Marking Dung Beetles (Coleoptera: Scarabaeidae):Retrospective and a Reliable “Scarification” Method

Authors: Wuerges, Mariah, and Hernández, Malva I. M.

Source: The Coleopterists Bulletin, 74(1) : 168-171

Published By: The Coleopterists Society

URL: https://doi.org/10.1649/0010-065X-74.1.168

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SCIENTIFIC NOTE

MARKING DUNG BEETLES (COLEOPTERA: SCARABAEIDAE): RETROSPECTIVE

AND A RELIABLE “SCARIFICATION” METHOD

MARIAH WUERGES AND MALVA I. M. HERNANDEZ

Department of Ecology and Zoology, Federal University of Santa CatarinaFlorianopolis, SC 88040-900, BRAZIL

mariahwuerges@gmail.com

DOI.org/10.1649/0010-065X-74.1.168

Scarabaeinae (Coleoptera: Scarabaeidae), withover 6,200 species described (Tarasov and Genier2015), are widely studied beetles often used asecological indicators in diversity studies (Halffterand Favila 1993). They also play important eco-logical roles in nutrient cycling, soil aeration, sec-ondary seed dispersal, and control of parasites(Nichols et al. 2008). However, some aspects of thebasic biology of these species are poorly investi-gated, such as life span, dispersal capacity, and otherpopulation parameters (Cultid-Medina et al. 2015;Hanski and Cambefort 1991; Villada-Bedoya andCultid-Medina 2017). One way to access some ofthis information is through mark, release, and re-capture studies (MRR) via permanent marks withthe least possible effect on individuals’ behavior andsurvival (Martınez-Quintero et al. 2013).An MRR study with dung beetles was carried out

by Peck and Forsyth (1982), which aimed to esti-mate population size for some species by markingthem with enamel paint spots on the pronotum andelytra, with a five color-code. Following just onerecapture event, with a recapture rate of 0.02%, theydescribed distances covered by some species. Favila(1988) presented an individual marking method forlaboratory and field studies, using paint and glue onthe dorsum to protect the marking from soil friction,highlighting the pronotum as the best part of thebeetle’s body to mark. Using mark-release methodsin fieldwork, Escobar and Chacon de Ulloa (2000)described the use of different habitats for somespecies, marking individuals of species greater than10 mm by using different colors of paint. Theirresults provided information about movement pat-terns and distances covered by Sulcophanaeusvelutinus (Murray, 1856) and Dichotomius cf.quinquedens (Felsche, 1910). Roslin (2000) pro-vided results about distances covered for species ofAphodiinae by using a mark that consisted of twosmall holes in different positions in the elytra, usingthe tip of the thinnest available insect pin, whichcould only be seen under a microscope. Larsen and

Forsyth (2005) performed amark-release study withCanthon acutus Harold, 1868, a small diurnal ball-rolling beetle (body width = 4.1 mm), by using asilver pen. Each group was identified with a uniquecombination of dots on the elytra and pronotum andreleased once, performing recaptures every 24 hoursfor four days, which allowed them to propose aspatial design to avoid pseudoreplicates in eco-logical studies of biodiversity. To understand theeffects of landscape structure on patterns of abun-dance and movement and the differences betweenmale and female movement, Arellano et al. (2008)painted individuals of Canthon cyanellus cyanellusLeConte, 1859, Phanaeus pyrois Bates, 1887, andDichotomius amplicollis (Harold, 1869) by usingsilver pens to make a unique combination of dots onthe elytra and/or pronotum. Based on Favila (2008),eachmark was covered with a fine layer of glue withcyanocrilate. Noriega andAcosta (2011) did amark-release experiment for over a month with Sulco-phanaeus leander (Waterhouse, 1891) by markingthe ventral surface of the metasternum of this largebodied species with a fast-drying latex paint. Thesame mark was used for all individuals. They ob-served high fidelity to the habitat and dispersalbetween different environments and answeredquestions such as daily activity and sex ratio.Recently, Martınez-Quintero et al. (2013) pro-

posed a novel method for marking dung beetles,which consisted of a permanent and individualscarificationmark. They used aMototol Stylus 1100Dremel® to scrape the dorsal surfaces of the elytraand pronotum, in companionwith an emerald 84922Dremel® stone that allowed a clean brand on thebeetle’s body. This mark was applied on individualsfrom 10 to 35 mm long because it was difficult toperform on individuals smaller than 10 mm, al-though they reported that they have been able tomark individuals as small as 6 mm as well. Theyreported no ecological, biomechanical, or repro-ductive implications on individuals, but, like pre-vious studies, they did not test for such.

