Walnut Twig Beetle Landing Rates Differ Between Host and Nonhost Hardwood Trees Under the Inﬂuence of Aggregation Pheromone in a Northern California Riparian Forest

Agricultural and Forest Entomology (2021), 23, 111–120 DOI: 10.1111/afe.12410 Walnut twig beetle landing rates differ between host and nonhost hardwood trees under the inﬂuence of aggregation pheromone in a northern California riparian forest

∗ † ‡ ∗ Crystal S. Homicz , Jackson P. Audley , Yigen Chen , Richard M. Bostock§, Catherine A. Tauber ¶ and ∗∗∗ Steven J. Seybold ∗ Department of Entomology and Nematology, University of California, Davis, CA, 95616, U.S.A., †Oak Ridge Institute for Science and Education, USDA Forest Service, Pacifc Southwest Research Station, Davis, CA, 95618, U.S.A., ‡Foundation Research, Analytics and Business Applications, E. & J. Gallo Winery, Modesto, CA, 95354, U.S.A., §Department of Plant Pathology, University of California, Davis, CA, 95616, U.S.A., ¶Department of Entomology, Cornell University, Ithaca, NY, 14853, U.S.A. and ∗∗ USDA Forest Service, Pacifc Southwest Research Station, Davis, CA, 95618, U.S.A.

Abstract 1 Host selection behaviour of the walnut twig beetle (WTB) among hardwood trees was investigated in a riparian forest in northern California by monitoring the landing rate of the beetle with sticky traps on branches baited with 3-methyl-2-buten-1-ol, the male-produced aggregation pheromone. 2 The assay was conducted over 7 days (22 May to 29 May 2017) and compared landing rates on branches of six nonhost species paired with northern California black walnut, Juglans hindsii (the host). 3 A total of 2242/1192 WTB were collected on branches of host/nonhost pairs, and more WTB landed on J. hindsii than on nonhosts in 42 of 58 instances. Female landing rate generally exceeded male landing rate, which underscores the infuence of the male-produced synthetic pheromone in this system. 4 Landing rates of WTB males, females, and the combined sexes on boxelder, Acer negundo, and valley oak, Quercus lobata, did not differ signifcantly from the landing rates on J. hindsii, suggesting that these two nonhost riparian hardwoods do not repel WTB (in the context of the aggregation pheromone). 5 Signifcantly fewer WTB landed on Oregon ash, Fraxinus latifolia, river red gum, Eucalyptus camaldulensis, Fremont cottonwood, Populus fremontii, and red willow, Salix laevigata, than on J. hindsii, which suggests that these four nonhosts may repel one or both sexes of WTB in the context of the aggregation pheromone. Future analysis of the volatiles from these four hardwood species may lead to the discovery of semiochemical repellents for WTB. Keywords Chrysoperla, Geosmithia morbida, host selection behaviour, Juglans hindsii, northern California black walnut, Pityophthorus juglandis, Scolytidae, thousand cankers disease, walnut pest, walnut twig beetle.

Introduction phloem occurs with each beetle attack causing thousand cankers disease (TCD), which results in the decline and mortality of wal- The walnut twig beetle (WTB), Pityophthorus juglandis Black- nut trees (Seybold et al., 2013b, 2019). man (Coleoptera: Scolytidae), vectors a phytopathogenic fun- Walnut twig beetle and G. morbida were frst described in gus, Geosmithia morbida Kolarík,̌ Freeland, Utley, & Tisserat the western U.S.A., and are thought to be native on Arizona (Ascomycota: Hypocreales: Bionectriaceae) to the phloem of walnut, Juglans major, in the southwestern U.S.A. and northern walnut and wingnut trees. A canker (e.g. tissue necrosis) in the Mexico (Bright, 1981; Kolaríǩ et al., 2011; Zerillo et al., 2014; Rugman-Jones et al., 2015; Seybold et al., 2016). Walnut twig Correspondence: Crystal S. Homicz. Tel.: (530) 524-2885; fax: (530) beetle was frst collected in New Mexico in 1896, followed by 752-1537; e-mail: [email protected] records in Arizona, California, and Chihuahua, Mexico (1907,

