Endophytes matter: Variation of dung beetle performance across different endophyte-infected tall fescue cultivars

Applied Soil Ecology 152 (2020) 103561

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Endophytes matter: Variation of dung beetle performance across different endophyte-infected tall fescue cultivars

T

Tatsiana Shymanovicha, , Grace Crowleya, Sammuel Ingramb, Chey Steenc, Daniel G. Panaccionec, Carolyn A. Youngd, Wes Watsone, Matt Pooreb ⁎

a

Biology Department, University of North Carolina at Greensboro, Greensboro, NC 27402, USA Animal Science, North Carolina State University, Raleigh, NC 27607, USA c Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506, USA d Noble Research Institute, LLC, Ardmore, OK 73401, USA e Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27607, USA b

ARTICLE INFO

ABSTRACT

Keywords: Epichloë coenophiala strains Ergot alkaloids Pasture ecology Cow dung Oviposition substrate choice Brood-balls

Traditional tall fescue cultivar, Kentucky 31, possesses a wild-type endophyte strain that produces several bioactive compounds including the ergot alkaloid, ergovaline, known to cause cattle toxicity and effects on insects. Novel cultivars, BarOptima PLUS E34, Jesup MaxQ, and Texoma MaxQ II, possess different endophyte strains that do not have negative effects on cattle but still protect from insect grazing. Our study investigated if different cultivars have different effects on insect dung decomposers such as dung beetles and determined if ergovaline could be detected in cow dung. Ergovaline at 0.04 and 0.27 μg/g was detected only from Kentucky 31 dung samples from 2017 and 2018 collections, respectively. From the 2017 dung collection, we tested Onthophagus taurus oviposition substrate preferences for each cultivar versus dung from uninfected pasture and also larval survival and development for each dung-type. From the 2018 dung collection, O. taurus and Digitonthophagus gazella oviposition substrate preferences, larval survival and development were tested for Texoma MaxQ II versus Kentucky 31 dung-types. Among the four cultivars, for making brood-balls O. taurus preferred dung from Texoma MaxQ II while dung from Kentucky 31 and BarOptima PLUS E34 were avoided. Both beetle species preferred dung from Texoma MaxQ II versus Kentucky 31 pasture. Larval survival was not affected by dung-type with the 2017 samples. However, with the 2018 samples both beetle species had reduced larval survival on Kentucky 31 than on Texoma MaxQ II brood-balls. Development time for O. taurus was shorter for larvae from Texoma MaxQ II versus Kentucky 31 or uninfected dung brood-balls. Adult mass was not affected in the 2017 collection but was reduced in 2018 Kentucky 31 samples when compared with Texoma MaxQ II. Finally, dung beetles can differentiate dung from pastures with different tall fescue cultivars. Novel cultivar, Texoma MaxQ II, provides more benefits for dung beetles. Pasture renovations with Texoma MaxQ II may improve pasture ecology by enhancing dung beetle populations.

1. Introduction Tall fescue [Lolium arundinaceum (Schreb.) Darbysh. = Schedonorus arundinaceus (Schreb.) Dumort., formerly Festuca arundunacea Schreb. var. arundincacea] serves as the major pasture grass for pasture-based livestock production in the east-central and mid-southern US regions (Ball et al., 2015). Total planted area in the US is estimated at 14 million ha (Bouton, 2007). Since the 1940s, the most commonly used cultivar across the US is Kentucky 31 (KY-31), which is known for persistence and high yields (Roberts et al., 2009). However, this cultivar is infected with wild-type fungal endophyte, Epichloë coenophiala ⁎

((Morgan-Jones & W. Gams) C.W. Bacon & Schard) [previously known as Neotyphodium coenophialum], which produces ergot alkaloids (Leuchtmann et al., 2014; Strickland et al., 2009). The ergot alkaloids, particularly ergovaline, have been linked to symptoms such as reduced weight gains, heat stress and poor reproductive performance in cattle. The symptoms of ergot alkaloid poisoning are collectively known as ‘fescue toxicosis’ and are estimated to cost beef cattle farmers $1 billion annually in lost revenue (Bouton, 2007; Waller, 2009). However, ergovaline and other ergot alkaloids produced by the endophyte are also known to negatively affect insect herbivores (Ball et al., 1997; Clay and Cheplick, 1989; Panaccione et al., 2014; Potter et al., 2008;

Corresponding author. E-mail address: [email protected] (T. Shymanovich).