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Cultid-Medina et al. (2015), based on the samemethod, carried out a population study of two dungbeetle species, Oxysternon conspicillatum (Weber,1801) and Dichotomius cf. alyattes (Harold, 1880),which were chosen for being abundant, large spe-cies (10 to 35mmbody length) with different habitatpreferences. They made a general marking on theelytra and pronotum, which allowed them toidentify sex, site, and type of vegetation in whichindividuals were captured. They proposed that theirdata were valid based on the consequence of thepermanent mark not adversely affecting the beetles.Using a similar permanent marking technique, Silvaand Hernandez (2015) redesigned the trap spacingprotocol proposed by Larsen and Forsyth (2005).They used the broken tip of a needle to scarify thecuticle of the pronotum and elytra and used a dotscheme resulting in a unique mark. Villada-Bedoyaand Cultid-Medina (2017) also used the scarifica-tion method of Martınez-Quintero et al. (2013) andcarried out a population study for sevenmonths withDichotomius cf. satanas (Harold, 1867) and D. cf.alyattes, obtaining a recapture rate of 0.04%.Our goal was to test if the scarification technique

affects any component of individual fitness, byassessing survival, behavior, and reproduction ofmarked and unmarked dung beetles. In addition, weassessed if markings could be applied individuallyand permanently on species of various sizes, andthus whether this technique can be used in labo-ratory experiments and in the field without harmingthese insects.We conducted a laboratory experiment with 26

pairs of Canthon rutilans cyanescens Harold, 1868,a Neotropical dung beetle about 4 mm in length.Thirteen pairs marked pairs and 13 unmarked pairswere tested to determine if markings affect survival,behavior, and reproduction. The marking was per-formed with a dental micromotor (Beltec LB100)with a 0.5 mm spherical drill and the aid of amagnifying glass or a stereoscope. We scraped theelytra of the beetles by drawing a scheme of pointsand lines in a numbering system used to provide aunique identifier to each individual (Fig. 1). Eachpoint represented one unit, and each line corre-sponded to five units. The right elytron corre-sponded to units, the left elytron to tens, the righthalf of the pronotum to hundreds, and the left half ofthe pronotum to thousands (Fig. 2). For example,the identifier 8 (Fig. 1A) resulted from one line andthree points in the right elytra. The identifier 13 (Fig.1B) is created by a point in the left elytron and threepoints in the right elytron.The beetles were collected with baited pitfall

traps in September 2017 at the Serra do TabuleiroState Park (27°430S, 48°480W, 300 m elevation), anarea of Atlantic rainforest located in southern Brazil,and taken to the Laboratory of Terrestrial Animal

Ecology at the Federal University of Santa Catarina.To avoid weight and age as confounding effectsin the experiment, we formed pairs of similarweight and age and distributed them between two

Fig. 1. Scarification marks on the elytra of Canthonrutilans cyanescens. A) Marks on the right elytroncorresponding to the number 8, in which was made oneline (value = 5) and three dots (value = 3, one dot = 1),B) Marks for number 13, in which a point was made inthe left elytron (value = 10) and three points in the rightelytron (value = 3).

THE COLEOPTERISTS BULLETIN 74(1), 2020 169

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treatments, systematically allocating similar pairs ofdifferent ages and sizes between the treatments. Agecategorization was performed according to theopacity of the elytra and thewear of the tarsal spur ofthe anterior tibia, thereby classifying individuals asnew, mature, and old. Very old or damaged indi-viduals were not used. All 26 pairs were placed inpots (8 × 11 × 6 cm) with 400 g of soil from thecollection site; before use, the soil was heated in amicrowave for five minutes to avoid contamination,especially by mites. The beetles were fed weeklywith 10 g of feces from domestic dogs bred withanimal feed at the Laboratory Animal House of theFederal University of Santa Catarina. The experimentstarted in November 2017 and lasted until the be-ginning of March 2018, completing four months.During this period the beetles were kept in the labo-ratory under ambient temperature and controlled lu-minosity (12 hours light and 12 hours dark). Deathsand births were counted weekly, and the number ofnest balls produced by the pairs was counted once inDecember 2017 and once in January 2018. Statisticalanalysis included t test and ANOVA for normal dataand GLM with Poisson distribution for counting data,all performed with R software.Scarified markings did not affect the survival of

the individuals under laboratory conditions. Nosignificant difference was detected between sexes(t = 0.51, df = 48.63, p = 0.61), as males lived onaverage 62.0 days and females lived on average