1959, and 1960, respectively) (Bright, 1981; Rugman-Jones have been studied widely, however little is known of WTB’s et al., 2015; Seybold et al., 2016, 2019). The frst published response to nonhost trees. The host colonization behaviour of incidence of TCD occurred in New Mexico in 2001; however, the WTB may be explained by one of two prevailing hypotheses disease complex was likely active in the early 1990s in Oregon that explain bark beetle host colonization: (i) Walnut twig beetle and Utah based on reports of walnut tree mortality in those U.S. may actively respond to long-range volatile cues and signals states (reviewed in Tisserat et al., 2011). Following these events, while in fight to locate an appropriate host tree; (ii) WTB mortality due to TCD was noted in 2003 in Colorado (Tisserat may be infuenced by gustatory cues and shorter-range olfactory et al., 2011), whereas TCD was frst detected in California signals/cues once it has landed on a tree to select host which in 2008 in Yolo Co. and Solano Co. (Seybold et al., 2019). indicates use of random landing (Raffa et al., 2016). It has been However, the disease had likely been present much longer in shown that WTB likely uses long-range signals and/or cues while California (Flint et al., 2010). Following these descriptions, in fight to select susceptible hosts (Hishinuma, 2017; Audley TCD was detected in the eastern U.S.A., with reports from et al., 2020; Lona et al., 2020). While feeding, the male beetle Tennessee, Virginia, Pennsylvania, Ohio, Maryland, Indiana, and releases an aggregation pheromone (3-methyl-2-buten-1-ol) to North Carolina (Tisserat et al., 2011; Seybold et al., 2013b). attract female and additional male beetles to the tree (Seybold Most recently (2013) TCD was discovered in fve regions in Italy et al., 2015). Male WTB then mate with several females and the (Faccoli et al., 2016; Moricca et al., 2018). cohort completes its life cycle in the phloem of the selected host Thousand cankers disease threatens walnut trees grown for tree. urban shade cover, agriculture, and wood production, as well In this study, we examined how WTB responded in fight to as trees in native North American riparian stands that provide tree volatile cues, in the presence of an aggregation pheromone benefts to wildlife (Hefty et al., 2018; Seybold et al., 2019). lure, to distinguish between J. hindsii (host trees) and nonhost In California, two native tree species, southern California black hardwood trees. Additionally, we were interested in testing the walnut, Juglans californica, and northern California black infuence of sex and branch aspect on behavioural responses walnut, Juglans hindsii, have particularly limited distributions as well. The sex ratio can be revealing of the infuence of (Jepson, 1917; Griffn & Critchfeld, 1972). These native wal- semiochemical constituents. For example, we typically observe nut tree species are found in riparian ecosystems and food approximately twice as many female beetles responding to the plains throughout California’s foothills and valleys. J. hindsii aggregation pheromone lure in trapping assays (Chen & Sey- is listed as critically imperilled by the California Native Plant bold, 2014; Seybold et al., 2015). Deviations from a 2:1 female Society (CNPS, 2018), therefore, conservation of the species to male ratio were interpreted as potentially different sensitiv- is a high priority. Even though their range has been signif- ities to volatile profles. We also assessed WTB preference for cantly reduced, they still occur throughout the state with similar branch aspect (top, bottom, left, right) because past studies have canopy and undergrowth structure as their historical counterparts found higher landing rates on the bottom side of the branch (Katibah, 1984). (Hishinuma, 2017). Bark beetles have been shown to use their antennae to Understanding host selection behaviour of WTB will likely sense semiochemicals (volatile chemical compounds that inform potential management strategies for this invasive bark elicit behavioural responses in receiving organisms) such as beetle and disease complex. This study also reports on the pheromones and kairomones to locate trees while in fight possible attraction of potential WTB predators and natural for colonization, feeding, and reproduction (Silverstein, 1981; enemies of foliar walnut pests to the pheromone lure. Studying Wood, 1982; Raffa et al., 1993; Borden, 1997; Raffa, 2001; these behavioural phenomena in a native riparian area provides Raffa et al., 2016; Seybold et al., 2018). Bark beetles follow a an opportunity to discover ecologically sourced volatiles that four-step process for host colonization: (i) the dispersal phase, repel the WTB. Knowledge of the action of these volatiles where (ii) selection phase, (iii) concentration phase, and (iv) establish- the WTB’s hosts and natural enemies are present, may form the ment phase (Wood, 1982). The beetles begin the dispersal phase basis for the development of a semiochemical repellent that can by responding to cues from the host trees and/or pheromones be used within an integrated pest management setting to protect after emerging from the brood tree. They then select a potential walnut trees in urban landscapes, native habitat, and commercial host tree by responding to stimuli from the host while in fight nut orchards. and/or after landing. Next, they initiate colonization by boring through the bark and into the phloem. During the concentration phase, they release aggregation pheromone to attract other Materials and methods conspecifcs. The establishment phase occurs when a tree has Site description been suffciently colonized by bark beetles and fungal inoculum. Past studies have shown that host colonization can be interrupted The experiment was conducted at Putah Creek Riparian Reserve by signals such as antiaggregation pheromones (e.g. repellence in Davis, California (Fig. 1). of Dendroctonus brevicomis by verbenone) (Silverstein, 1981; At this site, Putah Creek is accompanied by a riparian forest Borden, 1997; Seybold et al., 2018). habitat that consists mainly of seven tree species: J. hindsii (WTB Walnut twig beetle is a primary pest of walnut trees in host), and Acer negundo, Eucalyptus camaldulensis, Fraxinus California and also colonizes closely-related wingnut trees (Flint latifolia, Populus fremontii, Quercus lobata,and Salix laevigata et al., 2010; Hishinuma et al., 2016; Seybold et al., 2016; (all nonhosts) (Table 1). E. camaldulensis is not a native member Hefty et al., 2018). Colonization behaviours of WTB under the of this plant community. In a previous survey of the vegetation infuence of aggregation pheromone and in response to host trees along Putah Creek, Q. lobata was the most abundant species

