Brief Report Cannibalism in the Brown Marmorated Stink Bug Halyomorpha halys (Stål)

Giulia Papa and Ilaria Negri * Department of Sustainable Crop Production, Università Cattolica del Sacro Cuore Via Emilia Parmense 84, 29122 Piacenza, Italy; [email protected] * Correspondence: [email protected]

 Received: 7 August 2020; Accepted: 16 September 2020; Published: 19 September 2020 

Simple Summary: Overwintering populations of the crop pest Halyomorpha halys exhibit cannibalistic behaviour towards conspecifics. Depletion of metabolic reserves and desiccation occurring in winter can be overcome by intraspecific predation. This behaviour may be facilitated by the aggregation of individuals and the suppression of species-specific signals that prevent predation upon conspecifics.

Abstract: The phytophagous brown marmorated stink bug Halyomorpha halys (Stål) is known to exhibit cannibalistic behaviour towards eggs. Here, we provide evidence of cannibalism among overwintering H. halys adults. Since diapausing individuals have high physiological demands for surviving long periods under stressful conditions, including the risk of depletion of metabolic reserves and desiccation, we assumed that nutritional and water requirements can be met by intraspecific predation. The role of aggregative behaviour in promoting cannibalism is also discussed. Given its evolutionary advantage, this trait should be maintained over generations and may be more widespread than previously considered in species that display aggregative behaviour during adverse seasons.

Keywords: cannibalism; Halyomorpha halys; ; ; overwintering; phytophagous ; diapause; aggregation

1. Introduction The brown marmorated stink bug Halyomorpha halys (Stål, 1855) (Hemiptera: Pentatomidae) is native to East Asia, but has been spreading in Europe since 2004, first in Switzerland and then in the rest of Europe [1,2]. This species is a significant invasive agricultural pest that damages fruits trees (e.g., apples, peaches, and pears), vegetables, row crops, and ornamental plants [3,4]. H. halys feeds by penetrating a stylet bundle into plant tissues and injecting digestive enzymes to allow sucking of plant fluids, resulting in feeding injuries in the plant tissues, such as deformities, scars, discolouration, and pitting [4]. H. halys overwinter as adults and exhibit aggregative behaviour in thermally buffered microhabitats [5]. Aggregated individuals then enter facultative diapause for several months until specific environmental cues in spring trigger their re-emergence. For example, in northern Italy, adults exit diapause when the maximum temperature exceeds 14 ◦C and the photoperiod has 13 h of light [6]. Adults may become active through winter in response to warm temperatures and a poor nutritional state [7,8]. The metabolic strategies and physiological mechanisms used by H. halys to survive during winter diapause are not fully known [7]. According to previous studies, depletion of energy reserves and desiccation are the main causes of mortality in the overwintering generation, and adequate nutrition and sequestration of energy reserves (e.g., lipids, glycogen, and sugars) in individuals before entering diapause majorly influence diapause success [7–9].

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nutrition and sequestration of energy reserves (e.g., lipids, glycogen, and sugars) in individuals before entering diapause majorly influence diapause success [7–9]. Overwintering populations of herbivorous species displaying aggregative habits usually have few or no sources of food, other than dead conspecifics [10]. Conspecific necrophagy has been documented among overwintering adults, for example, in the boxelder bug Leptocoris trivittatus [10].

