In phytophagous insects, adult attraction and oviposition preference for a host plant is often positively correlated with their immature performance; however, little is known how this preference-performance relationship changes within insect populations utilizing different host plants. Here, we investigated differences in the preference and performance of two populations of a native North American frugivorous insect pest, the plum curculio (Conotrachelus nenuphar) ‒ one that utilizes peaches and another that utilizes blueberries as hosts ‒ in the Mid-Atlantic United States. For this, we collected C. nenuphar adult populations from peach and blueberry farms and found that they exhibited a clear preference for the odors of, as well as an ovipositional preference for, the hosts they were collected from, laying 67-83% of their eggs in their respective natal hosts. To measure C. nenuphar larval performance, a fitness index was calculated using data on larval weights, development, and survival rate from egg to fourth instars when reared on the parent’s natal and novel hosts. Larvae of C. nenuphar adults collected from peach had high fitness on peach but low fitness when reared on blueberry. In contrast, larvae from C. nenuphar adults collected in blueberry had high fitness regardless of the host they were reared on. In this study, we show that utilizing a novel host such as blueberry incurs a fitness cost for C. nenuphar from peaches, but this cost was not observed for C. nenuphar from blueberries, indicating that the preference-performance relationship depends on the particular insect population-host plant association.

Insect and fruit sources 

In early spring of 2019 and 2021, overwintered C. nenuphar adults utilizing peach as their host were collected from peach orchards at the Rutgers Agricultural Research and Extension Center (RAREC) (latitude 39°31'7.99"N, longitude 75°12'21.99"W) in Bridgeton, New Jersey (USA) (Figure 1B). Peach orchards were located in an ecosystem largely consisting of managed agricultural land (primarily apple, peach, soy, and corn), deciduous forest, and hedgerows. Surrounding forest edges were home to several Rosaceous hosts such as crabapple and wild cherry, as well as wild blueberry, potential wild hosts of C. nenuphar (Maier, 1990). Similarly, overwintered C. nenuphar adults utilizing blueberries as their host were collected from blueberry fields at an organic blueberry farm in Hammonton, New Jersey (USA) (latitude 39°39’37.53”N, longitude 74°45’14.75”W) (Figure 1B). These blueberry fields were located in the New Jersey Pinelands National Reserve, an environment dominated by several species of pine trees. Wild blueberry, huckleberry, and wild cherry occur in the forested areas adjacent to the crop plantings and could potentially be used as wild hosts by C. nenuphar. Overall, the area surrounding the blueberry fields contained mostly Ericaceous wild hosts and other blueberry plantings. The peach and blueberry sites were separated by 41.39 km (Figure 1B).

Conotrachelus nenuphar adults collected from these sites were used for all the following experiments. The collected adults were exclusively fed on the host plant of their origin. As a result, we collected adults from two populations with distinct origins (peach or blueberry). We chose to collect feral adults rather than adults reared from the laboratory because we were interested in the host preferences of the overwintered C. nenuphar adults, which would be difficult to produce under laboratory conditions. Overwintered adults are most ecologically relevant to our study than later generations because of their movement into the crop, which indicate that these adults make critical foraging decisions when choosing a host plant. All insects were collected using beat sheets or unbaited trunk traps (Lampasona et al., 2020), and kept in incubators at 25±1°C, 70±10% relative humidity and 16:8 light:dark cycle until used. Adult age was indeterminate since all insects were field collected, but based on the timing of captures, most insects were likely to be of the overwintered generation, and thus eclosed the previous year.

All peach and blueberry flowers and fruits used for rearing insects and in experiments were collected from RAREC and the organic blueberry farm, as they were not sprayed with conventional insecticides. All samples were collected the week of experiments.

Olfactory preference of Conotrachelus nenuphar

In 2019 and 2020, we collected 30 male and 30 female C. nenuphar adults from peaches and 30 male and 30 female C. nenuphar adults from blueberries from our two New Jersey locations (see above); for a total of 60 individuals for each sex and host plant. Collected insects were placed in incubators under the conditions described above for at least 72 h before olfactory trials began. Insects were additionally subjected to a 24-h starvation period with no food and only distilled water prior to testing.

Olfactory bioassays were conducted in a 40-mm-diameter × 36-cm-long glass Y-tube olfactometer that had a 50° inside angle (Sigma Scientific LLC, Florida, USA) (Figure 2). The Y-tube was placed in a particle board box in a dark room lit only with a 20-W red LED light and maintained at approximately 25° during the insect’s scotophase. Incoming laboratory-grade air (Airgas Company, Vineland, New Jersey, USA) was pushed through one of two customized 4.5-L stainless steel crock pots, each with two openings allowing air to flow in and out of a glass chamber (Figure 2). Each pot held fresh cuttings of either peach or blueberry plants (odor source). Each odor source consisted of 300 g of flowers and leaves or 600 g of fruit and leaves (all leaves collected from same plant as flowers or fruit) for each testing period and subsequently connected by tubing to an arm of the Y-tube. The airflow was modulated by an inline flowmeter (Gilmont Instr., Barnant Co., Barrington, Illinois, USA) set to 12 L/min to deliver 6 L/min to the olfactometer arm. Glass components of the Y-tube were cleaned with 70% ethanol and air dried between each replicate, and the left/right position of each basin was swapped to mitigate potential directional bias. Individual C. nenuphar adults were transferred using featherweight forceps and placed in the Y-tube specimen adapter, which was then attached to the Y-tube. After attachment, a stopwatch was started. If the insect spent 60 seconds in either arm, or after 12 minutes had elapsed (whichever came first), the timer was stopped. If the insect spent 60 seconds in an arm, it was considered a “choice” for the odor proximal to that arm and was recorded as such. If, after 12 minutes the insect did not spend 60 seconds in either arm, the insect was placed in a “no choice” category and removed. Insects that did not make a choice were excluded from the statistical analysis. All individuals were used only once for an experiment and new plant material was used for each experiment.

