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Trans-generational effects of ivermectin exposure in dung beetles
Accepted Manuscript Trans-generational effects of ivermectin exposure in dung beetles Fernanda Baena-Díaz, Imelda Martínez-M, Yorleny Gil-Pérez, Daniel GonzálezTokman PII:
S0045-6535(18)30526-5
DOI:
10.1016/j.chemosphere.2018.03.109
Reference:
CHEM 21053
To appear in:
ECSN
Received Date: 29 November 2017 Revised Date:
15 March 2018
Accepted Date: 17 March 2018
Please cite this article as: Baena-Díaz, F., Martínez-M, I., Gil-Pérez, Y., González-Tokman, D., Transgenerational effects of ivermectin exposure in dung beetles, Chemosphere (2018), doi: 10.1016/ j.chemosphere.2018.03.109. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Trans-generational effects of ivermectin exposure in dung beetles
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Fernanda Baena-Díaz1, Imelda Martínez-M.1, Yorleny Gil-Pérez1, Daniel González-
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Tokman1,2,*
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Mexico. 91070.
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CONACYT
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Corresponding author: [email protected] ; +52 228 842 1800 - 4144
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Instituto de Ecología A. C. Antigua Carretera a Coatepec 351. El Haya, Xalapa, Veracruz,
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Abstract
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Ivermectin is a powerful antiparasitic drug commonly used in cattle. Ivermectin residues
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are excreted in dung, threatening non-target coprophagous fauna such as dung beetles. This
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can have severe ecological and economic consequences for dung degradation and soil
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fertility. Even though the negative effects of direct ivermectin exposure on dung-degrading
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organisms are well known, effects could extend across generations. Here, we tested the
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effects of paternal or maternal exposure to ivermectin on offspring in the dung beetle
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Euoniticellus intermedius. This species is a classic study subject in ecotoxicology and
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sexual selection because males have a cephalic horn that is under intense selection via
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male-male competition. After confirming a negative effect of ivermectin on the number of
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emerged beetles, we found trans-generational effects of ivermectin exposure on the horn
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size of male offspring. Surprisingly however, this trans-generational effect only occurred
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when only the father was exposed. We detected no trans-generational effects of ivermectin
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exposure on offspring number, sex ratio or body size. Our results confirm that ivermectin
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not only has a strong effect on exposed individuals but also in their progeny. Our study
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opens new questions about the mechanisms responsible for parental effects and their long-
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term fitness consequences in contaminated habitats.
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Keywords: contamination, parental effects, Scarabaeinae, sexual selection
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1. Introduction
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The parental environment has very important effects on offspring. When parental effects
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transferred from the father or the mother define offspring viability, they are important
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drivers of evolution that are not linked to inherited genes (Badyaev and Uller, 2009; Leimar
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and McNamara, 2015; McNamara et al., 2016). The parental environment can define
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several phenotypic features in the offspring. For example, in insects, mothers and fathers
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that were infected by a pathogen can transfer resistance to their offspring (Moret, 2006;
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Roth et al., 2010). Also, mothers developed under high population densities can produce
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male offspring with larger sexual traits, which will likely increase their mating success
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(Buzatto et al., 2012). In some cases, the effects of maternal and paternal environments can
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be strikingly different: for example, in the fly Telostylinus angusticollis (Neriidae), mothers
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fed high protein diets have offspring with larger body size and elongated heads (a sexually-
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selected trait), whereas the opposite occurs in males, whose offspring body size and sexual
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traits are instead favored by high-carbohydrate diets (Bonduriansky et al., 2016). Most
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studies focus on maternal rather than paternal effects, despite the known importance of both
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effects in offspring phenotype (Crean and Bonduriansky, 2014). Trans-generational
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plasticity including maternal and paternal effects is an important tactic to deal with
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environmental change (Salinas et al., 2012) and has been interpreted as cryptic parental
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care (Jokela, 2010).
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Parental effects are particularly important under adverse conditions, even when the new
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generation grows in environments that are more favorable. This frequently occurs in
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habitats where human use of pesticides or other toxic compounds during certain times of
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the year, or in certain places, leads to temporary contamination which affects only some
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generations of short-lived organisms. Even though the pesticide is only intermittently
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present in the environment, trans-generational consequences of pesticide exposure may be
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observed in offspring viability and performance (Bonduriansky et al., 2012; Tassou and
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Schulz, 2009). This is the case of veterinary medications that are used in many countries
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despite their negative effect on the environment and biodiversity (Lumaret et al., 2012).
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Ivermectin is the most common parasiticide used in cattle at some regions of the world
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because it is both relatively inexpensive and is highly effective against nematodes, ticks and
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other parasites (Lumaret et al., 2012). Ivermectin binds to glutamate-gated and GABA
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receptors, modifying membrane permeability to chloride ions in invertebrates, therefore
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affecting the nervous and muscular functions; this causes paralysis and death in
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invertebrates but is of relatively low toxicity in mammals (Lumaret et al. 2012). Either
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applied topically or injected ivermectin is excreted in the dung over weeks or months
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during which it has lethal and negative non-lethal effects on non-target fauna, mainly dung
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flies and beetles (Lumaret et al., 2012). This causes severe economic losses given the high
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influence of coprophagous insects in burying and degrading dung in pastures and forests,
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contributing to increase soil fertility and control noxious fauna associated with the
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remaining dung (Beynon et al., 2015; Huerta et al., 2013; Wall and Beynon, 2012). Among
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the negative effects of ivermectin in dung beetles are reduced fecundity and reproductive
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success, delayed sexual maturation, reduced growth rate and body size, altered ovary
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morphology, reduced muscle development and reduced locomotor and olfactory capacity
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(González-Tokman et al., 2017; Martínez-M. et al., 2017; Verdú et al., 2015; Wardhaugh
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and Rodríguez-Menéndez, 1988).
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In the present study, we evaluated the trans-generational effects of maternal and paternal
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exposure to ivermectin in the dung beetle Euoniticellus intermedius (Coleoptera:
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Scarabaeinae). This species is negatively affected by ivermectin, and relatively small doses
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of 10-60 µg/kg of fresh dung cause important reductions in fecundity, development, muscle
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mass and body size (González-Tokman et al., 2017; Krüger and Scholtz, 1995; Martínez-
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M. et al., 2017). Euoniticellus intermedius is a classical model in sexual selection studies
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because males possess a cephalic horn whose size defines the outcome of male-male
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contests for mates (Pomfret and Knell, 2006a) and is an important indicator of immune
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condition and physical resistance (Lailvaux et al., 2005; Pomfret and Knell, 2006b).
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Counter-intuitively, there is evidence showing that developing in high doses of ivermectin
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causes male beetles of this species to emerge with larger horns, but this probably occurs
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because only high-quality, larger-horned males are able to survive developing in
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ivermectin, filtering small-horned males out of the measured population and biasing
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average horn size upward among the survivors (González-Tokman et al., 2017).
