DOI: 10.5061/dryad.69p8cz93j
title: Fitness costs and oviposition choice of C. nenuphar on blueberry and peach

description: 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.
description: <strong>Insect and
 fruit sources </strong> In early spring of 2019 and
 2021, overwintered <em>C. nenuphar</em> 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 <em>C. nenuphar
 </em>(Maier, 1990)<em>. </em>Similarly, overwintered
 <em>C. nenuphar</em> 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 <em>C. nenuphar. </em>Overall, the
 area surrounding the blueberry fields contained mostly Ericaceous wild
 hosts and other blueberry plantings.<em> </em>The peach and
 blueberry sites were separated by 41.39 km (Figure 1B). <em>Conotrachelus
 nenuphar</em> 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
 <em>C. nenuphar</em> 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. <strong> </strong>
 <strong>Olfactory
 preference of <em>Conotrachelus
 nenuphar</em></strong> In 2019 and 2020, we collected
 30 male and 30 female <em>C. nenuphar </em>adults from peaches
 and 30 male and 30 female <em>C. nenuphar </em>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 <em>C. nenuphar </em>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.   <strong>2.3
 Oviposition preference of <em>Conotrachelus
 nenuphar</em></strong> In 2020, we used fresh fruit to
 test for <em>C. nenuphar</em> 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 <em>C. nenuphar</em> 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, <em>C. nenuphar</em> 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. <strong> </strong>
 <strong>Offspring
 performance of <em>Conotrachelus
 nenuphar</em></strong><br>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 <em>C. nenuphar </em>adults.
 <em>Conotrachelus nenuphar </em>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 <em>C. nenuphar</em> 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
 DD<sub>10</sub> = Mean Daily Temperature – Base Temperature
 (10°C).   <strong>2.5 Data
 analyses</strong> All data were analyzed in JMP
 Pro 16 (SAS, Cary, NC). The Y-tube olfactometer data were analyzed using
 χ<sup>2 </sup>Goodness of Fit tests to determine if
 <em>C. nenuphar </em>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
 <em>C. nenuphar, </em>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<sup>th</sup> instar larvae (g), h = head capsule
 width (mm), d = DD<sub>10</sub> 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.