https://doi.org/10.1016/j.apsoil.2020.103561 Received 11 September 2019; Received in revised form 12 February 2020; Accepted 14 February 2020 Available online 21 February 2020 0929-1393/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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Shymanovich et al., 2015). Furthermore, the endophyte provides enhanced heat and insect tolerance, which is essential for pasture sustainability (Belesky and West, 2009). To overcome fescue toxicosis, new (novel) endophyte strains that provide fitness benefits similar to the wild-type but produce low or nil ergot alkaloids were identified and introduced into new tall fescue cultivars such as BarOptima PLUS E34 (trademark of Royal Barenbrug Group, Netherlands), Jesup MaxQ, and Texoma MaxQ II (trademarks of Pennington Seed Co, Madison, GA, USA). The endophyte strain E34 in BarOptima PLUS E34 may produce low ergovaline levels, but strains MaxQ (AR542) and MaxQ II (AR584) in Jesup MaxQ and Texoma MaxQ II, respectively, do not produce ergovaline (Bouton et al., 2002; Hopkins et al., 2010). Moreover, the wild-type endophyte strain in KY-31 and the strains E34, MaxQ, and MaxQ II also produces other bioactive compounds that possess insect deterrence (peramine) or insecticidal effects [N-acetylnorloline (NANL), N-acetylloline (NAL), N-formylloline (NFL)] (Bush and Fannin, 2009), and indole-diterpenes, such as terpendole C (Christensen et al., 1993; Takach and Young, 2014; Young et al., 2014). To achieve the plant persistence without the negative attributes of wild type tall fescue, persistent novel endophyte-infected tall fescue have been developed to increase animal performance (Bouton et al., 2002; Gunter and Beck, 2004; Hopkins et al., 2010; Parish et al., 2013). Costs to convert toxic tall fescue pastures are estimated to be $988.66 ha−1 (Gunter and Beck, 2004) to $1205.64 ha−1 (Zhuang et al., 2005). Conversion of KY-31 pastures to novel endophyte-infected tall fescue is estimated to be < 1% of the 14 Mha in total tall fescue (Chris Agee, personal communication). These high costs likely contribute to the reason why few farmers renovate toxic tall fescue pastures and still have to manage fescue toxicosis. Soil richness, aeration, and water permeability are essential for pasture productivity (Schnabel et al., 2001). The cattle inventory in the United States in July 2019 is at 103 million head (NASS, 2019). Grazing cattle compact soil but produce dung, about 9 kg dry matter per head daily (Church, 1979), which may improve soil nutrition. However, without dung decomposition, 12.3 cows would be able to cover a one hectare pasture with their pats in one year (Jones, 2017). Invertebrate decomposers, especially dung beetles, can accelerate dung decomposition rate but also enhance soil fertility, aeration and water permeability (Nichols et al., 2008). The importance of dung beetles in service to the pasture ecosystem cannot be overstated. Dung beetles exhibit several strategies in their nesting behaviors. Some use dung for egg laying without any manipulations, but others have developed various complex techniques. Endocoprid (dwelling) beetles manipulate dung within a dung pat, paracoprid (tunneling) relocate dung into tunnels below a dung pat, and telecoprid (rolling) move dung away from a dung pat before burying it (Bornemissza, 1976; Tonelli et al., 2019). These nesting activities result in either dung shredding or burial. Tunneling beetles are capable of burying dung deep into the soil and in doing so, increase soil percolation and nutrient recycling (Bertone et al., 2006). Dung beetles are projected to avert annually a $380 million loss in pasture fouling, nitrogen volatilization, parasitism and pest reduction simply through the burial of dung (Losey and Vaughan, 2006). Dung beetles have been shown to reduce methane emissions into the atmosphere (Hammer et al., 2016; Piccini et al., 2017; Slade et al., 2016) and also improve water quality in nearby streams (Brown et al., 2010; Vadas et al., 2011). Thus, dung beetles play an important role in pasture ecology and their wellbeing is essential for ecosystem sustainability. The direct effects of Epichloë infections and their specific alkaloids on insect herbivores has been under intense research investigation (e.g., Clay, 1996; Crawford et al., 2010; Hartley and Gange, 2009; Popay et al., 2009; Shymanovich et al., 2017; Shymanovich et al., 2018; Shymanovich and Faeth, 2018; Siegel et al., 1990; Tanaka et al., 2005). However, their indirect effects are still not completely understood. Nevertheless, the scientific literature has some interesting findings. Insect predators might be more negatively affected by endophytic alkaloids than insect herbivores (Faeth and Saari, 2012; Härri et al.,