InsectsNecrophagy2020, 11, 643does not occur in H. halys, and freshly dead individuals can even repel conspecifics 2[11]. of 7 Here, we provide evidence that supports the hypothesis of cannibalism, i.e., feeding on live conspecifics, among overwintering adults of H. halys. Cannibalism is defined as intraspecific predationOverwintering and has been populations observed ofin herbivorousmany , species including displaying protozoa, aggregative insects, birds, habits and usually mammals have few[12]. orCannibalism no sources may of food, play otherdifferent than roles dead in conspecifics the ecology [10of ].a population, Conspecific such necrophagy as stabilising has been the documentedhost–plant/insect among relationship, overwintering regulating adults, population for example, density, in the boxelderand providing bug Leptocoris a selective trivittatus advantage[10]. Necrophagyby removing doesconspecifics not occur that in H.are halys infected, and with freshly pathogens, dead individuals which reduces can even the repel rates conspecifics of parasitism [11 in]. subsequentHere, we generations provide evidence[13]. Cann thatibalistic supports behaviour the hypothesis may be influe of cannibalism,nced by both i.e., density-dependent feeding on live conspecifics,and -independent among factors; overwintering for example, adults developmenta of H. halys. Cannibalisml asynchrony is with defined the ashost intraspecific plant, sex or predation genetic andrelatedness has been of the observed participants, in many and animals, limitations including in the amount protozoa, or quality insects, of birds, food andand mammalsenvironmental [12]. Cannibalismfactors, such as may humidity play diandfferent high rolestemperature in the [13]. ecology of a population, such as stabilising the host–plantAlthough/insect cannibalism relationship, has regulatingbeen observed population in predatory density, insects, and providing it has also a been selective documented advantage in bymany removing phytophagous conspecifics species, that areincluding infected withmember pathogens,s of Lepidoptera, which reduces Coleoptera, the rates and of parasitism Hemiptera in subsequent[12,13]. In phytophagous generations [13 hemipter]. Cannibalisticans, cannibalism behaviour has may been be influencedreported in byadults both and density-dependent nymphs which andpredate -independent on eggs, factors;e.g., for example, japonensis developmental, Nezara asynchrony viridula, withand theH. hosthalys plant, [14–17], sex orwhereas genetic relatednesscannibalism ofamong the participants, adults has only and been limitations suggested in the but amount never documented or quality of [16]. food and environmental factors, such as humidity and high temperature [13]. 2. MaterialsAlthough and cannibalism Methods has been observed in predatory insects, it has also been documented in many phytophagous species, including members of Lepidoptera, Coleoptera, and Hemiptera [12,13]. During November 2017, about 400 H. halys adults in diapause at aggregation sites were In phytophagous hemipterans, cannibalism has been reported in adults and nymphs which predate on collected from barns in the Province of Cremona (northern Italy, 45°08′17.3′′ N, 10°26′10.9′′ E). After eggs, e.g., Parastrachia japonensis, Nezara viridula, and H. halys [14–17], whereas cannibalism among removing dead/damaged individuals, 215 live H. halys adults were selected and placed into a single adults has only been suggested but never documented [16]. cage (ca. 40 cm high × 20 cm diameter) filled with cardboard panels. The cage was kept under 2.outdoor Materials conditions, and Methods following the protocol adopted by Costi et al. [6]. The individuals were maintained in diapause without any food or water for about 14 weeks. In mid-February 2018, diapauseDuring was November terminated 2017, by about exposure 400 H. halysto 25adults °C/L16:D8 in diapause to start at aggregation laboratory sitescolonies. were collectedAmong fromindividuals barns in that the had Province just exited of Cremona diapause, (northern we ob Italy,served 45◦ 08one017.3 living00 N, 10adult◦26 010.9being00 E).cannibalised After removing by a deadconspecific/damaged (Figure individuals, 1A; Video 215S1). liveThisH. observation halys adults was were shortly selected followed and by placed two other into occurrences a single cage of (ca. 40 cm high 20 cm diameter) filled with cardboard panels. The cage was kept under outdoor this behaviour (Videos× S2 and S3). conditions,Dead specimens following left the in protocol the overwintering adopted by cage Costi were et al.counted [6]. The and individuals the bodies inspected were maintained using a instereomicroscope diapause without to check any food for the or presence water for of about predation 14 weeks. marks. In Scanning mid-February electron 2018, microscopy diapause (SEM; was terminatedZeiss Gemini by exposureSEM 500) to 25was◦C /usedL16:D8 to to confirm start laboratory the occurrence colonies. Amongof predation individuals marks. that For had SEM just exitedobservations, diapause, naturally we observed dehydrated one living specimens adult being were cannibalised gold-coated by and a conspecific mounted onto (Figure a carbon-coated1A; Video S1). Thisstub. observation was shortly followed by two other occurrences of this behaviour (Videos S2 and S3).

Figure 1. Prey and predators of Halyomorpha halys.(. (A) Frame Frame of Video Video S1 showing cannibalism in the metathoracic ventral part of prey. (B) PicturePicture showingshowing predation in dorsaldorsal partpart ofof prey.prey. MovementMovement responses ofof preyprey proddedprodded withwith aa brushbrush demonstrateddemonstrated thatthat theythey werewere stillstill alivealive (Video(Video S3).S3).