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Euoniticellus intermedius is one of the most important providers of ecosystem services in
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pastures by degrading dung in our study region. Ivermectin is a particularly serious threat to
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this species because it may actually be attracted to ivermectin-contaminated dung (Holter et
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al., 1993). Here, under controlled laboratory conditions, we exposed the male, the female or
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both beetle mates to a low concentration of ivermectin in dung during their whole
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development. These beetles were then allowed to mate in ivermectin-free dung and we
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evaluated offspring number, sex ratio, body size and male horn length in comparison with
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the offspring of control parents that were not exposed. We hypothesized that both maternal
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and paternal exposure to ivermectin would alter offspring reproductive success, sex ratio,
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body size and the size of the male cephalic horn. To the best of our knowledge, this is the
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first report of trans-generational effects of ivermectin in dung beetles.
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2. Materials and methods
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The present study was carried out using the dung beetle Euoniticellus intermedius (Reiche).
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This beetle is native to Africa, but invaded Mexico in the 1980’s after intentional
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introduction in the United States during the 1970’s and subsequent migration southwards; it
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is now one of the most abundant dung beetle species in southern Mexico, including our
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study site (Flota-Bañuelos et al., 2012; Montes de Oca and Halffter, 1998). Sixty-eight
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beetles were collected in Rancho El Salado, Acajete, Veracruz, Mexico (19º34’36’’N,
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96º23’39’’ W, altitude: 54 m asl), where cattle are not treated with ivermectin. Beetles were
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bred in an insectary at the Instituto de Ecología, Xalapa, Veracruz, Mexico (27.5±0.4°C,
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74±2.5% humidity). Beetles were kept in sterilized wet soil and fed ad libitum with
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homogenized cow dung collected at the same ranch. During the whole experiment, dung
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was replaced every third day. Before feeding the beetles, dung was frozen for at least two
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days at -20°C to kill parasites and other insect larvae.
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2.1 Experimental design
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The present study was designed to test the effect of paternal and maternal exposure to
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ivermectin (Fig. 1). Therefore, during the experiment we used two treatments spiked in
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cattle dung (83% humidity): Ivermectin and Control. As ivermectin is excreted largely 6
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unchanged in the dung of treated livestock, spiking ivermectin in dung is a recommended
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method for laboratory assays with this antiparasitic drug (González-Canga et al., 2009).
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The tests were performed with technical grade ivermectin (CAS-Number: 70288-86-7.
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Batch number: MKBS6097V; Sigma-18898). Since ivermectin is poorly soluble in water, it
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was dissolved in acetone (CAS- Number: 1567-89-1; Sigma purity > 99.8%). A
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concentration of 10 µg ivermectin in 10 mL of acetone solution was added per kg of fresh
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dung (corresponding to 59 µg ivermectin per kg of dry dung) in the experimental treatment.
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As a control treatment, 10 mL of acetone were used per kg of fresh dung. The acetone was
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allowed to evaporate from the dung before feeding the beetles. Dung was always conserved
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at 4°C. The ivermectin concentration used is ecologically realistic, as it resembles the
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concentration excreted by cattle treated topically with a recommended dose (500 µg
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ivermectin/kg of cattle body mass) 28 days earlier (Wohde et al., 2016). Despite being
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considered relatively low, this dose affects ovary development and the properties of the fat
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body of our study species (Martínez-M. et al., 2017).
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Beetles collected from the field were allowed to reproduce in two containers (34
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individuals per container) with untreated cow dung to obtain a laboratory population (F0,
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the grandparents) known to be unexposed to ivermectin during their whole life cycle (Fig.
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1). With beetles from the F0 we formed random pairs that were allowed to reproduce in
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either ivermectin-treated (N=10 pairs) or control (N=13 pairs) dung in 1-L plastic
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containers. The emerging individuals from each pair (full siblings) were considered a
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family (see below). Individuals emerged from the Ivermectin or Control treatment (F1, the
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parents) were randomly assigned to mate in untreated dung, with a mate of the same or
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different treatment, to test for parental effects of ivermectin exposure on the next generation
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(F2, the offspring). Mating pairs of males and females from different families from the F1
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resulted in the following combinations: Male Ivermectin-Female Ivermectin (N=26 pairs),
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Male Ivermectin-Female Control (N=20 pairs), Male Control-Female Ivermectin (N=13
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pairs) and Male Control-Female Control (N=16 pairs) (Fig. 1). Siblings were never crossed
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with each other. We recorded the number of emerged beetles and sex ratio in both the F1
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and F2 generations (F1=38 families, F2=75 families), and body size and male horn length
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in both F1 and in F2 generations (F1=61 males and 65 females and F2=295 males and 150
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females). Males were distinguished from females by the presence of the cephalic horn.
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Body size and horn length were measured from pictures taken at 10x under a Leica Z16
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microscope with Leica LAS EZ software. Body size was measured as pronotum width from
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pictures with a dorsal view. To avoid any potential bias, measurements were taken by the
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same person (YGP), who was blind to the beetle treatment at the time of measurement.
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Horn length was measured from lateral view pictures (Fig. 2).
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Figure 1. Experimental design used to test trans-generational effects of ivermectin
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exposure on the dung beetle Euoniticellus intermedius.
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Figure 2. Cephalic horn of male Euoniticellus intermedius dung beetles. The white line
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represents the measurement of horn length used.
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2.2 Statistical analyses
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Data were analyzed separately for each generation (F1 and F2) and for each response
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variable. Generalized linear models (GLM) were used to analyze the number and the sex
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ratio of emerged beetles. For analyzing the number of emerged beetles in the F1 we used a
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negative binomial GLM given the high overdispersion found for the Poisson model
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(Residual deviance/Residual d. f.=4.12), that is suggested for count data (Zuur et al., 2009).
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In the F2, we analyzed the number of emerged beetles with a Poisson GLM for count data
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without overdispersion (Residual deviance/Residual d. f.< 2). We analyzed the sex ratio
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with Binomial GLMs for both F1 and F2. Linear mixed models (LMM) controlling for
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genetic relatedness between siblings were used to analyze male and female body size and
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male horn length in both F1 and F2 (using family as a random variable with a random
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intercept) (Bolker et al., 2008; Bolker, 2016).
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For the analyses of the F1, we initially tested the effect of the treatment (ivermectin or
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control), the mother’s body size, the father’s body size, the father’s horn length and the
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interactions (treatment × mother size), (treatment × father size) and (treatment × father horn
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length). For the F2 we initially tested the effect of the mother’s treatment, the father’s
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treatment, the interaction (mother × father treatment), the mother’s body size, the father’s
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body size, the father’s horn length and the interactions (mother treatment × mother size),
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(father treatment × father size) and (father treatment × father horn length). For both
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generations, the analyses of horn length always included the individual’s own body size as
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a covariate and body size was exponentially transformed to improve normality.
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For F1 and F2 generations, the initial statistical models tested were reduced based on the
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Akaike Information Criterion (AIC) to obtain the best supported model. P-values for
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explanatory variables were obtained from Chi-squared tests for GLMs and from likelihood
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ratio tests for LMMs (Zuur et al., 2009). Normality of residuals was inspected in normal q-
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q plots and variance homogeneity was evaluated with Fligner-Killeen tests; the presence of
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outliers was tested with Cook’s distance (Crawley, 2013). All analyses were done
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following the procedures suggested by Zuur et al. (2009). Data were analyzed with R
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software version 3.2.3 (R Development Core Team, 2015) and mixed models were done
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using the nlme package (Pinheiro et al., 2016).