67.8 days. Nor was there a significant differencebetween treatments (t = 0.33, df = 50, p = 0.74), withmarked individuals living a mean of 66.8 days andunmarked individuals a mean of 63.1 days. More-over, the mark did not affect behavior, as measuredby the production of nesting balls. Marked pairsproduced a mean of 2.3 nest balls, and unmarkedones produced on average 1.6 nest balls, with nodifference between treatments (t = 1.25, df = 34.98,p = 0.22). Scarification also did not affect the re-production of marked pairs, since the number ofoffspring per pair was statistically the same in bothtreatments (z = 1.48, df = 11, p = 0.77). The totalnumber of offspring was 10 females and six malesfor all marked pairs, and four females and threemales for all unmarked pairs. Furthermore, larvaldevelopmental times were not significantly differentbetween treatments (F = 0.55, df = 1, p = 0.46), withan average of 86.6 days and 78.7 days for larvaefrom marked and unmarked parents, respectively.The fertility rates (calculated as the ratio betweenthe number of developed offspring per pair dividedby the maximum number of nest balls produced bythat pair) for the marked and unmarked pairs were0.53 and 0.54, respectively, with no significantdifference detected (F = 0.001, df = 1, p = 0.97).MRR experiments with insects are quite chal-

lenging tasks, yet they are improving with tech-nological advances, although still requiringcreativity and dexterity of the researcher. Beyondthe difficulties of application, mainly because of thegreat abundance and small size of these animals, anideal mark must be durable, cheap, non-toxic, andeasy to apply and observe (Hagler and Jackson2001). Paintings and tattoos have been a commontechnique for dung beetles (Arellano et al. 2008;Escobar and Chacon de Ulloa 2000; Favila 1988;Larsen and Forsyth 2005; Noriega and Acosta 2011;Peck and Forsyth 1982). Markings of this type aremainly used on large insects because of the logis-tical difficulties in applying the mark on small in-dividuals (Hagler and Jackson 2001), althoughsome studies managed to apply it on small dungbeetles (<10 mm) (Arellano et al. 2008; Larsen andForsyth 2005). Moreover, the use of ink makes itpossible to recapture marked dung beetles only overshort periods, since the markings have a limitedduration due to friction with the soil during thebeetle’s burial behavior (Martınez-Quintero 2013).Although Favila (1988) and Arellano et al. (2008)applied a drop of glue to fix the paint, this techniqueis difficult to perform because of the waxy epicuticleon the beetle’s integument and the small size ofsome species. Roslin (2000) was able to mark smallindividuals with elytral perforations visible onlywith a dissecting scope. Although it was a perma-nent mark, perforations in the elytra can exposeindividuals to parasites (Martınez-Quintero 2013)

Fig. 2. Marks on a specimen of Canthon rutilansrutilans, corresponding to the number 1428, whereas 1point at the left side of the pronotum (= 1,000), 4 pointson the right side of the pronotum (= 400, 1 point =100), 2 points on the left elytron (= 20, 1 point = 10),and 1 line (= 5) plus 3 points (= 3) on the right elytron.

170 THE COLEOPTERISTS BULLETIN 74(1), 2020

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and solar radiation, and the needs to be tested re-garding individual fitness.Scarification marks, more recently performed, are

durable, cheap, non-toxic, easy to identify, and do notcreate perforations. This method has been mostlyapplied on large individuals (>10 mm length) (Cultid-Medina 2015; Martınez-Quintero 2013; Villada-Bedoya and Cultid 2017), and more rarely appliedto small individuals (<10 mm length) (Silva andHernandez 2015), although it was not tested for po-tential negative effects it might have on beetle fitness.We have shown that labeling with a “scarificationmark” has no adverse influence on dung beetle sur-vival, behavior, and fertility. The marks are permanentand easy to seewith the naked eye, and themethod canbe applied to species of different sizes and shapes.Furthermore, a marking machine can be added to asmall mobile battery and used in the field. Therefore,we propose that scarification can be a reliable methodfor field and laboratory MRR studies.

ACKNOWLEDGMENTS

We appreciate the contributions made by EloisaAlves, Maristela Carpintero, and Luiz FernandoFerreira in the development of the laboratory ex-periment, and we appreciate the initial suggestionby Dr. Jorge Lobo of the method involved in thiswork. We thank the other members of the Labo-ratory of Terrestrial Animal Ecology for help andsupport and the biologist Fernando Bruggemann forproviding access to the collection site. This studywas financed in part by the Coordenaç~ao deAperfeiçoamento de Pessoal de Nıvel Superior -Brasil (CAPES) - Finance Code 001 and in partby the Conselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq) for a researchproductivity grant to the second author for theproject “Diversidade beta de assembleias debesouros Scarabaeinae: Efeito da temperatura nadistribuiç~ao espacial e temporal” (307437/2017-5).This research was funded by the “PVE” projectMEC/MCTI/CAPES/CNPq/FAPs No 09/2014 ofthe Ministry of Education of Brazil: “Efeito com-parado do clima e das mudanças no uso do solo nadistribuiç~ao espacial de um grupo de insetos indi-cadores (Coleoptera: Scarabaeinae) na MataAtlantica” (88881.068089/2014-01).

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(Received 31 August 2018; accepted 2 January 2020.Publication date 25 March 2020.)

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