Dead specimens left in the overwintering cage were counted and the bodies inspected using a stereomicroscope to check for the presence of predation marks. Scanning electron microscopy (SEM; Zeiss Gemini SEM 500) was used to confirm the occurrence of predation marks. For SEM observations, naturally dehydrated specimens were gold-coated and mounted onto a carbon-coated stub. In October 2018, a second population of H. halys adults (about 180) already triggered for diapause were collected from an overwintering site in the Province of Mantua (northern Italy, 45◦00014.900 N Insects 2020, 11, x FOR PEER REVIEW 3 of 7

In October 2018, a second population of H. halys adults (about 180) already triggered for diapause were collected from an overwintering site in the Province of Mantua (northern Italy, 45°00′14.9′′ N 11°04′38.5′′ E). After removing dead/unhealthy individuals, 147 live individuals were Insectsselected2020, 11and, 643 maintained in diapause as previously described. In January 2019, dead and live3 of 7 specimens were counted. Dead specimens were inspected under a stereomicroscope for predation marks. 11◦04038.500 E). After removing dead/unhealthy individuals, 147 live individuals were selected and maintained in diapause as previously described. In January 2019, dead and live specimens were 3. Results counted. Dead specimens were inspected under a stereomicroscope for predation marks. In the first overwintering population, 90 out of 215 (about 42%) individuals were found dead; in 3.the Results second population, 88 out of 147 (about 60%) were found dead. The stereomicroscope observationsIn the first of overwintering the dead H. halys population, from the 90 two out ofpopulations 215 (about 42%)identified individuals 63 out wereof 90found and 23 dead; out of in the88 secondspecimens, population, respectively, 88 out showing of 147 (about clear 60%) marks were of foundcannibalism dead. Theor feeding stereomicroscope activity. The observations marks of ofcannibalism the dead H. were halys infrom the theform two of populationsperforation holes identified in the 63 ventral out of part 90 and of the 23 outinsect, of 88while specimens, saliva respectively,residue on the showing stylet clearwas considered marks of cannibalism a mark of fe oreding feeding activity. activity. Of the The 63 marks specimens of cannibalism showing weremarks in theof formcannibalism, of perforation 46 were holes putative in the prey, ventral as they part had of the perforation insect, while holes saliva compatible residue with on thethe styletpredator was consideredstylet and awere mark coated of feeding with activity.coagulated Of thesubsta 63nce specimens compatible showing with markshaemolymph of cannibalism, spilling 46(Figure were putative2B,C). Almost prey, asall theypredation had perforation marks were holes in the compatible metathoracic with ventral the predatorpart at the stylet level andof the were peritreme, coated withmedian coagulated furrow, substance and ostiole compatible of the scent with gland haemolymph (Figure spilling2A–C) or (Figure nearby2B,C). joints Almost of dorsal all predation sclerites (Figure 1B). The remaining 17 specimens were putative predators since they showed presence of marks were in the metathoracic ventral part at the level of the peritreme, median furrow, and ostiole fluid exudates on the tip of the stylet. SEM observations confirmed the feeding holes in the fourth of the scent gland (Figure2A–C) or nearby joints of dorsal sclerites (Figure1B). The remaining 17 and fifth sternites (Figure 2E and Video S2) and the presence of the salivary sheath on the stylet specimens were putative predators since they showed presence of fluid exudates on the tip of the (Figure 3). The salivary sheath showed a typical bulbous morphology with sequentially stacked stylet. SEM observations confirmed the feeding holes in the fourth and fifth sternites (Figure2E and droplets (Figure 3). In the second population, SEM observations confirmed that all 23 specimens had Video S2) and the presence of the salivary sheath on the stylet (Figure3). The salivary sheath showed a predation holes. typical bulbous morphology with sequentially stacked droplets (Figure3). In the second population, Importantly, we also observed brownish faeces produced by allegedly starving individuals SEM observations confirmed that all 23 specimens had predation holes. exiting diapause.

FigureFigure 2. 2.Stereomicroscope Stereomicroscope (A (–AD–,DF),F and) and SEM SEM (E) images(E) images showing showing marks marks of cannibalism of cannibalism in the ventralin the partventral of overwintering part of overwinteringHalyomorpha Halyomorpha halys.(A halys) The. ( redA) The square red identifiessquare identifies the peritreme, the peritreme, median median furrow, andfurrow, ostiole and of ostiole the scent of glandthe scent area, gland and area, the green and the and green blue squaresand blue highlight squares abdominalhighlight abdominal segments. Letterssegments. within Letters squares within refer squares to the positionrefer to the ofthe position figures. of (theB) Holesfigures. with (B) tissue Holes and with haemolymph tissue and residues.haemolymph (C) Haemolymph residues. (C) clot.Haemolymph (D) The red clot. arrows (D) The show red the arrows holes show in the the abdomen. holes in the (E) abdomen. SEM image (E of) theSEM hole image in the of abdomen. the hole in This the pictureabdomen. represents This pictur thee same represents individual the same in Video individual S2. (F) in Close-up Video S2. image (F) ofClose-up the holes image in D. of the holes in D.