Figure 1 Study site for an experiment on host selection behavior of the walnut twig beetle, Pityophthorus juglandis, was located at the Putah Creek Riparian Reserve in Davis, Yolo Co., California. Each tree pair included in the study is indicated with a balloon marker. All study trees were within 45 m of the north shore of Putah Creek at the Putah Creek Riparian Reserve in Davis, California. The west end of the transect was located at 38∘31.62′ , −120∘48.3′ , and the east end of the transect was located at 38∘31.44′ , −121∘46.98′ .

Table 1 Common, binomial, and family names, as well as sizes of tree species included in an experiment to test the landing rates of walnut twig beetle, Pityophthorus juglandis, in a riparian forest habitat in northern California (Putah Creek Riparian Reserve, Davis, Yolo Co.)

Common name Binomial name Family Sample size Mean DBHa (cm) Range of DBHa (cm)

Northern California black walnut Juglans hindsii Jeps. Ex R.E. Sm. Juglandaceae 58 17.2 4.8–40.9 Fremont cottonwood Populus fremontii S. Watson Salicaceae 10 48.4 21.1–111.5 River red gum Eucalyptus camaldulensis Denh. Myrtaceae 8 15.8 8.9–26.4 Boxelder Acer negundo L. Sapindaceae 9 13.1 5.3–23.4 Oregon ash Fraxinus latifolia Benth. Oleaceae 10 16.0 5.1–23.4 Valley oak Quercus lobata Née Fagaceae 12 31.5 8.1–57.1 Red willow Salix laevigata Bebb Salicaceae 9 18.2 4.8–40.9 a DBH = diameter at breast height, measured on the tree stem at 1.37 m above the forest ﬂoor. followed by F. latifolia, A. negundo, E. camaldulensis, J. hindsii, along an east–west transect of approximately 2 km (Fig. 1; see S. laevigata,andP. fremontii (Hort Science, 1997). Supporting information, Table S1). Ten individuals of each nonhost tree species were paired with Primary peak fight of WTB typically occurs from May to July a selected individual of J. hindsii. To maximize sample size, with a secondary fight period from September to October (Chen nearly all J. hindsii present at the reserve were used in the study. & Seybold, 2014). We conducted our experiment from May 22 As such, paired host and nonhost branches were no more than to 29, 2017 during the initial period of peak fight in hopes of 5 m from one another. The proximity of the two trees within a utilizing the highest possible number of WTB for testing discrim- pairing probably made detecting signifcant differences between ination among the host and the hardwood nonhosts at the site. tree species conservative because at this distance WTB might exploit volatiles emitted from both tree species for orientation, in addition to the pheromone lures. On the other hand, if the Trap placement and handling two tree species were farther apart such that the WTB could only utilize volatiles from one tree species plus the pheromone On each host/nonhost tree, one translucent acetate sheet covered lure, then landing rate on host tree might be greater than that on with trap adhesive (Stick-em Special, Seabright Enterprises, nonhost trees, supposing WTB uses host volatile for orientation. Emeryville, California) was attached to a selected branch with A GPS coordinate was recorded at a point equidistant between a diameter ranging from 3 to 8 cm (Hishinuma, 2017). Traps the host and nonhost tree for each pairing (see Supporting were 1 to 4 m above the forest foor. Adhesive-coated traps were information, Table S1). Diameter at breast height of the study rectangular (21.6 cm by 28 cm), and they were attached to the tree trees ranged from 4.8 cm to 111.5 cm (Table 1). Host/nonhost by wrapping the trap around a branch so that the 21.6 cm side was pairs were at least 3 m from other tree pairings to avoid con- aligned with the axis of the branch. Pushpins were then used to founding variables. Audley et al. (2020) found the effect of affx the trap to two corks that had been glued on the outer bark of the pheromone lure to have a fairly localized effect on WTB the branch. Host and nonhost tree branches were baited with the behaviour, infuencing landing rates within 200 cm of the lure. WTB aggregation pheromone, 3-methyl-2-buten-1-ol (released Study trees were within 45 m of the north shore of Putah Creek at 1.2 mg/d) (Contech Enterprises Inc., Product #300000968, lot