2.3 Oviposition preference of Conotrachelus nenuphar

In 2020, we used fresh fruit to test for C. nenuphar oviposition preference between peach and blueberry in the laboratory. Small sections of peach and blueberry branches were thinned to one peach or five blueberries free of visible insect damage. Each branch section was then cut, inserted into soaked floral foam, and placed inside a mesh 30.5 × 30.5 × 30.5-cm insect rearing cage (BioQuip Products Inc., Rancho Dominguez, California, USA). In each cage, we alternated the left/right placement of the fruit between an equal number of replicates to mitigate directional bias. Insects were collected from our New Jersey sites and maintained in the laboratory as described above. Twenty-three C. nenuphar male/female pairs collected from peach, and 23 pairs collected from blueberry were placed inside the cages (one pair per cage). Each caged pair was provided with both one peach cutting and one blueberry cutting as a choice test. In addition, 10 pairs were provided only with one host as a no-choice check and 10 extra fruit cuttings were held in cages with no insects as “untreated” controls to determine if field oviposition had occurred but had gone unnoticed. Prior to the experiment, individual male/female pairs were given a 24-hour starvation period in microcentrifuge tubes. Following, C. nenuphar pairs were introduced into the cages and allowed 48 hours to oviposit freely on either host, after which all insects and fruit were removed, and the number of oviposition scars on each fruit was counted.

Offspring performance of Conotrachelus nenuphar
In 2019, we thinned 60 blueberry and 60 peach branches down in the field so that each branch held only three peaches or 10 blueberries each. These branches were covered with a sleeve netting made of 5-gallon paint strainer bags and secured at the base of the branch to prevent wild insects from colonizing the fruit before we introduced the C. nenuphar adults. Conotrachelus nenuphar adults were collected from peach trees and blueberry bushes during the week of 20 May 2019, as described above, and kept in 946-ml plastic deli containers with fruit collected from blueberry fields or peach orchards. Insects collected from peach and blueberry were not co-mingled and were grouped into male/female pairs and placed in microcentrifuge tubes for a 24-hour starvation period. Insects were then moved to the sleeve cages on their respective outdoor hosts. Mating occurred at any point after introduction of males and females, although since they were wild-caught, it was possible they had already mated. As such, inclusion of the males was only to ensure mated status during the experiment.

A total of 60 C. nenuphar adult pairs collected from blueberry were individually placed on blueberry branches (1 pair per branch), and 60 pairs were placed individually on peach branches. This was done over a 3-week period using 20 pairs/week for the first two weeks, then 15 pairs for the third week (due to lower insect captures) for a total of 55 replicates. Insects were allowed to oviposit for 4 days inside the sleeves and then removed. Fruit was kept on the branches for an additional 48 hours before removal and placement in rearing containers in the laboratory. The number of oviposition scars on each fruit was counted, and each fruit weighed to calculate the number of eggs per gram of fruit. All containers with fruit were kept in incubators at 25°C and a 16:8 light: dark cycle for 60 days. As larvae emerged from the fruit, they were weighed and head capsule width (mm) measured as a proxy for body mass and size, respectively. The accumulated degree-days (DDs; using a base temperature of 10°C) between adult introduction and larval emergence were recorded for each larva during the 60-day observation period. DDs were calculated using the following formula: Daily DD₁₀ = Mean Daily Temperature – Base Temperature (10°C).

2.5 Data analyses

All data were analyzed in JMP Pro 16 (SAS, Cary, NC). The Y-tube olfactometer data were analyzed using χ² Goodness of Fit tests to determine if C. nenuphar adults preferred one host-plant odor over the other. Each group of insects (based on their respective collected host) was tested separately. Because of the natural thanatosis response of C. nenuphar, some insects will “play dead” throughout the entire trial. As such insects that did not respond (i.e., insects that stayed in the release area or that stayed inside the main body of the olfactometer without moving into either arm for the 12-min test duration) were not included in the statistical analysis.

Choice oviposition data were analyzed using the non-parametric Steel-Dwass all-pairs test, as data did not meet assumptions of normality. The proportion of eggs laid on each host was compared between insects collected from peach and those collected from blueberry. No-choice oviposition data from the larval performance study were analyzed separately using Steel-Dwass all-pairs tests using the same combinations as above (i.e., eggs laid on each host was compared between insects collected from peach and blueberry), since data were not normally distributed.

To assess larval performance, we calculated a fitness index based on the method used by (Jallow & Zalucki, 2003). The fitness index = w × h × d × s, where w = weight of 4^th instar larvae (g), h = head capsule width (mm), d = DD₁₀ accumulation until larval emergence, and s = survival rate from egg to larval emergence. All values in the equation were means from each of the 55 replicates.

Each fitness metric (weight, head capsule size, development time in DDs, and survival), and the overall fitness index were tested separately to determine which collected/novel host combination (i.e., blueberry/blueberry, blueberry/peach, peach/blueberry, peach/peach) were different from each other; each combination of collected and novel host was treated as an independent variable. We analyzed these data using the non-parametric Kruskal-Wallis test after data did not meet assumptions of normality. Post-hoc comparisons were made using the Dunn Method for joint ranking.