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3. Results
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F1 generation
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Results from the F1 are summarized in Table 1. Ivermectin treatment caused a 50%
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reduction in the number of emerged beetles (mean=19 beetles) compared to the control
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treatment (mean=38 beetles) (Table 1; Fig. 3). There was no effect of any of the tested
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covariates to explain the number of emerged beetles (Table 1). Despite the reduction in
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brood size, we did not find differences in the sex ratio caused by ivermectin treatment or
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the tested covariates (Table 1).
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Regarding morphological variables in the F1, male body size was affected by treatment and
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by the interaction of treatment and father horn length (Table 1). Males that developed in the
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ivermectin treatment emerged 5% larger than males in the control treatment (Table 1; Fig.
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4). In addition, males that developed in the ivermectin treatment had larger body size when
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their fathers (F0 generation) had larger horns. In F1 females, ivermectin had no effect on
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body size, but females whose fathers (F0 generation) had larger horns were larger
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independent of treatment (Table 1; supplementary Fig. S1).
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Male horn length was highly positively related to a male’s own body size (Table 1).
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Maternal and paternal body size (from the F0) had no effect on any of the tested variables
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in the F1 (Table 1).
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Table 1. Effect of ivermectin treatment and covariates on characteristics of the F1
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generation of the dung beetle Euoniticellus intermedius. Significant effects are shown in
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bold. Dev.=Deviance from GLMs; L. Ratio=Result from the likelihood ratio tests obtained
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from LMMs to calculate P-values. NS=Effect not retained in the best supported model.
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NA=Covariate not used in the analysis.
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Explanatory Variables
Number of emerged beetles PDev. value
Treatment
20.017 <0.001 0.478 0.488 14.639 0.002
NS
NS
NS
NS
Mother size
1.842
0.174
0.309 0.578
NS
NS
NS
NS
2.802
0.094
Father size
0.381
0.536
1.195 0.274
2.466
0.291
NS
NS
NS
NS
1.839
0.175
NS
6.898
0.031
NS
NS
4.281
0.038
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Pvalue
Male body size L. PRatio value
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NS
Male horn length L. PRatio value
Female body size L. PRatio value
2.886
0.089
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
2.122
0.145
NS
NS
NS
NS
2.230
0.135
NS
NS
4.421
0.035
NS
NS
NS
NS
NA
NA
NA
NA
NA
NA
NA
NA
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Own body size
Sex ratio
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Father horn length Treatment × Mother size Treatment × Father size Treatment × Father horn length
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Figure 3. Effect of ivermectin on the number of emerged Euoniticellus intermedius dung
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beetles in the F1. Bars represent means ± 95% confidence intervals. Numbers in
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parentheses are the sample sizes.
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Figure 4. Effect of ivermectin treatment on the body size of emerged Euoniticellus
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intermedius dung beetles in the F1 generation. Bars represent means ± 95% confidence
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intervals. Numbers in parentheses are the sample sizes (grouped in 10 families in the
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control and 13 families in the ivermectin treatment).
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F2 generation: trans-generational effects of ivermectin exposure
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Results from the F2 generation are summarized in Table 2. Parental exposure to ivermectin
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had no effect on the number or sex ratio of emerged offspring or offspring body size (Table
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2). However, we found a significant effect of parental treatment on male offspring’s horn
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length (Table 2). A reduction in horn length was observed when the father was exposed to
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ivermectin but not when the mother or both parents were exposed (see the significant
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interaction between mother treatment and father treatment in Table 2 and Fig. 5). There was
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a significant 4.3% reduction in horn length when only the father was exposed and a
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marginally-significant 3.4% reduction when only the mother was exposed; the reduction in
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offspring’s horn length was only 0.5% when both parents were exposed (Table 2; Fig. 5).
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Interestingly, unlike in the F1, in the F2 generation body size in both sexes increased with
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maternal body size but was not affected not by the father’s horn length (Table 2;
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supplementary Fig. S2). The number of offspring that emerged was positively affected by
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the father’s horn length (Table 2; supplementary Fig. S3). Male offspring (F2) horn length
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decreased with the father’s horn length in the ivermectin treatment but had no effect on the
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control treatment (Table 2; supplementary Fig. S4). Also, male offspring horn length
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increased with the male’s own body size (Table 2).
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Table 2. Trans-generational effects of ivermectin treatment and covariates in the F2
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generation of the dung beetle Euoniticellus intermedius. Significant effects are shown in
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bold. Dev.=Deviance from GLMs; L. Ratio=Result from the likelihood ratio tests obtained
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to calculate P-values of LMMs. NS=Effect not retained in the best supported model.
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NA=Covariate not used in the analysis.
Explanatory Dev. P-value Variables Mother 2.236 0.134 treatment Father treatment Mother treatment × Father treatment
Sex ratio
L. Ratio
Male horn length
Female body size
Pvalue
L. Ratio
Pvalue
L. PRatio value
Dev. NS
Pvalue
Male body size
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Number of emerged beetles
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NS
NS
NS
5.125
0.077
3.480 0.175
NS
4.033
0.133
8.861
0.031
2.718 0.256
NS
NS
NS
4.717
0.029
2.198
0.138
6.463 0.039
NS
NS
NS
NS
NS
Mother size
1.032
0.309
NS
NS
17.679 <0.001
Father size
NS
NS
NS
NS
5.165
0.075
NS
NS
2.550 0.279
2.770
0.095
NS
NS
NS
NS
NS
NS
3.479 0.062
NS
NS
NS
NS
3.767
0.052
NS
NS
2.329 0.126
3.914
0.047
NS
NS
NS
NS
10.745
0.004
NS
NS
NS
NS
NS
NS
NS
NS
5.057
0.024
NS
NS
NA
NA
NA
NA
NA
NA
390.507 <0.001
NA
NA
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NS
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NS
NS
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Figure 5. Effect of parental (F1 generation) exposure to ivermectin on male offspring’s
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(F2) horn length in Euoniticellus intermedius dung beetles. Bars represent mean model
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estimates ± 95% confidence intervals. Numbers in parentheses are the sample sizes
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(grouped in 26, 20, 13 and 16 families respectively). The asterisk indicates significant
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differences.
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4. Discussion
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Parental effects can enhance offspring fitness, particularly under adverse environmental
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conditions (Mousseau and Fox, 1998). However, trans-generational effects can also have
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inevitable negative consequences on offspring survival and reproduction (Uller et al.,
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2013). In the present study, we show that parental (mainly paternal) exposure to the toxic
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contaminant ivermectin had negative trans-generational effects on the horn length of male
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offspring but did not affect offspring number, sex ratio or body size. By emerging with
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smaller horns, the offspring of exposed males or females will likely face reproductive
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disadvantages during male-male competition.
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In our studied species, the observed 4% mean reduction in horn length caused by paternal
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exposure to ivermectin (an average decrease of 0.05 mm) may be highly relevant for
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offspring fitness. In E. intermedius, horn length is an accurate indicator of male quality (i.
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e. physical strength, immune reactivity, fat content and reproductive success) that is highly
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determinant of the result of male-male contests for access to females, mainly in evenly-
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matched contests between large males (Pomfret and Knell, 2006a). In this situation, even a
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decrease as small as 0.05 mm can reduce the probability of winning an intrasexual contest
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by 20-30% (Pomfret & Knell 2006a).