#14342, Victoria, BC, Canada) (Seybold et al., 2013a). The lure For this analysis, host status, branch aspect, and their interaction was centred and pinned on the top of the branch and immediately were fxed terms, and tree pair location was the only random beneath the trap. A total of 116 acetate sticky traps were used term. in the experiment (one trap per tree). In the feld, we decided For both analyses, likelihood ratio tests [the function ‘anova()’ to include two extra pairs for Q. lobata because the eleventh in the package ‘lme4’] were used to compare models with and twelfth pairs were readily accessible. Additionally, two various complexities and if the P-value of the likelihood ratio J. hindsii/E. camaldulensis trap pairs, one J. hindsii/A. negundo test between two models was less than 0.05, then the more trap pairs and one J. hindsii/S. laevigata pair were lost in the feld. complex model was chosen. Otherwise, the simpler model was The sticky traps were in place from May 22 to May 29, 2017 (7 chosen (Zuur et al., 2009). Scatter plots of model residuals d). When sticky traps were removed from the tree, the aspect against ftted values, block, and location were visually checked of the trap on the tree branch (top, right, bottom, and left, from for independence and variance homogeneity. Signifcance of the perspective of the main stem of each tree) was labelled on model terms in the fnal model was determined by a Type III the trap along with a tree identifcation code, and then each trap analysis of deviance (the function ‘Anova()’ in the package was pinned in a corresponding cardboard box for storage prior to ‘car’). Analyses were conducted separately for female, male, insect removal in the laboratory. and total (= female + male) WTB. All analyses were conducted using the R statistical software (v. 3.3.1) (R Core Team, 2018). Statistical tests were considered signifcant for P-values ≤0.05. Handling of insects and traps in the laboratory To roughly gauge if WTB landing had any spatial pattern, a correlation between WTB trap catches at each tree pair location All insects of interest were cleaned and identifed at least to and distance from the western terminus of the transect was order, and to family or species level when possible. In addition to conducted. Since WTB catches were counts, we chose the WTB, other subcortical insects, suspected WTB predators, and nonparametric Kendall rank correlation coeffcient [the function potential natural enemies of foliar walnut pests were collected ‘cor()’ in the R base package]. (Seybold et al., 2016; Grant et al., 2017). All WTB were sexed and counted by sex. Results

Insect and tree identiﬁcation Landing rates of WTB on adhesive-coated traps Coleoptera was generally identifed by SJS. Some speci- A total of 2242/1192 WTB (host/nonhost) were collected on the mens of bark and ambrosia beetles were sent to Dr. Donald branch pairs (n = 116 traps) during the experimental trapping E. Bright, Colorado State University, Fort Collins, Colorado, for period (Table 3). WTB had a higher landing rate on J. hindsii fnal confrmation. In this note, we have elected to use the orig- in 42 of 58 (∼72%) instances and an approximate sex ratio of inal nomenclature for bark and ambrosia beetles (Coleoptera: 2 females to 1 male across all pairings (Fig. 2). Female landing Scolytidae) based on the arguments presented in Bright (2014, rate generally exceeded male landing rate, which underscores 2019). Voucher specimens of scolytid adults were accessioned the infuence of the male-produced synthetic pheromone in into the Entomology Department at the California Academy of this system (Table 3). When all nonhost tree species were Sciences, San Francisco, California. CAT identifed specimens pooled, there was a signifcant effect of host/nonhost on the 2 of Chrysopidae and Hemerobiidae. Voucher specimens of these landing rates of male, female, and total WTB (X1 = 35.97, < 2 < 2 < adults were deposited in the UCD Bohart Insect Museum. P 0.001; X1 = 47.69, P 0.001; and X1 = 52.72, P 0.001, Ellen Dean, University of California Davis, Davis, California, respectively). When nonhost tree species were separately anal- identifed E. camaldulensis and S. laevigata trees. ysed, there was no signifcant difference in the landing rate of male, female, or total WTB between J. hindsii and A. negundo 2 2 2 (X1 = 2.26, P = 0.133; X1 = 2.41, P = 0.121; and X1 = 20.64, 2 P = 0.074, respectively) or Q. lobata (X1 = 1.10, P = 0.294; Data handling and statistical analysis 2 2 X1 = 3.44, P = 0.064; and X1 = 5.80, P = 0.092, respectively) To study the effects of host status and branch surface aspect on (Table 2, Fig. 2). Signifcantly fewer male, female, and total 2 2 WTB landing, two generalized linear mixed-effects model [the WTB landed on S. laevigata (X1 = 5.52, P = 0.019; X1 = 9.76, 2 function ‘glmer.nb()’ in the package ‘lme4’] were ft to the data. P = 0.002; and X1 = 9.78, P = 0.002, respectively), P. f re - 2 < 2 < For the frst analysis, data from all six nonhost tree species were montii (X1 = 31.89, P 0.001; X1 = 27.92, P 0.001; and 2 < 2 pooled. For this analysis, host status (2 levels: host and nonhost), X1 = 35.58, P 0.001, respectively), F. latifolia(X1 = 7.31, 2 2 branch aspect (4 levels: top, bottom, left, and right), and the P = 0.007; X1 = 8.79, P = 0.008; and X1 = 5.07, P = 0.005, 2 interaction between tree pair location and branch aspect were the respectively), and E. camaldulensis (X1 = 7.95, P = 0.005; 2 < 2 fxed terms. Non-host tree species (i.e. six) and tree pair location X1 = 2.15, P 0.001; and X1 = 0.65, P = 0.001, respectively) nested within nonhost species (i.e. 10 pairs) were the two random versus J. hindsii (Table 2, Fig. 2). terms. The distribution of WTB was modelled as a negative WTB landing by trap aspect (top, right, bottom, left) was binomial distribution to account for potential over-dispersion and signifcant only for males landing on P. fremontii versus J. hindsii 2 the link function between WTB catches and the linear predictor (X3 = 13.07, P = 0.005) in the original model. Aspect was not was the default natural logarithm. In the second analysis, WTB signifcant in any of the other host/nonhost tree pairs. In general, landing was separately analysed for each nonhost tree species. fewer WTB landed on the top side versus bottom, left, and right.

