Chaboo, C.S., E. Grobbelaar, \u0026 A. Larsen. 2007. Fecal ecology in leaf beetles: novel records in the African arrow-poison beetles, Diamphidia Gerstaecker and Polyclada Chevrolat

$Page 1: Chaboo, C.S., E. Grobbelaar, \u0026 A. Larsen. 2007. Fecal ecology in leaf beetles: novel records in the African arrow-poison beetles, Diamphidia Gerstaecker and Polyclada Chevrolat$
FECAL ECOLOGY IN LEAF BEETLES: NOVEL RECORDS IN THE AFRICAN

ARROW-POISON BEETLES, DIAMPHIDIA GERSTAECKER AND POLYCLADA

CHEVROLAT (CHRYSOMELIDAE: GALERUCINAE)

CAROLINE S. CHABOO

Cullman Molecular LaboratoryAmerican Museum of Natural History

Central Park West @ 79th StNew York, NY, 10024–5192, U.S.A.

[email protected]

ELIZABETH GROBBELAAR

South African National Collection of InsectsPlant Protection Research Institute

Agricultural Research CouncilPrivate Bag X134, Queenswood 0121

Pretoria, SOUTH [email protected]

AND

ARNE LARSEN

Ingrid Marie Vej 3DK-5683 Haarby, DENMARK

[email protected]

Abstract

Immature stages in five chrysomelid clades, Cassidinae, Criocerinae, Cryptocephalinae,Lamprosomatinae and Galerucinae, use their feces as a significant part of their defense. InGalerucinae, only two genera, Blepharida Chevrolat and Polyclada Chevrolat have beenknown to carry larval fecal coats. We report for the first time that immature stages of twospecies in the African arrow-poison genus Diamphidia Gerstaecker, as well as an additionalspecies of Polyclada Chevrolat cover themselves with their feces. In Diamphidia femoralisGerstaecker, Diamphidia nigroornata Stal and an undetermined species of Polyclada, femalesoviposit masses on stems of Commiphora (Burseraceae) and Sclerocarya birrea (A. Richt.)Hochst. (Anacardiaceae), and they coat their eggs with sticky olive-green feces that hardeninto a dark-brown covering. All larval instars retain their feces, as semi-solid pellets or a wetmass that partially or completely covers the dorsum, or as long anal strands. The final instarloses its fecal coat prior to descending the host stem or dropping to the ground to enter thesoil for pupation. These behaviors further support a close evolutionary relationship betweenBlepharida, Diamphidia and Polyclada, and suggest similar morphological features formaintaining fecal coats.

Resumen

Los estadios inmaduros en cinco cladas de crisomelidos, Cassidinae, Criocerinae,Cryptocephalinae, Lamprosomatinae y Galerucinae, usan sus excrementos como parteimportante de sus defensas. En Galerucinae, solo se conocen dos generos, BlepharidaChevrolat y Polyclada Chevrolat, que usen cubiertas fecales en las larvas. Reportamos porprimera vez que estadios inmaduros de tres especies en los generos africanos de flechasenvenenadas, Diamphidia Gerstaecker y Polyclada Chevrolat, se cubren con susexcrementos. En Diamphidia femoralis Gerstaecker, Diamphidia nigroornata Stal y unaespecie desconocida de Polyclada, las hembras ovipositan masas en troncos de Commiphora

The Coleopterists Bulletin, 61(2):297–309. 2007.

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(Burseraceae) y Sclerocarya birrea (A. Richt.) Hochst. (Anacardiaceae), y cubren sus huevoscon excrementos verde-oliva pegajosos que se transforman al endurecerse en una cubiertamarron oscura. Todas los estadios larvales retienen sus excrementos como bolas semi-solidas, o como una masa humeda que parcial o completamente cubre el dorso, o comolargos filamentos anales. El ultimo estadio larval pierde su cubierta fecal antes de descenderdel tronco huesped o caerse al suelo para entrar en fase de pupa. Estos comportamientosapoyan aun mas una relacion evolucionaria entre Blepharida, Diamphidia y Polyclada, ysugieren elementos morfologicos similares para el mantenimiento de cubiertas fecales.

Fecal ecology of insects is gaining some attention (Weiss 2006). Most animalsput distance between themselves and their feces; some caterpillars use unusualprojectile methods (Frohawk 1913). Other animals mark territory boundarieswith feces, e.g., badgers (Stewart et al. 2001). Chrysomelid beetles displaya remarkable array of defenses including crypsis, cycloalexy, saltation,stridulation, thanatosis, buccal regurgitations, glandular and anal secretions,distasteful or toxic hemolymph, and plant-sequestered or derived offensivechemicals (often accompanied by aposematism) (Begossi and Benson 1988;Ferguson and Metcalf 1985; Jolivet 1988; Hilker 1992). In addition, immaturestages in five chrysomelid clades treat their feces as a material resource forconstructing domiciles and protective shields (Eisner et al. 1967; Root andMessina 1983; Damman and Cappucino 1991; Olmstead 1994; Morton and Vencl1998; Vencl and Morton 1999; Nogueira-de-Sa 2004; Nogueira-de-Sa and Trigo2002, 2005; Chaboo, in press).