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Our findings in the F2 confirm that father horn length may be an important determinant of
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reproductive success (Pomfret and Knell, 2006a), even though male-male competition was
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not involved in our experiment. However, father horn length did not always have the same
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effects on the offspring; it did not affect offspring number in the F1 and had contrasting
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effects on offspring horn length in the F2. The same is true for maternal body size, which
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had no effect on the offspring characteristics measured in F1 and affected offspring body
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size more strongly in males than females in the F2 generation. We are currently carrying
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out quantitative genetic analyses to determine if modification of the heritability of offspring
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number, horn length and body size under ivermectin exposure may explain our contrasting
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results across treatments and generations. For example, in E. intermedius, horn length has
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low genetic basis and is highly defined by environmental conditions (broad sense
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heritability H2=0.12; Reaney and Knell, 2015), but this heritability could change under
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challenging environmental conditions such as ivermectin contamination.
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The mechanisms of parental transfer of environmental information to offspring are not well
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known. However, both maternal and paternal environments can define self-investment in
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reproduction (McNamara et al. 2009) or cause epigenetic changes in gametes resulting in
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differential gene expression in the offspring (Bonduriansky et al., 2016; García-González
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and Simmons, 2007; Polak et al., 2017; Sirot et al., 2006). The extent to which ivermectin
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may cause epigenetic changes in gametes or changes in reproductive investment by males
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and females remains to be studied with molecular analyses such as DNA methylation (Lyko
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and Maleszka, 2011). However, the fact that the combined effect of ivermectin in both
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parents did not affect offspring horn length makes it more plausible that males and females
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evaluate each other and invest differentially according to their perception of self- and mate
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condition.
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The reduction in brood size caused by ivermectin treatment in the F1 was about 50%,
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suggesting very strong selection promoted by the contaminant. Surprisingly, this reduction
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in the number of offspring was accompanied by an increase in male body size, which could
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have been driven by a trade-off between offspring number and quality (Stahlschmidt and
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Adamo, 2015). However, this seems unlikely given that direct exposure to ivermectin did
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not affect female body size or male horn length. Further studies should analyze the hatching
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success of exposed and control beetles to know the extent to which our studied beetles
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favor offspring quality over number under stressful conditions such as contamination with
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ivermectin.
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Here we found no effects of ivermectin exposure on sex ratio, either within or across
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generations, which could impact sexual selection processes in nature (Ancona et al., 2017;
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Carmona-Isunza et al., 2017). In previous experiments using a higher dose, ivermectin has
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proven to differentially affect males and females, causing biases in the sex ratio of emerged
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beetles (Desneux et al., 2007; Garric et al., 2007; González-Tokman et al., 2017). The lack
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of effect here is thus likely the consequence of using a dose of ivermectin which is below
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the threshold at which differential effects occur (González-Tokman et al., 2017).
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Our study shows only a marginally-significant trans-generational effect transferred by
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mothers exposed to ivermectin. This is surprising, as the mothers in our experiment
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survived the whole development in ivermectin, and could have transferred genetic
340
resistance as well as non-genetic maternal effects to the offspring (Crean and
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Bonduriansky, 2014). In our study, the offspring only grew in untreated dung, but we
342
cannot discard that more evident maternal effects could be observed if the offspring were
343
exposed too. Evidence in other insects shows that a stressor in the parental environment can
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have protective effects in the offspring even when they are exposed to a different stressor
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(Piiroinen et al., 2013; Plautz et al., 2013). In the future, we should also evaluate the
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parental effects when the offspring is exposed to ivermectin or other stressors to test the
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hypothesis that parental environments have larger effects on offspring when the parental
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and offspring environments match (Marshall, 2008; Uller et al., 2013).
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Our findings in the laboratory are relevant under natural situations, where the studied beetle
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is one of the most abundant species in cattle pastures (Cruz-Rosales et al., 2012; Montes de
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Oca and Halffter, 1998). Ivermectin is applied intermittently, exposing only some
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generations of dung beetles. Moreover, the contrasting cattle management practices in our
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study region, with farmers either using excessive ivermectin (and other contaminants) or
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not using it at all (local farmers, personal communication), provide potential natural
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scenarios for dung beetles to suffer trans-generational effects of ivermectin exposure. Even
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when testing for trans-generational effects of ivermectin in dung beetles under natural
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conditions may be logistically challenging, environmental risk assessment for ivermectin
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and other contaminants should consider trans-generational effects and their potential
359
impacts on wildlife and ecosystem function. Further studies could evaluate to what extent
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trans-generational effects of ivermectin exposure also alter ecosystem services provided by
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dung beetles in pastures (Beynon et al., 2015; Manning et al., 2017). Our study generates
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new insight into the importance of parental effects during the contemporary evolution of
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wild dung beetles, and probably many other animals, in contaminated habitats.
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5. Conclusions
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Parental effects are fundamental drivers of evolution in rapidly changing environments.
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Here we show that exposure to ivermectin in parental dung beetles defines offspring 23
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phenotype in ways that may affect offspring through sexual selection processes. Our results
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highlight the importance of considering trans-generational effects of contaminant exposure
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in wild animals, particularly insects, and generate new insights about the mechanisms and
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consequences of parental effects in the evolution of animals exposed to anthropogenic
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disturbance.
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Acknowledgements
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The authors acknowledge Ricardo Madrigal Chavero for help in the field and the
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laboratory. Lynna Kiere provided helpful comments that improved language and
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manuscript quality. The present study was funded by Consejo Nacional de Ciencia y
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Tecnología (CONACYT project Ciencia Básica 257894) granted to DGT.
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Competing interests
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Declarations of interest: none.
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cornicina (F.) and the scarabaeine dung beetles, Copris hispanus L., Bubas bubalus
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(Oliver) and Onitis belial F. Journal of Applied Entomology, 106, 381–389.
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Wohde, M., Blanckenhorn, W. U., Floate, K. D., Lahr, J., Lumaret, J. P., Römbke, J., … Düring, R. A. (2016). Analysis and dissipation of the antiparasitic agent ivermectin in
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cattle dung under different field conditions. Environmental Toxicology and Chemistry,
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35(8), 1924–1933. http://doi.org/10.1002/etc.3462
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Supplementary figure S1. Effect of the father’s horn length on the body size of F1 males
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and females of Euoniticellus intermedius dung beetles exposed to different treatments.
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C=Control, IV=Ivermectin, F=Females, M=Males. The effect of the father’s (F0) horn
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length on offspring (F1) body size was significant in F1 females of both treatments and F1
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males only in the Ivermectin treatment (see results text, Table 1).
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Supplementary figure S2. Effect of the mother (F1) size on offspring (F2) body size of
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Euoniticellus intermedius dung beetles. F=Female offspring, M=Male offspring. The
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relationship was significant in both sexes (Table 2).
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Supplementary figure S3. Effect of father (F1) horn length on the number of emerged
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offspring (F2) of Euoniticellus intermedius dung beetles.
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Supplementary figure S4. Effect of father horn length (F1) on male offspring’s (F2) horn
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length depending on the father’s treatment in Euoniticellus intermedius dung beetles.
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C=Control, IV=Ivermectin.