2008; Jani et al., 2010; Saari et al., 2014). Lehtonen et al. (2005) reported pests on root hemiparasitic plants having increased protection from aphids via Epichloë alkaloids transferred from infected meadow fescue. A few studies tested soil mediated effects of Epichloë infected plants on reduction of other plant species growth (Matthews and Clay, 2001), on reduced insect pollination of thistle plants (Casas et al., 2016), on changes in soil microbial community structure and activities (Casas et al., 2011). Siegrist et al. (2010) detected a reduced decomposition rate for infected tall fescue litter in comparison to endophytefree litter, but they did not attribute it just to alkaloids. In their study plant tissue was enclosed in fiberglass mesh bags, so most likely this effect is mainly attributed to soil microbes with less effect attributed to soil invertebrates which had limited access to the samples. Finally, little is known about the effects of Epichloë alkaloids on insect decomposition, and some papers provide confusing results. It was reported that KY-31 reduced horn fly survival (Parra et al., 2016), but surprisingly the indole-diterpene, lolitrem B, was detected, which has never been found in KY-31 (Bush and Fannin, 2009), but it is common for infected ryegrass (Fuchs et al., 2013). For this project we used two very abundant dung beetle species, Onthophagus taurus (Schreber 1759) and Digitonthophagus gazella (Fabricius 1787) [previously Onthophagus gazella (Génier and Krell, 2017)], that dig deep tunnels for their brood balls, because tunnelers or paracoprids are considered as the most beneficial for pasture ecology (Bertone et al., 2005). Tunnelers disrupt the pat and remove dung from the surface which decreases suppression of plants under a dung pile and provide resource competition for cattle pest fly larvae, species that blood feed or cause nuisance and eye infections for cattle (Bornemissza, 1970). Right under a cow pat, such species as O. taurus and D. gazella dig multiple tunnels 20–30 cm below ground level where females combine dung pieces with soil into a brood-ball and lay a single egg into a specially made air cavity. Dung serves as a food source for the larvae, and decomposition of dung facilitated by soil microbes helps to fertilize lower soil levels (Yamada et al., 2007). Moreover, the larval tunnels provide additional aeration, friability, and water permeability for soil (Nichols et al., 2008). In this study we are testing the hypothesis that residual endophytic insecticidal or insect deterring compounds that remained after grass digestion may negatively affect invertebrate dung decomposers. A study on digestion of tall fescue endophytic alkaloids by lambs has reported that 35.4% of ergovaline intake was recovered in feces (De Lorme et al., 2007). Moreover, due to microbial degradation of ergovaline in the rumen, lysergic acid, a precursor to hallucinogenic compounds, has been detected in sheep feces. Only about 4% of loline alkaloids intake was detected in sheep feces (Gooneratne et al., 2012). Thus, endophyte strains from tall fescue cultivars that vary in alkaloid production may have different effects on invertebrate dung decomposers and that may affect dung decomposition on pastures. In this project we compared the prevailing traditional tall fescue cultivar, KY-31, with several cultivars that are newer and increasing in popularity including BarOptima PLUS E34 (here after BarOptima), Jesup MaxQ (here after MaxQ), and Texoma MaxQ II (here after MaxQ2), in regard to their effects on dung beetles. We hypothesized that: 1) dung beetles can differentiate dung that originates from pastures with different cultivars, 2) due to high retention of ergot alkaloids in dung from the KY-31 cultivar pasture, this dung-type has negative effects on dung beetles, 3) due to low retention of loline alkaloids in dung and no or low levels of ergot alkaloids, novel cultivars have minimal effects and are in general beneficial to dung beetles. 2. Materials and methods 2.1. Field-plot studies First, we assessed North Carolina State University (NCSU) tall fescue field plots that had been established with different cultivars located at 2

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Butner Beef Cattle Field Laboratory (36°17′ N; 78°80′ W) in Bahama, North Carolina, USA. Established in 2011, four 0.6 ha experimental plots endophyte-free (E-), BarOptima, MaxQ, and MaxQ2, and one 2 ha with KY-31 were used for this study in 2017 and 2018. We had to ensure that after many years these plots are still valid with respect to their initial status. Plant samples were collected from 30 plants located by a plot diagonal and about 5 m apart. From two tillers per plant, we cut 10 cm pieces near soil level and preserved them in the refrigerator before immunoblot testing with the Phytoscreen Immunoblot Kit (Agrinostics, GA) to test Epichloë infection rate (%) per plot. The rest of these samples were freeze-dried and used for DNA testing for endophyte strain identification by genotyping of alkaloid genes. For ergot alkaloid analyses, we collected all tall fescue plant material from randomly selected 30 × 30 cm area per each cultivar plot, and this material was also freeze-dried prior to testing.

for internal parasite control for 2017 and 2018. Dung collection was at least 60 d post ivermectin application to avoid affecting larval survival (Hempel et al., 2006; Römbke et al., 2007). Thirteen Angus and Angus-cross dry brood cows were utilized in 2017, and twelve Angus and Angus-cross dry brood cows were utilized in 2018 for dung collection on E-, MaxQ, MaxQII, and BarOptima plots. Cattle were allowed to graze plots for four days. Ayers et al. (2009) observed no detectable concentration of ergot alkaloids in ruminal fluid 3 d post removal of steers grazing endophyte-infected tall fescue. Further, Drewnoski and Poore (2012) observed mean retention time of tall fescue hay fed to cannulated steers to be 50 h. To ensure digestion and passage of previously grazed plots and removal of ergot alkaloids from ruminal fluid, cows began grazing (07/06/2017, 07/26/2018) E- plots first and continued in a following order MaxQ2, MaxQ, and BarOptima. BarOptima was grazed last as it is the only variety in this series that might contain trace levels of ergot alkaloids. On the fourth day, dung was collected immediately after visual observation of defecation and from a minimum of six animals. Dung was mixed and preserved for subsequent analyses. Small subsamples (100 g) for each plot were preserved and freeze-dried later for alkaloid analysis. The rest of dung samples were preserved at -20 °C before the insect experiments and tests for nutrient analyses (Cumberland Valley Analytical Services, Waynesboro, PA). The KY-31 plot was located on a different portion of the farm; therefore a different set of cattle, 15 Angus heifers, were utilized for this dung collection in 2017 and 2018. The dung collection protocol was the same for the KY-31 plots and was conducted immediately following the last dung collection on BarOptima plots.

