Insects

A colony of Carpophilus spp. was established from approximately 100 beetles collected from an unidentified pear cultivar, Pyrus pyrifolia (Burman f.) Nakai. Beetles were housed in a 19.0 × 13.5 × 9.5-cm plastic container with a vented lid (Pioneer Plastics, Dixon, KY). A moist layer of autoclaved potting soil, 2.5 cm deep, lined the bottom of the container. Halved, pears (Pyrus communis L.) purchased in a local store were placed on the soil for food as needed. These containers were kept in an environmental chamber (Model I-36VL, Percival Scientific, Inc., Perry, IA) at 27 ± 1°C and a 14:10 light:dark (L:D)-h photoperiod. Adults fed and oviposited on the pears. Larvae fed on the pears and pupated in the soil. As the population increased, more containers were used. When adults or late instars were needed for experiments, they were collected from the pear halves.

Entomopathogenic nematodes

For the experiments, entomopathogenic nematodes (Heterorhabditis bacteriophora Vs strain, H. indica HOM1 strain, H. megidis UK211 strain, Steinernema carpocapsae All strain, S. feltiae Sn strain, and S. riobrave 355 strain) were reared on last instar Galleria mellonella (L.) (Lepidoptera: Pyralidae) at 25°C according to procedures described in Shapiro-Ilan et al. (2016). All of the nematode species tested in our study are currently available commercially. Galleria mellonella larvae were obtained from Webster's Waxieworms (Webster, WI). After harvesting the nematodes, they were aerated and kept at 13°C for ≤2 weeks before being used in experiments.

Microbial-based and synthetic insecticides

Microbial-based insecticides used in this study included Grandevo^® and Venerate™ bioinsecticides (Marrone BioInnovations, Davis, CA) (Table 1). Synthetic insecticides used in this study included thiamethoxam (Actara^® , Syngenta Crop Protection, LLC, Greensboro, NC), acetamiprid (Assail^® 30SG, United Phosphorous, Inc., King of Prussia, PA), indoxacarb (Avaunt^® , E.I. du Pont de Nemours and Company, Wilmington, DE), β-cyfluthrin (Baythroid^® XL, Bayer CropScience LP, Research Triangle Park, NC), clothianidin (Belay^® , Valent U.S.A. Corporation, Walnut Creek, CA), carbaryl (Carbaryl 4L, Loveland Products, Inc., Greeley, CO), chlorantraniliprole (Coragen^® , E.I. du Pont de Nemours and Company, Wilmington, DE), fenpropathrin (Danitol^® 2.4EC, Valent U.S.A. Corporation, Walnut Creek, CA), spinetoram (Delegate^® WG, Dow AgroSciences, LLC, Indianapolis, IN), phosmet (Imidan^® 70-W, Gowan Company, Yuma, AZ), malathion (Malathion 5EC, Drexel Chemical Company, Memphis, TN), and imidacloprid (MANA Alias^® 4F, Makhteshim Agan of North America, Inc., Raleigh, NC) (Table 1). All treatments were prepared in 50-ml total volume at rates equivalent to using 935.4 l/ha, as commonly used by peach growers using airblast sprayers in southeastern orchards (Table 1).

Table 1 Microbial-based and synthetic insecticides used in assays against adult Carpophilus spp.

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Assays using entomopathogenic nematodes

Infective juvenile nematodes of the previously described entomopathogenic nematode species were assayed twice in parallel against late instar Carpophilus spp. in 30-ml portion cups (Comet, WNA Inc. division of The Waddington Group, Covington, KY). These cups were filled with 10 g of sterile soil classified as a loamy sand (80:16:4 [sand:silt:clay]; pH, 6.1). The moisture content of the soil was brought to field capacity (17%) by adding 1 ml of nematodes in solution and 700 µl of water. Each nematode species was tested at two rates: 1,000 and 2,000 infective juveniles. A small piece of apple fruit was partially submerged into the soil and five larvae were added. This assay was completed using five replicates of each treatment with five larvae in each treatment. A nontreated control was included. Cups were placed on trays with the treatments randomly arranged within each of the five replicates. Replicates were housed in darkness at 25°C within an environmental incubator. Based on preliminary testing (to determine optimum duration of the assay, unpublished data), the cups were sampled for larvae 7-d postinoculation. Soil was removed from the cup and sifted, and the piece of apple was examined for the presence of any larvae. Larvae were recorded as alive or dead. The entire experiment was repeated once in time (i.e., two full trials were completed).