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Trans-generational effects of ivermectin exposure in dung beetles
Highlights Ivermectin is an antiparasitic drug that threatens dung beetles in cattle pastures
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We explored the effects of parental exposure to ivermectin in a dung beetle
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Paternal exposure caused a reduction on male offspring’s sexual traits
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We found no effect of parental treatment on offspring number or body size
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Parental effects are fundamental drivers of evolution in contaminated habitats
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S0045-6535(18)30526-5
DOI:
10.1016/j.chemosphere.2018.03.109
Reference:
CHEM 21053
To appear in:
ECSN
Received Date: 29 November 2017 Revised Date:
15 March 2018
Accepted Date: 17 March 2018
Please cite this article as: Baena-Díaz, F., Martínez-M, I., Gil-Pérez, Y., González-Tokman, D., Transgenerational effects of ivermectin exposure in dung beetles, Chemosphere (2018), doi: 10.1016/ j.chemosphere.2018.03.109. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Trans-generational effects of ivermectin exposure in dung beetles
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Fernanda Baena-Díaz1, Imelda Martínez-M.1, Yorleny Gil-Pérez1, Daniel González-
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Tokman1,2,*
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Mexico. 91070.
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CONACYT
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Corresponding author: [email protected] ; +52 228 842 1800 - 4144
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Instituto de Ecología A. C. Antigua Carretera a Coatepec 351. El Haya, Xalapa, Veracruz,
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Abstract
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Ivermectin is a powerful antiparasitic drug commonly used in cattle. Ivermectin residues
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are excreted in dung, threatening non-target coprophagous fauna such as dung beetles. This
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can have severe ecological and economic consequences for dung degradation and soil
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fertility. Even though the negative effects of direct ivermectin exposure on dung-degrading
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organisms are well known, effects could extend across generations. Here, we tested the
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effects of paternal or maternal exposure to ivermectin on offspring in the dung beetle
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Euoniticellus intermedius. This species is a classic study subject in ecotoxicology and
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sexual selection because males have a cephalic horn that is under intense selection via
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male-male competition. After confirming a negative effect of ivermectin on the number of
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emerged beetles, we found trans-generational effects of ivermectin exposure on the horn
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size of male offspring. Surprisingly however, this trans-generational effect only occurred
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when only the father was exposed. We detected no trans-generational effects of ivermectin
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exposure on offspring number, sex ratio or body size. Our results confirm that ivermectin
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not only has a strong effect on exposed individuals but also in their progeny. Our study
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opens new questions about the mechanisms responsible for parental effects and their long-
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term fitness consequences in contaminated habitats.
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Keywords: contamination, parental effects, Scarabaeinae, sexual selection
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1. Introduction
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The parental environment has very important effects on offspring. When parental effects
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transferred from the father or the mother define offspring viability, they are important
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drivers of evolution that are not linked to inherited genes (Badyaev and Uller, 2009; Leimar
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and McNamara, 2015; McNamara et al., 2016). The parental environment can define
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several phenotypic features in the offspring. For example, in insects, mothers and fathers
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that were infected by a pathogen can transfer resistance to their offspring (Moret, 2006;
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Roth et al., 2010). Also, mothers developed under high population densities can produce
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male offspring with larger sexual traits, which will likely increase their mating success
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(Buzatto et al., 2012). In some cases, the effects of maternal and paternal environments can
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be strikingly different: for example, in the fly Telostylinus angusticollis (Neriidae), mothers
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fed high protein diets have offspring with larger body size and elongated heads (a sexually-
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selected trait), whereas the opposite occurs in males, whose offspring body size and sexual
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traits are instead favored by high-carbohydrate diets (Bonduriansky et al., 2016). Most
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studies focus on maternal rather than paternal effects, despite the known importance of both
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effects in offspring phenotype (Crean and Bonduriansky, 2014). Trans-generational
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plasticity including maternal and paternal effects is an important tactic to deal with
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environmental change (Salinas et al., 2012) and has been interpreted as cryptic parental
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care (Jokela, 2010).
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Parental effects are particularly important under adverse conditions, even when the new
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generation grows in environments that are more favorable. This frequently occurs in
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habitats where human use of pesticides or other toxic compounds during certain times of
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the year, or in certain places, leads to temporary contamination which affects only some
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generations of short-lived organisms. Even though the pesticide is only intermittently
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present in the environment, trans-generational consequences of pesticide exposure may be
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observed in offspring viability and performance (Bonduriansky et al., 2012; Tassou and
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Schulz, 2009). This is the case of veterinary medications that are used in many countries
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despite their negative effect on the environment and biodiversity (Lumaret et al., 2012).
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Ivermectin is the most common parasiticide used in cattle at some regions of the world
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because it is both relatively inexpensive and is highly effective against nematodes, ticks and
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other parasites (Lumaret et al., 2012). Ivermectin binds to glutamate-gated and GABA
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receptors, modifying membrane permeability to chloride ions in invertebrates, therefore
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affecting the nervous and muscular functions; this causes paralysis and death in
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invertebrates but is of relatively low toxicity in mammals (Lumaret et al. 2012). Either
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applied topically or injected ivermectin is excreted in the dung over weeks or months
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during which it has lethal and negative non-lethal effects on non-target fauna, mainly dung
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flies and beetles (Lumaret et al., 2012). This causes severe economic losses given the high
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influence of coprophagous insects in burying and degrading dung in pastures and forests,
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contributing to increase soil fertility and control noxious fauna associated with the
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remaining dung (Beynon et al., 2015; Huerta et al., 2013; Wall and Beynon, 2012). Among
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the negative effects of ivermectin in dung beetles are reduced fecundity and reproductive
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success, delayed sexual maturation, reduced growth rate and body size, altered ovary
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morphology, reduced muscle development and reduced locomotor and olfactory capacity
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(González-Tokman et al., 2017; Martínez-M. et al., 2017; Verdú et al., 2015; Wardhaugh
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and Rodríguez-Menéndez, 1988).
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In the present study, we evaluated the trans-generational effects of maternal and paternal
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exposure to ivermectin in the dung beetle Euoniticellus intermedius (Coleoptera:
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Scarabaeinae). This species is negatively affected by ivermectin, and relatively small doses
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of 10-60 µg/kg of fresh dung cause important reductions in fecundity, development, muscle
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mass and body size (González-Tokman et al., 2017; Krüger and Scholtz, 1995; Martínez-
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M. et al., 2017). Euoniticellus intermedius is a classical model in sexual selection studies
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because males possess a cephalic horn whose size defines the outcome of male-male
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contests for mates (Pomfret and Knell, 2006a) and is an important indicator of immune
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condition and physical resistance (Lailvaux et al., 2005; Pomfret and Knell, 2006b).
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Counter-intuitively, there is evidence showing that developing in high doses of ivermectin
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causes male beetles of this species to emerge with larger horns, but this probably occurs
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because only high-quality, larger-horned males are able to survive developing in
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ivermectin, filtering small-horned males out of the measured population and biasing
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average horn size upward among the survivors (González-Tokman et al., 2017).