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Figure 3. SEM images of the stylet of overwinteringoverwintering Halyomorpha halys associated with cannibalism. (A) Stylet Stylet with with salivary salivary sheath. sheath. (B) (Close-upB) Close-up image image of a salivary of a salivary sheath sheathshowing showing the typical the bulbous typical bulbousmorphology. morphology.

4. DiscussionImportantly, we also observed brownish faeces produced by allegedly starving individuals exiting diapause. In the present study, we provide evidence of previously undocumented behaviour in 4.overwintering Discussion adults of H. halys that cannibalised other adults. Nutrient intake in diapausing adults has been previously documented in other insect species, such as the black blow fly Phormia regina, the mosquitoIn the present Culex study, pipiens we, provide and L. evidencetrivittatus of [9,10]. previously Overwintering undocumented individuals behaviour undergo in overwintering a costly adultsphysiological of H. halys demandthat cannibalisedfor surviving otherlong adults.periods Nutrientof stressful intake conditions, in diapausing including adults depletion has been of previouslymetabolic documentedreserves and in desiccation. other insect species,The costs such of as diapause the black bloware commonly fly Phormia regina,reflectedthe mosquitoin lower Culexpost-diapause pipiens, andsurvivalL. trivittatus and fitness.[9,10 In]. H. Overwintering halys, it has been individuals estimated undergothat more a than costly 70% physiological of adults do demandnot survive for winter surviving and long only periods 14% can of survive stressful until conditions, reproduction including [6]. depletionAccording of to metabolic previous reservesstudies, andthe mortality desiccation. of Theadults costs is ofdue diapause to a combination are commonly of dehydration reflected in lowerand metabolic post-diapause depletion survival of their and fitness.energy InreservesH. halys [9]., it hasOur been study estimated supports that the more notion than that 70% ofnutritional adults do and not survivewater requirements winter and only of 14%overwintering can survive adults until reproductioncan be met by [6 intraspecific]. According predation, to previous and studies, this could the mortality also explain of adults why is H. due halys to aindividuals combination that of dehydrationhave just emerge and metabolicd from diapause depletion have of their exceptionally energy reserves high dispersal [9]. Our study capacity, supports and thecan notioneven survive that nutritional for many and days water without requirements food [18,19]. of overwintering adults can be met by intraspecific predation,Successful and thisfeeding could events also explain are demonstrated why H. halys byindividuals the formation that of have the justsalivary emerged sheath from at the diapause tip of havethe stylet exceptionally in some high dead dispersal individuals capacity, (i.e., and puta cantive even cannibals) survive for [20]. many Asdays in other without phytophagous food [18,19]. hemipterans,Successful feeding feeding in events H. halys are is demonstrated facilitated by by the the formation formation of of a thesalivary salivary sheath sheath that at encapsulates the tip of the styletthe stylet in some bundle dead [20,21]. individuals As the (i.e., stylet putative penetrates cannibals) the tissue, [20]. As H. in halys other secretes phytophagous liquid droplets hemipterans, that feedingsolidify to in formH. halys a solidis facilitated hollow tube by with the formation typical bulbous of a salivary morphology. sheath The that salivary encapsulates sheath themay stylet stay bundleattached [20 to,21 the]. Asfeeding the stylet site, penetrates but in some the tissue,cases, H.it may halys remainsecretes attached liquid droplets to the thatstylet solidify when to feeding form a solidceases, hollow being tube subsequently with typical expelled bulbous during morphology. grooming The [20,21]. salivary In sheath the present may stay work, attached the overwintering to the feeding site,populations but in some were cases, maintained it may remainin diapause attached withou to thet any stylet food when or feedingwater for ceases, about being 14–16 subsequently weeks, and expelledconspecifics during were grooming the sole [source20,21]. of In food. the present At pres work,ent, thewe overwinteringdo not know why populations these putative were maintained predators inthen diapause died, and without further any work food is or needed water forto clarify about 14–16this phenomenon. weeks, and conspecifics were the sole source of food.In At our present, opinion, we do cannibalism not know why is thesealso putativemediated predators by aggregative then died, behaviour. and further Aggregation work is needed is towidespread clarify this in phenomenon. the kingdom, especially in insects, and its ecological and evolutionary significanceIn our opinion, is based cannibalism on the assumption is also mediated that joining by aggregative a group increases behaviour. the Aggregationsurvival and isreproductive widespread insuccess the animal of its kingdom, members especially [22]. In in insects,insects, and aggregation its ecological occurs and evolutionary via species-specific significance aggregation is based on pheromones that induce group formation and attract and/or arrest conspecifics at the locality of release [22]. Aggregative behaviour in H. halys has been observed in both summer and