Microbial-based and synthetic insecticide assays

Three different approaches were used to assay insecticide treatments against adult Carpophilus spp. The first was an application of the treatment to filter paper, it was allowed to dry, and then adults were added. The second approach used a plug of peeled pear fruit dipped into the treatment, and it was allowed to dry and placed in a petri dish, and then adults were added. The third was done similarly when a green peach fruit was dipped into the treatment, allowed to dry, and placed in a cup, and then adults added.

The first approach was done using two experiments. For experiment 1, treatments were applied to filter paper (Fisherbrand^TM P8 Grade, Fisher Scientific, Pittsburgh, PA) by using an autoload Potter spray tower (Burkard Scientific, Ltd., Uxbridge, UK) set at 0.35 kg/cm² to deliver 2 ml of treatment, followed by a 5-s settling time. For the second experiment, 250 µl of treatment was pipetted directly onto the filter paper, ensuring that all of the filter paper was wetted. In both trials, the filter paper was allowed to air dry and then placed into the lid of a clean petri dish (6-cm diameter). Five adult Carpophilus spp. were added to the petri dish bottom and covered with the lid (housing the filter paper), and then the dish was inverted so the filter paper would be on the bottom. Each treatment was replicated four times, with treatments randomized within replicates. Treatments were held in an environmental chamber at 25 ± 1°C and a 14:10 (L:D)-h photoperiod. Humidity in the environmental chamber was increased by adding food trays filled with water to the bottom of the chamber and using paper towels as wicks. Beetles were examined after 72 h by recording the number that were considered incapable of feeding (i.e., moribund + dead). Moribund beetles were incapable of righting themselves when turned over.

The second approach was conducted using two trials in one experiment. In each trial, a 0.5-cm diameter cork borer was used to remove plugs from a store-bought pear fruit that had been washed. The plugs were then cut into 1-cm-long sections and no peel was ever included. A single section of pear was dipped into a treatment, added to a 30-ml portion cup (Comet, WNA Inc. division of The Waddington Group), and allowed to dry for 1 h. After the pear dried, five adult Carpophilus spp. were added to the cup and a lid was placed on each cup. Each treatment was replicated four times in the first trial and three times in the second trial, with treatments randomized within replicates. Treatments were held in an environmental chamber at 25 ± 1°C and a 14:10 (L:D)-h photoperiod, and beetles were examined after 72 h by recording the number that were nonfeeding.

The third approach was conducted using two trials of the same experiment, performed concurrently from 16–20 July 2015. Small, green peach fruit (3.3 ± 0.1 cm from pedicel to tip and a circumference of 6.2 ± 0.1 cm) were picked, taken to the laboratory, and each dipped in a treatment. Dipped fruit were placed on filter paper and allowed to dry overnight. Peaches were then placed in a cup (266 ml; Eco Products^® , Boulder, CO), five adult Carpophilus spp. were added, and a lid was placed on each cup. Each treatment was replicated four times, with treatments randomized within replicates. Treatments were held in an environmental chamber at 25 ± 1°C and a 14:10 (L:D)-h photoperiod, and beetles were examined after 72 h by recording the number that were nonfeeding (i.e., moribund + dead).