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Euoniticellus intermedius is one of the most important providers of ecosystem services in
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pastures by degrading dung in our study region. Ivermectin is a particularly serious threat to
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this species because it may actually be attracted to ivermectin-contaminated dung (Holter et
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al., 1993). Here, under controlled laboratory conditions, we exposed the male, the female or
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both beetle mates to a low concentration of ivermectin in dung during their whole
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development. These beetles were then allowed to mate in ivermectin-free dung and we
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evaluated offspring number, sex ratio, body size and male horn length in comparison with
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the offspring of control parents that were not exposed. We hypothesized that both maternal
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and paternal exposure to ivermectin would alter offspring reproductive success, sex ratio,
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body size and the size of the male cephalic horn. To the best of our knowledge, this is the
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first report of trans-generational effects of ivermectin in dung beetles.
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2. Materials and methods
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The present study was carried out using the dung beetle Euoniticellus intermedius (Reiche).
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This beetle is native to Africa, but invaded Mexico in the 1980’s after intentional
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introduction in the United States during the 1970’s and subsequent migration southwards; it
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is now one of the most abundant dung beetle species in southern Mexico, including our
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study site (Flota-Bañuelos et al., 2012; Montes de Oca and Halffter, 1998). Sixty-eight
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beetles were collected in Rancho El Salado, Acajete, Veracruz, Mexico (19º34’36’’N,
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96º23’39’’ W, altitude: 54 m asl), where cattle are not treated with ivermectin. Beetles were
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bred in an insectary at the Instituto de Ecología, Xalapa, Veracruz, Mexico (27.5±0.4°C,
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74±2.5% humidity). Beetles were kept in sterilized wet soil and fed ad libitum with
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homogenized cow dung collected at the same ranch. During the whole experiment, dung
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was replaced every third day. Before feeding the beetles, dung was frozen for at least two
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days at -20°C to kill parasites and other insect larvae.
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2.1 Experimental design
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The present study was designed to test the effect of paternal and maternal exposure to
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ivermectin (Fig. 1). Therefore, during the experiment we used two treatments spiked in
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cattle dung (83% humidity): Ivermectin and Control. As ivermectin is excreted largely 6
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unchanged in the dung of treated livestock, spiking ivermectin in dung is a recommended
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method for laboratory assays with this antiparasitic drug (González-Canga et al., 2009).
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The tests were performed with technical grade ivermectin (CAS-Number: 70288-86-7.
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Batch number: MKBS6097V; Sigma-18898). Since ivermectin is poorly soluble in water, it
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was dissolved in acetone (CAS- Number: 1567-89-1; Sigma purity > 99.8%). A
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concentration of 10 µg ivermectin in 10 mL of acetone solution was added per kg of fresh
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dung (corresponding to 59 µg ivermectin per kg of dry dung) in the experimental treatment.
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As a control treatment, 10 mL of acetone were used per kg of fresh dung. The acetone was
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allowed to evaporate from the dung before feeding the beetles. Dung was always conserved
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at 4°C. The ivermectin concentration used is ecologically realistic, as it resembles the
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concentration excreted by cattle treated topically with a recommended dose (500 µg
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ivermectin/kg of cattle body mass) 28 days earlier (Wohde et al., 2016). Despite being
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considered relatively low, this dose affects ovary development and the properties of the fat
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body of our study species (Martínez-M. et al., 2017).
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Beetles collected from the field were allowed to reproduce in two containers (34
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individuals per container) with untreated cow dung to obtain a laboratory population (F0,
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the grandparents) known to be unexposed to ivermectin during their whole life cycle (Fig.
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1). With beetles from the F0 we formed random pairs that were allowed to reproduce in
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either ivermectin-treated (N=10 pairs) or control (N=13 pairs) dung in 1-L plastic
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containers. The emerging individuals from each pair (full siblings) were considered a
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family (see below). Individuals emerged from the Ivermectin or Control treatment (F1, the
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parents) were randomly assigned to mate in untreated dung, with a mate of the same or
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different treatment, to test for parental effects of ivermectin exposure on the next generation
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(F2, the offspring). Mating pairs of males and females from different families from the F1
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resulted in the following combinations: Male Ivermectin-Female Ivermectin (N=26 pairs),
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Male Ivermectin-Female Control (N=20 pairs), Male Control-Female Ivermectin (N=13
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pairs) and Male Control-Female Control (N=16 pairs) (Fig. 1). Siblings were never crossed
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with each other. We recorded the number of emerged beetles and sex ratio in both the F1
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and F2 generations (F1=38 families, F2=75 families), and body size and male horn length
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in both F1 and in F2 generations (F1=61 males and 65 females and F2=295 males and 150
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females). Males were distinguished from females by the presence of the cephalic horn.
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Body size and horn length were measured from pictures taken at 10x under a Leica Z16
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microscope with Leica LAS EZ software. Body size was measured as pronotum width from
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pictures with a dorsal view. To avoid any potential bias, measurements were taken by the
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same person (YGP), who was blind to the beetle treatment at the time of measurement.
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Horn length was measured from lateral view pictures (Fig. 2).
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Figure 1. Experimental design used to test trans-generational effects of ivermectin
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exposure on the dung beetle Euoniticellus intermedius.
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Figure 2. Cephalic horn of male Euoniticellus intermedius dung beetles. The white line
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represents the measurement of horn length used.
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2.2 Statistical analyses
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Data were analyzed separately for each generation (F1 and F2) and for each response
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variable. Generalized linear models (GLM) were used to analyze the number and the sex
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ratio of emerged beetles. For analyzing the number of emerged beetles in the F1 we used a
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negative binomial GLM given the high overdispersion found for the Poisson model
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(Residual deviance/Residual d. f.=4.12), that is suggested for count data (Zuur et al., 2009).
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In the F2, we analyzed the number of emerged beetles with a Poisson GLM for count data
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without overdispersion (Residual deviance/Residual d. f.< 2). We analyzed the sex ratio
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with Binomial GLMs for both F1 and F2. Linear mixed models (LMM) controlling for
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genetic relatedness between siblings were used to analyze male and female body size and
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male horn length in both F1 and F2 (using family as a random variable with a random
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intercept) (Bolker et al., 2008; Bolker, 2016).
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For the analyses of the F1, we initially tested the effect of the treatment (ivermectin or
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control), the mother’s body size, the father’s body size, the father’s horn length and the
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interactions (treatment × mother size), (treatment × father size) and (treatment × father horn
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length). For the F2 we initially tested the effect of the mother’s treatment, the father’s
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treatment, the interaction (mother × father treatment), the mother’s body size, the father’s
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body size, the father’s horn length and the interactions (mother treatment × mother size),
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(father treatment × father size) and (father treatment × father horn length). For both
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generations, the analyses of horn length always included the individual’s own body size as
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a covariate and body size was exponentially transformed to improve normality.
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For F1 and F2 generations, the initial statistical models tested were reduced based on the
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Akaike Information Criterion (AIC) to obtain the best supported model. P-values for
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explanatory variables were obtained from Chi-squared tests for GLMs and from likelihood
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ratio tests for LMMs (Zuur et al., 2009). Normality of residuals was inspected in normal q-
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q plots and variance homogeneity was evaluated with Fligner-Killeen tests; the presence of
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outliers was tested with Cook’s distance (Crawley, 2013). All analyses were done
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following the procedures suggested by Zuur et al. (2009). Data were analyzed with R
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software version 3.2.3 (R Development Core Team, 2015) and mixed models were done
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using the nlme package (Pinheiro et al., 2016).