2.2. Endophyte strain identification DNA was extracted from freeze-dried plant material with the Qiagen Magattract 96 Plant DNA Core kit and PCR was used to determine the endophyte infection rate and strain identification. The PCR reactions contained 1× Green GoTaq reaction buffer (Promega), 200 μM of each deoxynucleoside triphosphate (dNTP), 200 nM each primer, and 1 U GoTaq DNA polymerase (Promega). Polymerase chain reaction conditions for all amplifications were as follows: 94C for 2 min; 30 cycles of 94C for 15 s, 56C for 30 s, and 72C for 1 min; and one cycle of 72C for 10 min. The following primer pairs were used in a multiplex reaction to amplify target fragments of five genes: (1) tefA with tef1-exon1d-1 5’GGGTAAGGACGAAAAGACTCA-3′ and tef1-exon4u-1 5’-CGGCAGCGA TAATCAGGATAG-3′, (2) perA-T2 with perA-T2-F 5’-TCTTCAGGCATC GCAGGAAC-3′ and perA-T2-R 5’-TCGGCCACCTCCAGCCTGATG-3′, (3) lolC with lolC3a 5’-GGTCTAGTATTACGTTGCCAGGG-3’and lolC-5b 5’TCTAAACTTGACGCAGTTCGGC-3′, (4) dmaW with dmaW-F4 5’-GTGT ACTTTACTGTGTTCGGCATG-3’and dmaW-6R 5’-GTGGAGATACACAC TTAAATATGGC-3′, and (5) idtG with idtG-F 5’-GTGTACTTTACTGTGT TCGGCATG-3′ and idtG-R 5’-GTGTACTTTACTGTGTTCGGCATG-3′. The PCR amplicons (10 μL) were visualized using electrophoresis with a 1.5% agarose gel in Tris-borate-EDTA buffer, followed by staining with ethidium bromide. The expected size fragments for each amplicon was as follows: tefA 860 bp, perA-T2 600 bp, lolC 442 bp, dmaW 282 bp, and idtG 113 bp. Each strain is recognized by a different banding pattern, where endophyte-infected KY-31 contains tefA, perA-T2, lolC and dmaW, BarOptima contains tefA, perA-T2, lolC and dmaW and MaxQ and MaxQ2 contained tefA, perA-T2, lolC and idtG. MaxQ and MaxQ2 could be further distinguished by PCR using primers designed to SNP in lolN and idtP. The primers were run as a multiplex reaction, where the lolN primers (lolN-F2 5’-ACGCTGCGTTTGAAGCAGCC-3′ and lolN-R5AR584 5’-CCAAAGCGTGCAGGAGCTTTTC-3′, 492 bp fragment) would amplify AR584, KY-31 and BarOptima and the idtP primers (idtP-F281 5’-TGGGAGATATGCCTGTCCGGGACGCT-3′ and idtP-R2-AR542 5’CTCACTATCTAGCTTAAAATCATGA-3′, 207 bp fragment) would only amplify AR542. The infection rates detected by PCR analyses were KY-31 70%, MaxQ2 70%, MaxQ 66.7%, and BarOptima 36.7%. Samples from the endophyte-free (E-) plot were not endophyte infected except one plant that had MaxQ2 strain.

2.4. Ergot alkaloids analyses Only tissue from tall fescue plants (other grasses were removed from the sample) were used for extraction of ergot alkaloids. For each plot sample, leaf blades and sheaths were cut into 5 mm pieces and mixed. Ergot alkaloids were extracted from tall fescue plant material by beadbeating 50 mg dried plant material per mL of 50% 2-propanol +1% lactic acid as described previously (Panaccione et al., 2012). Ergot alkaloids were analyzed by high performance liquid chromatography (HPLC) with fluorescence detection essentially as described by Panaccione et al. (2012). In brief, the solid phase was a Prodigy ODS3 column (150 mm × 4.6 mm, 5 μm particle size; Phenomenex, Torrance, CA), and the mobile phase was a multilinear, binary gradient from 5% acetonitrile +50 mM ammonium acetate to 75% acetonitrile +50 mM ammonium acetate. Analytes were detected in two serially-arranged fluorescence detectors. One detector measured lysergic acid derivatives by exciting at 310 nm and detecting emission at 410 nm; the other detector was set for measuring clavine precursors to lysergic acid, with excitation and emission wavelengths of 272 nm and 372 nm, respectively. Lysergic acid derivatives, such as ergovaline and ergine, were quantified by comparison to a known concentration of ergotamine (Sigma-Aldrich, St. Louis, MO, USA), which has the identical lysergyl fluorophore and which was included in the extraction solvent as an internal standard. Dung samples were extracted by suspending 1 g freeze-dried dung in 15 mL of 50% 2-propanol with 1 μg/mL ergotamine (as internal standard), vortexing, and then rotating, end-over-end, at 20 rpm for 18 h. After clarification by centrifugation (4500 rpm for 5 min), 0.5 volumes of water were added to the supernatant which was then loaded on an Isolute C18 column (500 mg, non-endcapped; Biotage, Charlotte, NC). The column was washed with 2 mL of 30% 2-propanol and 2 mL of 50% 2-propanol, before eluting analytes with 2 mL 100% 2-propanol. The eluate was then concentrated to 200 μL in a centrifugal vacuum concentrator, and 20 μL was analyzed by HPLC with fluorescence detection, as described above.