Results

Carpophilus spp. larvae were susceptible to all entomopathogenic nematode species and strains tested. Data from both rates of application (1,000 and 2,000 infective juveniles) were combined because there was no treatment × rate interaction (P = 0.9359). Data 7 d after treatment always resulted in significantly lower survival than nontreated larvae (F = 28.36; df = 6, 116; P < 0.0001). Within the entomopathogenic nematodes, survival was lower for S. riobrave and H. megidis than for H. bacteriophora and H. indica. Also, survival of larvae treated with S. carpocapsae was lower than those treated with H. bacteriophora (Fig. 1).

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Applying insecticides (microbial-based and synthetic) to filter paper and allowing treatments to dry had a significant effect on the number of Carpophilus spp. adults rated as nonfeeding 3 d later when applied by a Potter spray tower (F=10.48; df= 14, 41; P < 0.0001) and by a micropipette (F=8.45; df=14, 42; P < 0.0001). When treatments were applied via the spray tower, a significantly higher number of nonfeeding beetles were recorded from the synthetic insecticides indoxacarb and phosmet than all other treatments except spinetoram (Fig. 2A). In fact, significantly more nonfeeding beetles were also recorded from the spinetoram treatment than from the control and imidacloprid treatments. No other treatments differed significantly from the control, including the microbial-based insecticides. Similarly, when treatments were applied to filter paper via a micropipette, more beetles were recorded as nonfeeding when exposed to phosmet than all other treatments, except indoxacarb and β-cyfluthrin (Fig. 2B). Additionally, except for these three treatments, none of the other treatments were significantly different from the nontreated control. In fact, no mortality was recorded for chlorantraniliprole, imidacloprid, or the microbial-based products derived from the metabolites of C. subtsugae and Burkholderia spp.

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Dipping pear plugs into the microbial-based and synthetic insecticides, allowing the plugs to dry, and then exposing beetles to them had a significant effect on the number of nonfeeding beetles 3 d later for each of the two trials (F=48.34; df=14, 42; P < 0.0001 and F=42.45; df=14, 42; P < 0.0001, respectively). During the first trial, all treatments resulted in a similar, significantly higher number of nonfeeding beetles than the control except for the similarly lower carbaryl, chlorantraniliprole, C. subtsugae, malathion, and Burkholderia spp. treatments (Fig. 3A). The synthetic insecticides chlorantraniliprole and malathion, except carbaryl, resulted in a significantly higher number of nonfeeding beetles than either of the microbialbased insecticides. During the second trial, all treatments except chlorantraniliprole, C. subtsugae, and Burkholderia spp. resulted in a significantly higher number of nonfeeding beetles (Fig. 3B). Again, the synthetic insecticides, except chlorantraniliprole, resulted in a significantly higher number of nonfeeding beetles than either of the microbial-based insecticides.

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Dipping green peach fruit into the microbial-based and synthetic insecticides, allowing the fruits to dry, and then exposing beetles to the fruit had a significant effect on the number of nonfeeding beetles 3 d later (F = 17.48; df = 14, 87; P < 0.0001). The indoxacarb, β-cyfluthrin, fenpropathrin, and phosmet treatments resulted in a significantly higher number of nonfeeding beetles than all other treatments except for spinetoram (Fig. 4). In fact, only these four treatments resulted in numbers of nonfeeding beetles that were significantly higher than the control.

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Discussion

The results of this study show that different options are available for the management of larval and adult sap beetles. All entomopathogenic nematodes assayed in the current study were capable of decreasing the survival of late instar Carpophilus spp. in soil cups. This is consistent with the findings of Vega et al. (1994) and Glazer et al. (1999, 2007) that showed that larval C. hemipterus and C. humeralis are susceptible to entomopathogenic nematodes. We tested three of the nematode species used by Vega et al. (1994), and in both studies, virulence was highest for S. riobrave. In contrast, we showed virulence for S. feltiae and S. carpocapsae, whereas Vega et al. (1994) demonstrated that those species were pathogenic to Carpophilus spp.