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3. Results
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F1 generation
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Results from the F1 are summarized in Table 1. Ivermectin treatment caused a 50%
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reduction in the number of emerged beetles (mean=19 beetles) compared to the control
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treatment (mean=38 beetles) (Table 1; Fig. 3). There was no effect of any of the tested
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covariates to explain the number of emerged beetles (Table 1). Despite the reduction in
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brood size, we did not find differences in the sex ratio caused by ivermectin treatment or
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the tested covariates (Table 1).
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Regarding morphological variables in the F1, male body size was affected by treatment and
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by the interaction of treatment and father horn length (Table 1). Males that developed in the
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ivermectin treatment emerged 5% larger than males in the control treatment (Table 1; Fig.
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4). In addition, males that developed in the ivermectin treatment had larger body size when
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their fathers (F0 generation) had larger horns. In F1 females, ivermectin had no effect on
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body size, but females whose fathers (F0 generation) had larger horns were larger
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independent of treatment (Table 1; supplementary Fig. S1).
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Male horn length was highly positively related to a male’s own body size (Table 1).
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Maternal and paternal body size (from the F0) had no effect on any of the tested variables
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in the F1 (Table 1).
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Table 1. Effect of ivermectin treatment and covariates on characteristics of the F1
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generation of the dung beetle Euoniticellus intermedius. Significant effects are shown in
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bold. Dev.=Deviance from GLMs; L. Ratio=Result from the likelihood ratio tests obtained
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from LMMs to calculate P-values. NS=Effect not retained in the best supported model.
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NA=Covariate not used in the analysis.
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Explanatory Variables
Number of emerged beetles PDev. value
Treatment
20.017 <0.001 0.478 0.488 14.639 0.002
NS
NS
NS
NS
Mother size
1.842
0.174
0.309 0.578
NS
NS
NS
NS
2.802
0.094
Father size
0.381
0.536
1.195 0.274
2.466
0.291
NS
NS
NS
NS
1.839
0.175
NS
6.898
0.031
NS
NS
4.281
0.038
227 228
Pvalue
Male body size L. PRatio value
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NS
Male horn length L. PRatio value
Female body size L. PRatio value
2.886
0.089
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
2.122
0.145
NS
NS
NS
NS
2.230
0.135
NS
NS
4.421
0.035
NS
NS
NS
NS
NA
NA
NA
NA
NA
NA
NA
NA
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Sex ratio
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Father horn length Treatment × Mother size Treatment × Father size Treatment × Father horn length
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54.221 <0.001
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Figure 3. Effect of ivermectin on the number of emerged Euoniticellus intermedius dung
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beetles in the F1. Bars represent means ± 95% confidence intervals. Numbers in
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parentheses are the sample sizes.
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Figure 4. Effect of ivermectin treatment on the body size of emerged Euoniticellus
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intermedius dung beetles in the F1 generation. Bars represent means ± 95% confidence
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intervals. Numbers in parentheses are the sample sizes (grouped in 10 families in the
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control and 13 families in the ivermectin treatment).
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F2 generation: trans-generational effects of ivermectin exposure
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Results from the F2 generation are summarized in Table 2. Parental exposure to ivermectin
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had no effect on the number or sex ratio of emerged offspring or offspring body size (Table
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2). However, we found a significant effect of parental treatment on male offspring’s horn
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length (Table 2). A reduction in horn length was observed when the father was exposed to
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ivermectin but not when the mother or both parents were exposed (see the significant
251
interaction between mother treatment and father treatment in Table 2 and Fig. 5). There was
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a significant 4.3% reduction in horn length when only the father was exposed and a
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marginally-significant 3.4% reduction when only the mother was exposed; the reduction in
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offspring’s horn length was only 0.5% when both parents were exposed (Table 2; Fig. 5).
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Interestingly, unlike in the F1, in the F2 generation body size in both sexes increased with
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maternal body size but was not affected not by the father’s horn length (Table 2;
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supplementary Fig. S2). The number of offspring that emerged was positively affected by
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the father’s horn length (Table 2; supplementary Fig. S3). Male offspring (F2) horn length
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decreased with the father’s horn length in the ivermectin treatment but had no effect on the
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control treatment (Table 2; supplementary Fig. S4). Also, male offspring horn length
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increased with the male’s own body size (Table 2).
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Table 2. Trans-generational effects of ivermectin treatment and covariates in the F2
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generation of the dung beetle Euoniticellus intermedius. Significant effects are shown in
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bold. Dev.=Deviance from GLMs; L. Ratio=Result from the likelihood ratio tests obtained
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to calculate P-values of LMMs. NS=Effect not retained in the best supported model.
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NA=Covariate not used in the analysis.
Explanatory Dev. P-value Variables Mother 2.236 0.134 treatment Father treatment Mother treatment × Father treatment
Sex ratio
L. Ratio
Male horn length
Female body size
Pvalue
L. Ratio
Pvalue
L. PRatio value
Dev. NS
Pvalue
Male body size
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Number of emerged beetles
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NS
NS
NS
5.125
0.077
3.480 0.175
NS
4.033
0.133
8.861
0.031
2.718 0.256
NS
NS
NS
4.717
0.029
2.198
0.138
6.463 0.039
NS
NS
NS
NS
NS
Mother size
1.032
0.309
NS
NS
17.679 <0.001
Father size
NS
NS
NS
NS
5.165
0.075
NS
NS
2.550 0.279
2.770
0.095
NS
NS
NS
NS
NS
NS
3.479 0.062
NS
NS
NS
NS
3.767
0.052
NS
NS
2.329 0.126
3.914
0.047
NS
NS
NS
NS
10.745
0.004
NS
NS
NS
NS
NS
NS
NS
NS
5.057
0.024
NS
NS
NA
NA
NA
NA
NA
NA
390.507 <0.001
NA
NA
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Mother treatment × Mother size Father treatment × Father size Father horn length Father horn length × Father treatment Own body size
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Figure 5. Effect of parental (F1 generation) exposure to ivermectin on male offspring’s
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(F2) horn length in Euoniticellus intermedius dung beetles. Bars represent mean model
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estimates ± 95% confidence intervals. Numbers in parentheses are the sample sizes
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(grouped in 26, 20, 13 and 16 families respectively). The asterisk indicates significant
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differences.
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278 279
4. Discussion
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Parental effects can enhance offspring fitness, particularly under adverse environmental
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conditions (Mousseau and Fox, 1998). However, trans-generational effects can also have
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inevitable negative consequences on offspring survival and reproduction (Uller et al.,
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2013). In the present study, we show that parental (mainly paternal) exposure to the toxic
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contaminant ivermectin had negative trans-generational effects on the horn length of male
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offspring but did not affect offspring number, sex ratio or body size. By emerging with
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smaller horns, the offspring of exposed males or females will likely face reproductive
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disadvantages during male-male competition.
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In our studied species, the observed 4% mean reduction in horn length caused by paternal
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exposure to ivermectin (an average decrease of 0.05 mm) may be highly relevant for
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offspring fitness. In E. intermedius, horn length is an accurate indicator of male quality (i.