2.3. Plot grazing and dung collection All cattle care, handling and sampling procedures were approved by the North Carolina State University Animal and Care Use Committee. The Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999) was used for animal care during these experiments, and the protocol was approved by the Institutional Animal Care and Use Committee (IACUC protocol # 13-124-A and 16212-A). All cattle received a pour-on application of ivermectin in May 3

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assess air cavity or larvae/pupae presence. If air camera is formed this indicates that brood ball construction was completed, and an egg might have been laid, if the air camera is missing, no oviposition was expected (Floate et al., 2015). For survival estimates, only brood balls with air camera were counted, and Survival = n(adults) / n (brood balls with air camera) * 100. 2.8. Statistical analyses All analyses were performed with RGui 64-bit 3. 5. 1. software with R Commander and Dunn Test packages “(R Development Core Team, 2008)”. Oviposition pots without brood balls laid were excluded from statistical analyses. To compare oviposition substrate preferences for brood ball number and total brood ball mass among four cultivar groups, ANOVA tests with multiple comparisons of means by Tukey contrasts were used for cultivar minus E- differences, and all assumptions, tested by Shapiro-Wilk normality test and Levene's test for homogeneity of variance, were met. To compare cultivar vs. E- and KY31 vs. MaxQ2 dung-type choices, paired t-tests were used for brood ball numbers and total brood-ball mass; all assumptions met. To compare binomially distributed larvae survival, Kruskal-Wallis rank sum tests with Bonferroni adjustments for pairwise comparisons, if needed, were implemented. To assess the effect of sex for O. taurus KY-31 vs. MaxQ2 survival, we used Pearson's Chi-squared test via contingency tables. For D. gazella development time and adult mass, no statistical analyses were made because of complete mortality in KY-31 group. For O. taurus, development time data distributions were not normal, so non-parametric, Kruskal-Wallis rank sum test with Bonferroni adjustments for pairwise comparisons, if needed, were used. For KY-31 vs. MaxQ2 experiment to assess the effect of sex on development time, we also used two-way ANOVA tests with sex and dung-type as factors. To compare adult mass among five groups, we used one-way ANOVA and multiple comparisons of means by Tukey contrasts with dung-type as a factor; all assumptions met. To compare adult mass between KY-31 and MaxQ2, two-way ANOVA test with dung-type and sex as factors was used first, and because the effect of sex was not significant (F = 19.0434, df = 1, P = .72) we continued with paired t-tests with only dung-type as a factor; all assumptions met.

Fig. 1. Paracoprid or tunneling beetles abundant in North Carolina, USA: Onthophagus taurus (left) and Digitonthophagus gazella (right). Photos by Matt Bertone.

2.5. Dung beetles For experiments we used wild O. taurus and D. gazella beetles (Fig. 1). Both species are very abundant in the south-eastern US but not native. Onthophagus taurus was introduced from southern Europe and D. gazella from Africa (Hoebeke and Beucke, 1997). Naive to endophytic alkaloids, adult dung beetles were collected at the Cherry Research Farm, Goldsboro, NC. Pastures there have no tall fescue infected with wild type Epichloë. 2.6. Oviposition substrate preference Two experiments were performed: (Experiment 1) To assess oviposition substrate preferences among four cultivar dung-types, binarychoice experiments were set up where dung beetles were provided with one cultivar dung-type and dung from the endophyte-free plot from 2017-year collection. Thus, four dung-types, KY-31, BarOptima, MaxQ, and MaxQ2, were tested in comparison to E- dung. (Experiment 2) To assess oviposition substrate preference between the most preferred versus the least preferred dung-types from 2018-year collection. For both experiments 2.7 L (19 cm deep) pots were divided in half with a paper-foam insert and filled with potting soil:sand:vermiculite 1:1:1 wet mix (modified from Floate et al. (2015) and Aschenborn et al. (1989)). At the start of the experiment in each pot, two 50 g dung pieces were placed on opposite sides of a divider: (Experiment 1) cultivar vs. E- dung or (Experiment 2) preferred cultivar, MaxQ2, vs. avoided cultivar, KY-31, based on the results from the Experiment 1 (Fig. S1A). Five females (Experiment 1) or three females (Experiment 2) and one male beetle were isolated per pot and incubated in a growth-chamber at 26 °C, 16/8 h light/dark period, and 80% relative humidity. Experiment 1 continued for 7 days and another 2 × 50 g of corresponding dungtypes was added on the third day. In two years, a total of 35 replicates with O. taurus were performed. Experiment 2 continued for 2.5 days. A total of 40 replicates were performed with each O. taurus and D. gazella. At termination, all brood balls were recorded by pot and dung-type and weighed (Fig. S1B).