Figure 2 Comparative mean landing rates (±SE) of (A) females of walnut twig beetle and (B) males of walnut twig beetle, Pityophthorus juglandis,on host/nonhost pairs of branches over a seven-day period (22–29 May, 2017) at the Putah Creek Riparian Reserve in Davis, (Yolo Co.), California. The host was northern California black walnut, Juglans hindsii. NS indicates no significant difference; (*) indicates significant difference at 0.001 < P < 0.05; and (**) indicates significant difference at P < 0.001.

We found no signifcant correlation between total number of Ptilinus sp. (all Anobiidae); the minute bark beetle, Hypothen- WTB trapped per pair (host + nonhost) and the distance of emus eruditus, a willow bark beetle, Trypophloeus thatcheri, each tree pair from the west end of the transect (r2 = 0.179, the fruit-tree pinhole borer, Xyleborinus saxeseni, Dendrocran- SE = 83.84) indicating there is no signifcant spatial pattern. ulus curcurbitae (all Scolytidae); and Nathrius brevipennis There was evidence of WTB boring dust accumulating on the (Cerambycidae). acetate trap sheets from 25 of the 60 traps (43%) on branches of Potential natural enemies of walnut pests were also found in the J. hindsii (Table 3). This suggests that volunteer male WTB may adhesive coated traps. Adults of green lacewings (Neuroptera: have colonized these branches during the 7-day fight period. Chrysopidae) and Hemerobius sp. (Neuroptera: Hemerobiidae) Surprisingly, boring dust was also evident on two of the nonhost were the most numerous (Table 4). A subset of the Chrysopidae branches in the study (one each of A. negundo and F. latifolia) (n = 47, 23.6% of the total chrysopids trapped) were examined (Table 3). by CAT; all of these were in the Chrysoperla carnea species group, and 46 of the 47 specimens examined were male. Therefore, we analysed landing rate data for C. carnea because Landing rates of associated insects on adhesive-coated a suffcient number of individuals were trapped, unlike for traps the other associated insects. We found that the Chrysoperla The most abundant insects collected were beetles from three fam- species were trapped signifcantly more often on J. hindsii ilies, Anobiidae, Cerambycidae, and Scolytidae (Table 4). The branches versus the branches of the other six sampled tree species 2 following species are those thought be associated with J. hind- (X1 = 7.11, P = 0.008). sii (Seybold et al., 2016) and the nonhost hardwoods (Furniss & Chrysopid larvae are generalist predators often preying on Carolin, 1977): Petalium californica, Priobium punctatum,and aphids (Albuquerque et al., 2012). Although these neuropteran

Table 2 Signiﬁcance of best model terms as determined by a type III analysis of deviance from an experiment to test the landing rates of walnut twig beetle, Pityophthorus juglandis, in a riparian forest habitat in northern California (Putah Creek Riparian Reserve, Davis, Yolo Co.)