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e. physical strength, immune reactivity, fat content and reproductive success) that is highly
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determinant of the result of male-male contests for access to females, mainly in evenly-
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matched contests between large males (Pomfret and Knell, 2006a). In this situation, even a
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decrease as small as 0.05 mm can reduce the probability of winning an intrasexual contest
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by 20-30% (Pomfret & Knell 2006a).
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Our findings in the F2 confirm that father horn length may be an important determinant of
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reproductive success (Pomfret and Knell, 2006a), even though male-male competition was
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not involved in our experiment. However, father horn length did not always have the same
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effects on the offspring; it did not affect offspring number in the F1 and had contrasting
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effects on offspring horn length in the F2. The same is true for maternal body size, which
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had no effect on the offspring characteristics measured in F1 and affected offspring body
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size more strongly in males than females in the F2 generation. We are currently carrying
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out quantitative genetic analyses to determine if modification of the heritability of offspring
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number, horn length and body size under ivermectin exposure may explain our contrasting
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results across treatments and generations. For example, in E. intermedius, horn length has
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low genetic basis and is highly defined by environmental conditions (broad sense
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heritability H2=0.12; Reaney and Knell, 2015), but this heritability could change under
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challenging environmental conditions such as ivermectin contamination.
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The mechanisms of parental transfer of environmental information to offspring are not well
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known. However, both maternal and paternal environments can define self-investment in
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reproduction (McNamara et al. 2009) or cause epigenetic changes in gametes resulting in
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differential gene expression in the offspring (Bonduriansky et al., 2016; García-González
314
and Simmons, 2007; Polak et al., 2017; Sirot et al., 2006). The extent to which ivermectin
315
may cause epigenetic changes in gametes or changes in reproductive investment by males
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and females remains to be studied with molecular analyses such as DNA methylation (Lyko
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and Maleszka, 2011). However, the fact that the combined effect of ivermectin in both
318
parents did not affect offspring horn length makes it more plausible that males and females
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evaluate each other and invest differentially according to their perception of self- and mate
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condition.
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The reduction in brood size caused by ivermectin treatment in the F1 was about 50%,
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suggesting very strong selection promoted by the contaminant. Surprisingly, this reduction
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in the number of offspring was accompanied by an increase in male body size, which could
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have been driven by a trade-off between offspring number and quality (Stahlschmidt and
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Adamo, 2015). However, this seems unlikely given that direct exposure to ivermectin did
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not affect female body size or male horn length. Further studies should analyze the hatching
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success of exposed and control beetles to know the extent to which our studied beetles
328
favor offspring quality over number under stressful conditions such as contamination with
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ivermectin.
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Here we found no effects of ivermectin exposure on sex ratio, either within or across
331
generations, which could impact sexual selection processes in nature (Ancona et al., 2017;
332
Carmona-Isunza et al., 2017). In previous experiments using a higher dose, ivermectin has
333
proven to differentially affect males and females, causing biases in the sex ratio of emerged
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beetles (Desneux et al., 2007; Garric et al., 2007; González-Tokman et al., 2017). The lack
335
of effect here is thus likely the consequence of using a dose of ivermectin which is below
336
the threshold at which differential effects occur (González-Tokman et al., 2017).
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Our study shows only a marginally-significant trans-generational effect transferred by
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mothers exposed to ivermectin. This is surprising, as the mothers in our experiment
339
survived the whole development in ivermectin, and could have transferred genetic
340
resistance as well as non-genetic maternal effects to the offspring (Crean and
341
Bonduriansky, 2014). In our study, the offspring only grew in untreated dung, but we
342
cannot discard that more evident maternal effects could be observed if the offspring were
343
exposed too. Evidence in other insects shows that a stressor in the parental environment can
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have protective effects in the offspring even when they are exposed to a different stressor
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(Piiroinen et al., 2013; Plautz et al., 2013). In the future, we should also evaluate the
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parental effects when the offspring is exposed to ivermectin or other stressors to test the
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hypothesis that parental environments have larger effects on offspring when the parental
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and offspring environments match (Marshall, 2008; Uller et al., 2013).
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Our findings in the laboratory are relevant under natural situations, where the studied beetle
350
is one of the most abundant species in cattle pastures (Cruz-Rosales et al., 2012; Montes de
351
Oca and Halffter, 1998). Ivermectin is applied intermittently, exposing only some
352
generations of dung beetles. Moreover, the contrasting cattle management practices in our
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study region, with farmers either using excessive ivermectin (and other contaminants) or
354
not using it at all (local farmers, personal communication), provide potential natural
355
scenarios for dung beetles to suffer trans-generational effects of ivermectin exposure. Even
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when testing for trans-generational effects of ivermectin in dung beetles under natural
357
conditions may be logistically challenging, environmental risk assessment for ivermectin
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and other contaminants should consider trans-generational effects and their potential
359
impacts on wildlife and ecosystem function. Further studies could evaluate to what extent
360
trans-generational effects of ivermectin exposure also alter ecosystem services provided by
361
dung beetles in pastures (Beynon et al., 2015; Manning et al., 2017). Our study generates
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new insight into the importance of parental effects during the contemporary evolution of
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wild dung beetles, and probably many other animals, in contaminated habitats.
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5. Conclusions
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Parental effects are fundamental drivers of evolution in rapidly changing environments.
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Here we show that exposure to ivermectin in parental dung beetles defines offspring 23
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phenotype in ways that may affect offspring through sexual selection processes. Our results
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highlight the importance of considering trans-generational effects of contaminant exposure
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in wild animals, particularly insects, and generate new insights about the mechanisms and
372
consequences of parental effects in the evolution of animals exposed to anthropogenic
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disturbance.
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Acknowledgements
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The authors acknowledge Ricardo Madrigal Chavero for help in the field and the
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laboratory. Lynna Kiere provided helpful comments that improved language and
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manuscript quality. The present study was funded by Consejo Nacional de Ciencia y
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Tecnología (CONACYT project Ciencia Básica 257894) granted to DGT.
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Competing interests
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Declarations of interest: none.
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Supplementary figure S1. Effect of the father’s horn length on the body size of F1 males
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and females of Euoniticellus intermedius dung beetles exposed to different treatments.
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C=Control, IV=Ivermectin, F=Females, M=Males. The effect of the father’s (F0) horn
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length on offspring (F1) body size was significant in F1 females of both treatments and F1
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males only in the Ivermectin treatment (see results text, Table 1).
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Supplementary figure S2. Effect of the mother (F1) size on offspring (F2) body size of
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Euoniticellus intermedius dung beetles. F=Female offspring, M=Male offspring. The
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relationship was significant in both sexes (Table 2).
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Supplementary figure S3. Effect of father (F1) horn length on the number of emerged
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offspring (F2) of Euoniticellus intermedius dung beetles.
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Supplementary figure S4. Effect of father horn length (F1) on male offspring’s (F2) horn
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length depending on the father’s treatment in Euoniticellus intermedius dung beetles.
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C=Control, IV=Ivermectin.
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Trans-generational effects of ivermectin exposure in dung beetles
Highlights Ivermectin is an antiparasitic drug that threatens dung beetles in cattle pastures
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We explored the effects of parental exposure to ivermectin in a dung beetle
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Paternal exposure caused a reduction on male offspring’s sexual traits
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We found no effect of parental treatment on offspring number or body size
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Parental effects are fundamental drivers of evolution in contaminated habitats
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