parasitoids consumed about 54% less leaf area than beetles in the absence of parasioitds [N = 58, W = 243, P = 0.011]. All statistical analyses were conducted using R and RStudio (Ver. 4.2.1; R Core Team, 2022). All figures were generated in RStudio using the ggplot2 package.](https://projects.sare.org/wp-content/uploads/fig6-5.jpg)
Education & Outreach Activities and Participation Summary
Participation Summary:
The outreach plan for this proposal is simple but direct and effective. By October 2023, the results of this project will be summarized and electronically distributed to cucurbit growers across New York State, in addition to being presented at ESA 2023 post-fellowship. New York State growers will be accessed using the NOFA-NY database [12].