Nonhost species Sexa Factora d.f. X2 P-value

All nonhosts Total Species 1 52.72 <0.001** All nonhosts Male Species 1 35.97 <0.001** All nonhosts Female Species 1 47.69 <0.001** Salix laevigata Total Species 1 9.78 0.002* Salix laevigata Male Species 1 5.52 0.019* Salix laevigata Female Species 1 9.76 0.002* Quercus lobata Total Species 1 5.80 0.092 Quercus lobata Male Species 1 1.10 0.294 Quercus lobata Female Species 1 3.44 0.064 Populus fremontii Total Species 1 35.58 <0.001** Populus fremontii Male Species 1 31.89 <0.001** Populus fremontii Male Trap aspect 3 13.07 0.005* Populus fremontii Female Species 1 27.92 <0.001** Fraxinus latifolia Total Species 1 5.07 0.005* Fraxinus latifolia Male Species 1 7.31 0.007* Fraxinus latifolia Female Species 1 8.79 0.008* Acer negundo Total Species 1 20.64 0.074 Acer negundo Male Species 1 2.26 0.133 Acer negundo Female Species 1 2.41 0.121 Eucalyptus camaldulensis Total Species 1 0.65 0.001* Eucalyptus camaldulensis Male Species 1 7.95 0.005* Eucalyptus camaldulensis Female Species 1 2.15 <0.001** aThe full models included species (host vs. nonhost), trap aspect (top, right, bottom, left), and their interaction as fixed terms. Block and tree pair location nested within a block were the two random factors. Since model selection showed no significant interaction between species and trap aspect in any of the models, the final models only included species and aspect (in one instance). In all models the host species was Juglans hindsii and the nonhost species of the model is specified in the left column. A model was built for each of the WTB sexes; male, female, and total (= male + female). Significance values are indicated with asterisks; (*) indicates significant difference at 0.001 < P < 0.05; and (**) indicates significant difference at P < 0.001.

Table 3 Number of walnut twig beetle (WTB), Pityophthorus juglandis, trapped on by tree species during landing rate assays (the host was Juglans hindsii), sex ratio of trapped WTB and evidence of colonization of branches by WTB during landing rate assays (22–29 May 2017), at the Putah Creek Riparian Reserve, Davis, Yolo Co., California

No. WTB WTB sex ratio No. traps Proportion of traps Common name Binomial name No. traps trapped (female: male) with boring dust with boring dust

Northern California black walnut Juglans hindsii Jeps. ex R.E. Sm. 58 2242 1496:746 25 0.43 Fremont cottonwood Populus fremontii S. Watson 10 59 39:20 0 0 River red gum Eucalyptus camaldulensis Denh. 8 39 29:10 0 0 Boxelder Acer negundo L. 9 560 347:213 1a 0.11 Oregon ash Fraxinus latifolia Benth. 10 152 104:48 1a 0.10 Valley oak Quercus lobata Née 12 308 213:95 0 0 Red willow Salix laevigata Bebb 9 73 48:25 0 0 aIn tree pairs where boring dust was present on traps on branches of the nonhost tree, boring dust was also present on traps on branches of the host tree. predators have not been reported to prey on bark beetles, we A. negundo and Q. lobata from J. hindsii. Our results here are observed a chrysopid larva feeding on a WTB adult in a funnel similar to recent fndings of Audley (2020) who, in a study of host trap catch in El Dorado Co., California, below an aphid-infested attraction in the absence of aggregation pheromone lures, also Juglans nigra tree (Fig. 3). From this observation, we suspect found signifcant difference between host and nonhost landing that chrysopid larvae may, at least occasionally and perhaps rates of WTB in four of six pairings, including no difference in opportunistically, prey on WTB. landing rates in the J.hindsii versus A. negundo and the J. hindsii versus P. fremontii pairings. We hypothesize A. negundo and Q. lobata may either have Discussion similar volatile profles to J. hindsii, or they may have different Our results show that, in the presence of aggregation pheromone, profles primarily composed of compounds which WTB does not WTB distinguish F. latifolia, E. camaldulensis, S. laevigata, perceive. Possibly, volatiles from F. latifolia, E. camaldulensis, and P. fremontii from J. hindsii, but they do not differentiate S. laevigata,andP. fremontii may repel both sexes of WTB.

Table 4 Subcortical insect associates and potential natural enemies of walnut twig beetle, Pityophthorus juglandis, trapped on host and nonhost paired branches during landing rate assays (22–29 May 2017), Putah Creek Riparian Reserve, Davis, Yolo Co., California