Insects 2020, 11, 643 5 of 7 the assumption that joining a group increases the survival and reproductive success of its members [22]. In insects, aggregation occurs via species-specific aggregation pheromones that induce group formation and attract and/or arrest conspecifics at the locality of release [22]. Aggregative behaviour in H. halys has been observed in both summer and overwintering generations [5]. The sex-specific aggregation pheromone is regularly released by males during the warmer months of the year to attract females [23,24]. Then, vibrational communications between the sexes facilitate mating [25]. In winter generations, sexually immature individuals aggregate in thermally buffered microhabitats that might also promote local moisture and water-conserving group effects [26,27]. Once diapause is initiated, H. halys males stop emitting aggregation pheromones and overwintering individuals cease to respond to experimental pheromone releases [28,29]. The unique volatiles released by diapausing adults consist of defensive compounds not specific to H. halys but commonly found in all stink bugs [29]. Since species-specific signals are known to prevent cannibalism in many animal taxa and may also help to avoid predation upon close relatives [30], we argue that the suppression of self-recognition mechanisms facilitates cannibalism in H. halys. Our results suggest the novel conclusion that intraspecific predation also represents a strategy to cope with nutrient and water depletion. The ability to feed on conspecifics would also increase the probability of individuals surviving until host plants become available. We argue that, given its evolutionary advantage, this trait should be maintained over generations and might be more widespread in species displaying aggregative behaviour during adverse seasons than previously considered. During diapause, insects are not always totally inactive [31]. H. halys adults, for example, may become active through winter at temperatures above 9 ◦C[7,8]. In northern Italy, both 2017 and 2018 were characterised by exceptionally mild winters, with daily maximum temperatures in January below 13–14 ◦C. Our experiment did not include continuous monitoring of the cages, hence it is possible that a proportion of those specimens that we classified as ‘cannibalised’ were subjected to necrophagy. In overwintering populations of species displaying aggregative behaviour, conspecific necrophagy may indeed provide resources during the winter months [10]. However, according to Chambers et al. [11], necrophagy is highly uncommon in H. halys. In their research, no evidence of necrophagy or survival advantage was recorded in overwintering individuals exposed to dead conspecifics during winter. In their experiments, the authors provided one-year-old dead H. halys to live individuals, but necrophagy was never observed [11]. In our opinion, the poor nutrient and water content of desiccated corpses cannot provide an appropriate food source, especially for a piercing-sucking species such as H. halys. Chambers et al. also reported that freshly killed individuals generated a repellent effect in live conspecifics [11]. The consumption of live conspecifics was observed in the present study. To the best of our knowledge, our study is the first to document cannibalism among adults in the phytophagous H. halys. Specific experiments are necessary to assess the extent of this behaviour, its sex bias, and the optimal microclimatic conditions that may trigger it. We believe that shedding light on this undocumented behaviour might open the field to new and exciting research on the topic.

5. Conclusions Cannibalism, i.e. intraspecific predation, is known to constitute an important component of the nutritional ecology of many species. In insects, cannibalism has been reported in non-carnivorous and strictly herbivorous species, where this behaviour provides a substantial nutritional benefit. Our results suggest that nutritional and water requirements of overwintering adults of the crop pest Halyomorpha halys can be met by intraspecific predation. Cannibals may therefore increase their probability to survive until host plants become available. This study provides a theoretical basis for a range of future studies to better understand the role of cannibalism in the biology and ecology of this invasive pest. Insects 2020, 11, 643 6 of 7

Supplementary Materials: The following are available online at http://www.mdpi.com/2075-4450/11/9/643/s1, Video S1: Observation 1, Video S2: Observation 2, Video S3: Observation 3. Author Contributions: G.P. and I.N. contributed equally to this work. All authors have read and agreed to the published version of the manuscript. Funding: This research is part of the DEBUG project funded by the “Bando per il finanziamento dei progetti di ricerca in campoagricolo e forestale-D.D.S. 28 marzo 2018, n. 4403 CUP: J34I20000770009”. Acknowledgments: Giulia Papa was partially supported by the Doctoral School on the Agro-Food System (Agrisystem) at the Università Cattolica del Sacro Cuore (Italy). We thank Mario Barbato for his helpful comments on the first draft of the manuscript. We thank Filippo Raguzzoni for his support in the lab and Lara Maistrello for the fruitful discussion. Conflicts of Interest: The authors declare that there is no conflict of interest regarding the publication of this article. The data that support the findings of this study are available from the corresponding author upon request.

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