Although Carpophilus spp. larvae typically feed within fruits, these fruits may occur on a plant above ground and may not be amenable to applications of entomopathogenic nematodes due the nematodes' sensitivity to ultraviolet radiation and desiccation (Shapiro-Ilan et al. 2018). Additionally, larvae can occur within fallen fruits on the ground that likely provide refuge from soil-dwelling entomopathogenic nematodes. However, when larvae leave fruits to pupate in the soil, our results indicate that they are susceptible, and even highly susceptible, to all species and strains of the nematodes we tested, with S. riobrave and H. megidis appearing to show the most promise for control. Although soil-applied nematodes would be directed against the pest after crop damage had occurred, such an approach could be used to reduce numbers of emerging adults. Similarly, insect growth regulators have provided control of larval Carpophilus spp. (Blumberg 2008), but these were not included in the current study.

Our results from assaying adult Carpophilus spp. with microbial-based and synthetic insecticides revealed that exposure of adult beetles to the same treatment by different methods (i.e., on a dry surface or likely ingested) affected beetle survival. Indoxacarb and phosmet provided the highest and most consistent effect against the beetles when treatments were applied to filter paper. Indoxacarb and phosmet stood out again, as did β-cyfluthrin and fenpropathrin, when treatments were applied to green peach fruit. No obvious evidence of feeding on the fruit during the assay was detected. Differences in the surfaces of the filter paper and peach fruit possibly led to differences in the numbers of nonfeeding beetles between these two exposure methods. Peach fruit are generally pubescent, and this characteristic may have elevated the treatment above the fruit skin, allowing for more contact with beetles. If so, the nonpubescent, glabrous skin of nectarines could be expected to have similar results as with the filter paper. However, pear plugs dipped into the treatments were likely fed upon by the beetles and this exposure method led to even more treatments having significantly higher numbers of nonfeeding beetles. The insecticides thiamethoxam, acetamiprid, clothianidin, spinetoram, and imidacloprid went from little activity on dry surfaces to high activity against the beetles when they were exposed to the treated pear plug. It is likely that providing the beetles with a treated food source played a large role in this observed difference. Blumberg (2008) reports that the pyrethroids λ-cyhalothrin and bifenthrin and the neonicotinoids imidacloprid and thiacloprid were effective in controlling sap beetles attacking date palm. Nault and Speese (2000) found a significant reduction in the number of adult Carpophilus spp. (but not larvae) attacking sweet corn (Zea mays L.) when treated with λ-cyhalothrin and β-cyfluthrin. The microbial-based insecticides, along with chlorantraniliprole and to a lesser extent carbaryl and malathion, did not have a significant effect on the sap beetles regardless of the exposure method. Miller and Williams (1983) achieved similarly poor results by using carbaryl against C. hemipterus when figs were dipped in treatments, similar to what was done with pear in the current study, and then exposed to beetles. In that same study, the authors report high mortality of C. hemipterus exposed to malathion that is in contrast to the results of the current study. Our results with malathion also contrast with those of Blumberg (2008) who states that malathion has provided the most satisfactory control of nitidulids attacking date palms in Israel.

Our results concerning the high level of activity of phosmet on dry surfaces against adult sap beetles strongly suggests that the decreased use of this insecticide in southeastern U.S. peach orchards can contribute to sap beetle damage, as previously documented in Australia (James et al. 2000). Although clothianidin has good activity against plum curculio and is used instead of phosmet at some points during the season, it is not likely to control adult sap beetles that contact it on dry surfaces. The greatest exposure of adult beetles to insecticides in peach orchards would be on surfaces such as bark, foliage, and fruit when applied as dilute applications via airblast orchard sprayers. Once inside a fruit, beetles would not be exposed to prior or future insecticide applications, unless they moved out of the fruit. This indicates that the efficacy of insecticides used to manage sap beetle adults should be considered when applied to surfaces that the beetle will contact but not feed upon. Additionally, treating the orchard floor with entomopathogenic nematodes prior to when larvae leave fruit to pupate in the soil could alleviate populations building up in orchards. Field trials confirming these laboratory results are needed.