Family Species Host Nonhost Total

Anobiidae Ptilinus sp. 7310 Anobiidae Petalium californicum Fall 10 19 29 Anobiidae Priobium punctatum LeConte 11 23 34 Cerambycidae Nathrius brevipennis Mulsant male 19 12 31 Cerambycidae Nathrius brevipennis Mulsant female 7 15 22 Cerambyicdae Phymatodes vulneratus LeConte 0 1 1 Chrysopidae Chrysoperla carnea Stephens species group 136 63 199 Hemerobiidae Hemerobius sp. 21 31 52 Raphidiidae Agulla sp. 3 0 3 Scolytidae Dendrocranulus curcubitae LeConte 2 0 2 Scolytidae Hypothenemus eruditus Westwood 0 8 8 Scolytidae Trypophloeus thatcheri Wood 1 0 1 Scolytidae Xyleborinus saxeseni Ratzeburg 2 0 2

may be worthy of investigation, given the relatively high WTB landing rates on this species that we recorded in this study and Audley (2020) (Fig. 2). Interestingly, the difference in landing rates between J. hindsii and P. fremontii was the largest (according to the Type III analysis of deviance) in the experiment (Fig. 2). One hypothesis to explain this result is that P. fremontii may produce a novel compound(s) recognizable by WTB, which indicates the tree is not a host species. An alternative hypothesis is that P. fremontii may also produce a volatile that blocks the WTB’s receptors, inhibit- ing the beetles’ ability to detect the male-produced aggregation pheromone. The native range of P. fremontii (Taylor, 2000) overlaps with that of J. major (USGS, 2014), the purported evolutionary host of WTB (Rugman-Jones et al., 2015). How- ever, the native distributions of E. camaldulensis (Australia) and F. latifolia (the Pacifc Coast of North America) do not overlap with J. major, and the range of S. laevigata overlaps very little. Thus, WTB likely evolved in the presence of both its host and the Figure 3 Scanning electron micrograph of a Chrysopidae larva with nonhost, P. fremontii, and natural selection may have honed its an adult female walnut twig beetle, Pityophthorus juglandis,heldin ability to distinguish the volatiles released by the host and non- its mandibles. The specimen was collected from a Lindgren funnel host tree species. In contrast, the WTB did not evolve alongside trap sample below an aphid infested Juglans nigra in July 2017 in E. camaldulensis and F. latifolia, and therefore, it may not have Georgetown, (El Dorado Co.), California. evolved the ability to recognize the components of the volatile profles released by these species. Future research should focus on the repellent potential of the nonhost P. fremontii, (either as Conversely, in the presence of the aggregation pheromone, active repellents or receptor blockers). Eucalyptus spp. volatiles volatiles from Q. lobata and A. negundo did not repel WTB or have already been quantifed and analysed in other studies and may attract them. The volatiles these tree species do produce shown to be repellent in other insect systems, so there is potential may also be in low quantities in comparison to the aggregation that volatiles could also repel WTB (Thacker & Train, 2010). pheromone used. Ultimately, WTB does not appear to be able Landing rate by aspect on the branches was signifcant only to distinguish a difference between the semiochemical cues of for males landing on J. hindsii versus P. fremontii,which wasa J. hindsii, A. negundo,andQ. lobata when the male-produced small subset of our data. Hishinuma (2017) reported that WTB aggregation pheromone is present. As such, there is little poten- preferred the bottom aspect of identical traps in an orchard tial to discover a novel compound from these two species. setting. In our experiment, fewer beetles landed on the top side Furthermore, little is known of WTB’s reproductive success of the branches, although no statistically signifcant trend was in the nonhost species that we surveyed. Hefty et al. (2018) found. It is interesting that the beetles may have avoided the found WTB was able to reproduce in branch sections of wingnut branch aspect where the pheromone lure was pinned. We predict (Juglandaceae: Pterocarya spp.) but not in hickory or pecan placement of the pheromone lure on the branch likely does (Juglandaceae: Carya spp.). Thus, WTB reproductive success not infuence WTB landing behaviour. Based on our results, on tree species outside of the Juglandaceae is unlikely. However, we conclude that WTB does not have a strong preference for a study to assess WTB’s reproductive capacity in A. negundo landing on any branch aspect (i.e. top, right, bottom, or left) in