3. Results 3.1. Evaluation of the field-plots and plant and dung samples After 6 years, the novel endophyte and endophyte free tall fescue field-plots used for this study remained consistent with the cultivar initially planted, while the KY-31 was an old stand that was highly infected with the wild-type endophyte (100%). Genotyping of alkaloid genes from plant samples from each plot assigned them respectively to the initial KY-31, BarOptima, MaxQ, and MaxQ2 cultivar strains as expected. Epichloë infection frequency was 87% or greater for KY-31, MaxQ, and MaxQ2 plots (Table 1), while the BarOptima plot had a lower infection rate at 57%. Despite cattle grazing and maintenance, the endophyte-free plot remained at a very low infection rate. Just a single plant at the edge of the plot was found to have the MaxQ2 endophyte strain. Ergot alkaloid testing detected ergovaline or ergine only in the KY-31 plant and dung samples (Table 1). No ergot alkaloids were detected from any other sample. Ergot alkaloid levels were lower in KY31 2017 plant samples compared to 2018 samples. In the dung samples our analyses recovered 8.3% (for 2017) and 12.3% (for 2018) of ergovaline in comparison to ergovaline plus ergine levels found in the plant samples. For ergovaline only, recovery in dung was 15.4% and 17.8% for 2017 and 2018, respectively. Dry matter content ranged from 18.1% in E- to 13.5% in KY-31 dung samples. The highest crude protein content was 12.3% and 12.4% in KY-31 and BarOptima samples, respectively. MaxQ and MaxQ2 had similar protein content of 11.8%, and the lowest 10.7% was found in

2.7. Larval survival and development time Each individual brood ball from the choice experiments was placed into a 50 mL Falcon tube, covered with wet substrate described above, and sealed with mesh. Brood balls were incubated at 26 °C, 16/8 light/ dark period, and 80% relative humidity. For adult eclosion, tubes were checked daily, and development time to adult was recorded. Newly eclosed adults were weighed and sex recorded. One week after eclosion stopped, the rest of the brood balls were checked under a microscope to 4

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Table 1 Characterization of the tall fescue field-plots used for the study: Epichloë infection frequencies and strains, predicted and detected alkaloids. Cultivar

Epichloë frequency per plota, %

E. coenophiala strain

Kentucky 31 (KY-31)

100 30/30

BarOptima PLUS E34 (BarOptima)

57 17/30

E. coenophiala wild-type Confirmed E34 Confirmed

Jesup MaxQ (MaxQ) Texoma MaxQ II (MaxQ2) Endophyte free (E-)c

87 26/30 93 28/30 3 1/30

a b c

Expected alkaloids (Takach and Young, 2014; Young et al., 2014)

AR 542 Confirmed AR 584 Confirmed Endophyte-free

High levels of ergot alkaloids (ergovaline, ergine), peramine, lolines (NFL, NAL, NANL)b − profile 2 Low levels of ergot alkaloids (ergovaline, ergine), peramine, lolines (NFL, NAL, NANL), indole-diterpenes (terpendoles) – profile 3 Peramine, lolines (NANL), indolediterpenes (terpendoles) – profile 4 Peramine, lolines (NFL, NAL, NANL), indole-diterpenes (terpendoles) – profile 4 None

Ergot alkaloids (ergovaline, ergine) in leaf, μg/g

Ergot alkaloids (ergovaline, engine) in dung, μg/g

2017

2018

2017

2018

EV = 0.26 EG = 0.2

EV = 1.52 EG = 0.68

EV = 0.04 EG = ND

EV = 0.27 EG = ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

Detected by immunoblot method. NFL = N-formylloline, NAL = N-acetylloline, NANL = N-acetylnorloline, EV = ergovaline, EG = ergine, ND = not detected. The one positive sample was Texoma MaxQ II.

the E- dung samples. The non-fiber carbohydrate content was the lowest in the uninfected dung-type at 1.7% compared to 16.6–23.1% in other dung-types. Surprisingly the iron content was 4 to 11 times higher in uninfected dung samples, which might be due to a soil ingestion or accidental contamination during collection (Table S1).

MaxQ2 experiment (χ squared).

Development time to adult was shorter for O. taurus larvae from MaxQ2 brood balls made from 2017 dung collection when compared to KY-31, BarOptima, MaxQ, and E- groups (Fig. 4A, B). Similarly, development time was shorter by about 10 days for O. taurus larvae from MaxQ2 brood balls from 2018 dung collection when compared with KY31. No effect of sex on development time was observed with the 2018 dung collection (F = 0.0608, df = 1, P = .8). Eclosed beetle mass was not different for all dung-types from the 2017 collection (Fig. 4D). However, from the 2018 dung collection, adult mass was reduced from KY-31 when compared to the MaxQ2 group (Fig. 4E). No effect of sex on eclosed adult mass was found with 2018 dung collection (F = 0.1328, df = 1, P = .7). D. gazella larvae from MaxQ2 brood balls had successfully developed into adults (Fig. 4C, F).

Comparison among four cultivar dung-types showed that the oviposition substrate O. taurus dung beetles had significant preference for MaxQ2 dung-type, while KY-31 and BarOptima were avoided when they were provided in paired-choices with endophyte-free dung (Fig. 2). Comparisons within each pair-choice showed significant preference for MaxQ2 versus E- dung. When two dung beetle species, O. taurus and D. gazella, were provided with a choice between KY-31 and MaxQ2 dung, they both made more brood balls by number and by total mass from MaxQ2 dung-type (Fig. 3). 3.3. Larval survival

4. Discussion

O. taurus larvae from brood balls made with different 2017 dungtypes showed some difference in survival (χ2 = 10.2157, df = 4, P = .04, KeW test). Larvae from BarOptima brood balls had slightly lower survival in comparison to larvae from MaxQ2 brood balls (Table 2). From 2018 dung brood-balls, larval survival was significantly lower in dung from KY-31 than MaxQ2 in both beetle species. There was complete mortality for D. gazella from KY-31 brood balls. No effect of sex on larval survival was detected for O. taurus from KY-31 vs.

KY-31 BarOptima MaxQ MaxQ2

Number of brood-balls (Cultivar minus E- )

4 3

**b

2 ab

1 0 -1 -2 -3

(B) Total brood-ball mass (Cultivar minus E-), g

5

a

Profitable beef and milk farming depends on productive and sustainable pastures with healthy ecology where multi-species interactions are in balance (Kumm, 2003; Michalk et al., 2003; Muir et al., 2011). However, agronomic ecosystems are often plant monocultures where low plant diversity affects other species performance and survival (Jones et al., 2005; Marshall et al., 2003; Songa et al., 2007). To partially remediate these problems, good agronomic practices should

15

**b

10 ab

5 0 -5

a

-10

= 2.366, df = 1, P = .124, Pearson chi-

3.4. Larval development

3.2. Oviposition substrate preferences

(A)

2

a a

5

Fig. 2. Mean ± SE number (A) and total mass (B) of brood-balls O. taurus beetles made from each cultivar minus brood balls made from endophyte-free (E-) pasture dung in binary-choice experiments with 2017 dung collection. Kentucky 31 (KY-31) n = 20, BarOptima PLUS E34 (BarOptima) n = 18, Jesup MaxQ (MaxQ) n = 18, Texoma MaxQ II (MaxQ2) n = 21. Different letters indicate significant difference among dung-types, P < .01, ANOVA tests with multiple comparisons of means by Tukey contrasts. Asterisks indicates significant difference within paired choices (cultivar vs. E- dung), P < .01, paired t-tests.

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O. taurus

Brood-ball number

(A) 7

(B) 7

6

6

5

5

4

4

3

3

***

2

2 1

0

0 40 (D) 35 30 25 20 15 10 5 0

(C)

30

Total brood-ball mass, g

1

25 20 15 10

***

5 0

KY-31

MaxQ2

avoided using dung from the pasture with KY-31, the traditional cultivar containing ergot alkaloids. Moreover, when ergovaline level in dung was at 0.27 μg/g, O. taurus larvae from KY-31 brood-balls had lower survival, smaller body weight, and delayed development than in the MaxQ2 group, and D. gazella larvae had complete mortality. Even though we did not chemically test for loline alkaloids in the dung, based on our bioassays we can assume that lolines do not negatively affect dung beetles as beetles preferred MaxQ2 dung-type when compared to uninfected dung, despite loline alkaloids being produced in this cultivar (Johnson et al., 2013; Young et al., 2013). Moreover, larvae from brood-balls made with MaxQ2 dung-type had shorter development when compared to larvae from the three other cultivars and uninfected pasture groups. Surprisingly, beetles also differentiated dung from the novel cultivar pastures. O. taurus strongly preferred MaxQ2 and did not differentiate MaxQ from E- grass, but BarOptima dung-type had a negative ratio when compared to dung from the uninfected pasture. Larvae from BarOptima have lower survival when compared to the MaxQ2 group. No ergovaline and ergine were detected in the BarOptima tall fescue in our study. The endophyte E34 in this cultivar is known to produce peramine, lolines, and indole-diterpenes (similar to MaxQ and MaxQ2) (Young et al., 2013), so factors in BarOptima affecting beetle choices and larval survival remain unknown. Unfortunately, we were unable to test for lolines, peramine, and indole-diterpene alkaloids. Nutrition analyses of dung samples showed no obvious reasons to explain adult preferences and advanced larval development in MaxQ2 dung-type. From this study we could not explicitly state all the mechanisms that affected dung beetle oviposition substrate choices and larval survival, but we suggest the effects of ergovaline alkaloid as one of the factors. There may also be differences in plant compounds or in dung microbial communities that have effects on dung beetles.