© 2020 The Royal Entomological Society, Agricultural and Forest Entomology, 23, 111–120 118 C. S. Homicz et al. a riparian forest habitat. We assumed that WTB would have a Ptilinus sp., Petalium californicum,andPriobium punctatum. random distribution with no concentrations in one area across the Included among our catch were the ethanol-responsive beetles landscape of our study site, assuming we sampled in the vicinity Xyleborinus saxeseni and Hypothenemus eruditus, but in rela- of the host tree, J. hindsii. Our results support this prediction tively small numbers compared with results reported by Hish- given that we found no signifcant correlation between WTB inuma (2017). These beetles may prefer fermenting tissues in landing rate and distance from the west end of the transect. declining trees. Notably, Hishinuma (2017) used cut branches (in However, there were areas along the transect where we did not contrast to our branches on live trees), so an absence or low lev- survey due to an absence of J. hindsii. els of ethanol in our study system may explain why we trapped All traps in our experiment were baited with one WTB few of these insects. aggregation pheromone lure attached to the top of the branch Our study provides important context for the future man- with the goal of attracting an even density of WTB to the agement of thousand cankers disease. It provides feld-based vicinity of the host and nonhost branches. The female to male evidence that WTB responds to long-distance cues to identify ratio of our total WTB trappings was approximately 2:1 which and locate host trees during dispersal (Audley et al., 2020). further underscores the effect of the male-produced aggregation Additionally, it provides a foundation for future investigation of pheromone. Additionally, we found that WTB had a signifcantly potentially repellent nonhost plant volatiles. As WTB continues higher landing rate on J. hindsii versus all nonhost trees (n = 58). to kill native walnut species, as well as ornamental and orchard Hypothetically, the host branches had a higher probability of walnut species, the development of a WTB semiochemical repel- receiving attempted colonization by volunteer male WTB over lent becomes more crucial. Given the success of semiochemical the 7 days of the experiment (likely enough time to elicit new repellents in other bark beetle systems and WTB’s response to attacks by WTB). We found boring dust on 43% of J. hindsii long-range cues, we suggest further research and development individuals further indicating that these branches may have been of a semiochemical management strategy (Seybold et al., 2018). colonized by WTB. Thus, another interpretation of the higher landing rates on the host branches in the pairs could be an elevated level of pheromone emanating from the host branches Acknowledgments (i.e. from the lures and from the attacks by volunteers), which This project was supported by the U.S. Department of Agri- may have caused a biased landing rate on the J. hindsii branches. culture’s (USDA) Agricultural Marketing Service through Grant We found that the neuropteran species C. carnea had a 16-SCBGP-CA-0035 to RMB and SJS. Its contents are solely signifcantly higher landing rate on J. hindsii versus the other the responsibility of the authors and do not necessarily represent six tree species in the study, and the majority of C. carnea the offcial views of the USDA. We also thank the USDA Forest trapped were male. There may be numerous explanations for Service, Pacifc Southwest Research Station, and the UCD Col- this fnding, including: (i) Chrysoperla carnea-like species in lege of Agricultural and Environmental Sciences for their fnan- the Putah Creek area are attracted to the stress volatiles induced cial support. We thank Noah Christe and Megan Siefker (UCD by WTB attack on J. hindsii (ii) the other tree species were Department of Entomology and Nematology) for their assistance more repellant than J. hindsii, (iii) J. hindsii supports higher in the feld and laboratory. We thank Andrew Fulks for granting C. populations of attractive prey than the other trees, (iv) us access to the Putah Creek Riparian Reserve feld site. This carnea-like males respond to the WTB aggregation pheromone, project was part of an undergraduate practicum for CSH under or (v) any combination of the above possibilities. Past research the Animal Biology major in the College of Agricultural and has found that C. carnea and related green lacewing species Environmental Sciences at UCD. respond to many volatile cues including those released from honeydew, and herbivore-induced plant volatiles (Hagen, 1987; Tauber et al., 2000; Koczor et al., 2010; for Chrysopa species, Data availability statement also see Jones et al., 2011, 2016; Aldrich & Zhang, 2016). Likely a combination of the explanations above contribute to the Data available upon request from the authors. high numbers of C. carnea-like males in our traps on J. hindsii relative to the other nonhost plants. Thus, given C. carnea’s importance as a predator in this and other agricultural systems, Supporting information further research into these fndings may be justifed. Other associated insects landed at similar rates on the non- Additional supporting information may be found online in the host tree species and J. hindsii (Table 4). Besides C. carnea, Supporting Information section at the end of the article. we trapped more Hemerobius sp. (Neuroptera: Hemerobiidae) Table S1 GPS coordinates of pairs of hardwood trees at the than any other associated insect species. This indicates that Neu- Putah Creek Riparian Reserve in Davis, (Yolo Co.), California, ropterans may be present in high numbers throughout the Putah generally presented from west to east. Creek Riparian Reserve. We also trapped a considerable number (53 specimens, Table 4) of Nathrius brevipennis,aninvasivecer- ambycid, a fnding that is consistent with other recent trapping References studies (Audley et al., 2020; Lona et al., 2020). Additionally, Albuquerque, G.S., Tauber, C.A. & Tauber, M.J. (2012) Green lacewing we trapped two individuals of notable, relatively rare Coleoptera (Neuroptera: Chrysopidae): predatory lifestyle. Insect Bioecology and species, specifcally Phymatodes vulneratus (Cerambycidae) and Nutrition for Integrated Pest Management (ed. by A. R. Panizzi and Trypophloeus thatcheri (Scolytidae), three Anobiidae species; J. R. P. Parra), pp. 593–631. CRC Press, Boca Raton, Florida.

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