D. gazella

**

** KY-31

MaxQ2

Fig. 3. Mean ± SE number (A, B) and total brood-ball mass (C, D) of broodballs made by dung beetles in a binary-choice experiments with 2018 dung collection from tall fescue pastures with Kentucky-31 (KY-31) and Texoma MaxQ II (MaxQ2) cultivars. n (O. taurus) = 40, n (D. gazella) = 15. Asterisks indicates significant differences, P < .001 and P < .01 in paired t-tests.

consider variation in plant genotypes and cultivars and promote the most suitable for the ecological community at the field site (Tooker and Frank, 2012). Our study is the first to demonstrate differential effects of E. coenophiala strains in tall fescue on insect dung decomposers. This project has shown another strong reason for pasture renovation with novel tall fescue cultivars, especially MaxQ2. Findings from our study may suggest that pastures with the MaxQ2 cultivar could have higher abundances of dung beetles, which may enhance dung decomposition rate and improve soil properties to provide higher forage yield. In general, our hypothesis that dung beetles will differentiate dung that originated from pastures with different cultivars and that KY-31 dung may have negative effects on beetle larvae was supported with two dung beetle species bioassays and was also reinforced with ergot alkaloid testing. For making brood-balls, dung beetles differentiated dung from pastures with several tall fescue cultivars, and their larval survival and development were affected based on oviposition substrate used. As predicted based on the alkaloid retention in dung, beetles

5. Conclusions In conclusion, the prevalence of KY-31 tall fescue in mid-Atlantic and mid-south USA pastures might have negative effects on insect dung decomposers, especially dung beetles, and on pasture ecology in general. Our study suggests that pasture renovation with novel cultivar, Texoma MaxQ II, may provide more benefits by enhancing dung decomposer community. However, more studies with different species of invertebrate decomposers are necessary to validate this finding. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsoil.2020.103561. Declaration of competing interest None.

Table 2 Dung beetle larvae survival from Onthophagus taurus and Digitonthophagus gazella experiments with 2017 and 2018 dung collections from tall fescue pastures with Kentucky-31 (KY-31), BarOptima PLUS E34 (BarOptima), Jesup MaxQ (MaxQ), and Texoma MaxQ II (MaxQ2) cultivars or endophyte-free (E-). Beetle species

O. taurus

D. gazella

a

Dung-type

EKY-31 BarOptima MaxQ MaxQ2 MaxQ2 KY-31

Survival 2017 dung

Survival 2018 dung

n Survived/Total(%)

K-Wa with Bonferroni pairwise tests

n Survived/Total (%)

K-W

115/147(78%) 28/34 (82%) 16/24 (67%) * 27/37 (73%) 88/98 (90%) * -

BarOptima≤MaxQ2 P = .052 χ 2 = −2.563848

6/43 (11%) 74/227 (33%) 16/61 (26.2%) 0/15 (0%)

KY-31 < MaxQ2 P = .002 χ 2 = 11.041

K-W = Kruskal-Wallis rank sum test, “-”= not tested. 6

KY-31 < MaxQ2 P = .027 χ 2 = 4.918

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(A)

55

O. taurus

(B)

55

O. taurus

***

(C) 50

50

45

45

45

40

40

35

35

35

30

30

30

25

25

25

20

20

(D) 0.06

(E) 0.06

0.055

0.055

0.1

0.05

0.05

0.09

0.045

0.045

0.04

0.04

0.035

0.035

0.03

0.03

0.025

0.025

0.02

0.02

Adult mass, g

Development time, days

50

*

40

KY-31

BarOptima

MaxQ

MaxQ2

E-

D. gazella

20

(F)

0.08 0.07 0.06

***

0.05 0.04 0.03 0.02

KY-31

MaxQ2

KY-31

MaxQ2

Fig. 4. Larval development time (median ± interquartile range) (A, B, C) and adult mass (mean ± SE) (D, E, F) from dung beetle exepriments with 2017 dung collection (A, D) and 2018 dung collection (B, C, E, F) from pastures with Kentucky-31(KY-31), BarOptima PLUS E34 (BarOptima), Jesup MaxQ (MaxQ), and Texoma MaxQ II (MaxQ2) tall fescue cultivars or endophyte-free (E-). For O. taurus experiment (A, D), KY-31 n = 28, BarOptima n = 16, MaxQ n = 27, MaxQ2 n = 88, En = 115. For O. taurus experiment (B, E), MaxQ2 n = 74, KY-31 n = 6. For D. gazella experiment (C, F), MaxQ2 n = 16, KY-31 n = 0. Asterisks and their sizes indicates significant differnces P < .001, and P < .05, respectively. Kruskal-Wallis rank sum test with Bonferroni pairwise comparisons were used for development time analyses and ANOVA and t-tests for adult mass analyses. No comparisons were made for D. gazella experiments with no beetles emerged from KY-31 brood-balls.

Acknowledgements

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