In Cryptocephalinae (Chapuis and Candeze 1855; Fiebrig 1910; Erber 1968,1969, 1988; Hinton 1981; LeSage and Steifel 1996; Schmitt 1988; Reid 1990;Scholler 1999; Flinte and Macedo 2004a, b; Flinte 2005; Brown and Funk 2005;Chaboo et al. in press) and Lamprosomatinae (Erber 1988; Lee and Morimoto1991) females create a fecal case around solitary eggs; this case or ‘‘scatoshell’’ isthen carried and enlarged by successive larval instars which live inside it. Exuvialskins and trichomes may be incorporated into the fecal matrix (Brown and Funk2005; Chaboo et al. in review). Cases are the outcome of a complex interplay ofmorphology and behavior and between the mother and the offspring that inherither initial construction. The cryptocephaline case serves as both domicile anda mechanical protection against abiotic (e.g., desiccation) and biotic (e.g.,predators and parasitoids) dangers.

In other fecal-retaining chrysomelids, feces are retained as shields offeringmechanical, offensive or chemical protection against desiccation, insolation,predators and parasitoids (Olmstead 1994). Many cassidine larvae and somepupae carry an elaborate mobile exuvio-fecal shield (Fiebrig 1910; Eisner et al.1967; Takizawa 1980; Chaboo and Nguyen 2004; Chaboo in press). Larvae ofCriocerinae maintain wet feces directly on the dorsum (Chapuis and Candeze1855; Schmitt 1988; Morton 1997; Morton and Vencl 1998; Mathews and Reid2002; Chaboo, Vencl, Grobbelaar, unpubl. data). In the largest clade ofchrysomelids, Galerucinae (5 Galerucinae + Alticinae) (,14,000 species)(Lawrence 1982), only two genera have been reported with fecal retention.Larvae of at least 26 of the 73 described species of Blepharida Chevrolat retain wetfeces directly on the dorsum (Riley 1869, 1874; Frost 1972, 1973; Furth 1982,2004; Evans et al. 2000; Jolivet and Verma 2002). In the African genus PolycladaChevrolat, a single species, Polyclada flexuosa Baly, was noted as ‘copropherous’as a third instar (Koch 1958) but no other details were given. Several studies have

298 THE COLEOPTERISTS BULLETIN 61(2), 2007

shown that the offensiveness of chrysomelid feces may be enhanced with plantchemicals (Gomez 1997, 2004; Gomez et al. 1999; Morton and Vencl 1998; Evanset al. 2000).

In the present paper we report novel fecal retention records in a secondPolyclada species and in two species of a third genus, Diamphidia Gerstaecker.Polyclada comprises 12 species that are distributed in Namibia, Kenya,Mozambique, Senegal, South Africa, Zanzibar, and on the Arabian Peninsula(Heikertinger and Csiki 1940; Bryant 1942). Diamphidia comprises nine describedspecies that are distributed in Angola, Burundi, Ethiopia, Kenya, Mozambique,Namibia, Rwanda, South Africa, and Tanzania (including Zanzibar) (Heikertin-ger and Csiki 1940). The biology of both genera is poorly known (Furth and Lee2000), but Koch’s (1958) brief outline of the life cycle of P. flexuosa indicates thategg masses are covered with feces, and the larval period altogether lasts up tothree weeks, before pupation. Pupation is underground, within a sandy cocoon,and can last several years.

Apart from their unusual fecal defense, Diamphidia and Polyclada are of ethno-entomological interest—southern African San (Bushmen) dig up the pupae anduse their body fluids as an arrow poison (Figs. 1–2) (Maulik 1931; Skaife 1953;Koch 1958; Kundig 1978; Kao et al. 1989). The active molecule is a toxalbumintermed diamphotoxin, which acts like some snake venoms, inducing generalparalysis, hemolysis and death (Breyer-Brandwijk 1937; de la Harpe and Dowdle1980).

We observed and photographed individuals of three species, Diamphidiafemoralis Gerstaecker (Figs. 5–11) and Diamphidia nigroornata Stal (Figs. 12–13)on several Commiphora species (Burseraceae; frankincense and myrrh family) inBotswana and South Africa, and one Polyclada species on marula, Sclerocaryabirrea (A. Richt.) Hochst. (Anacardiaceae; mango and cashew family) in Namibia(Figs. 14–19). Host plants with live beetles currently are maintained in thelaboratory for further observations. Phylogenetic revisions with morphologicaldescriptions of these immature stages will be presented elsewhere.

Materials and Methods

Behavioral observations of various life stages of D. femoralis and D.nigroornata on their host plants, Commiphora species (Burseraceae), were madeby the authors (initials below) in Botswana: Nata, Botswana (February 2006 byCSC), Republic of South Africa: North-West and Limpopo Provinces (December2004–January 2005 by CSC) and KwaZulu-Natal Province (January 2006 by CSCand EG). The KwaZulu-Natal sites are located in the Savanna Biome with thevegetation of the Mkuze and Ndumu sites classified as Natal Lowveld Bushveldand the Tembe site classified as Sub-humid Lowveld Bushveld (Granger 1996).The Northwest and Limpopo sites localities included Jakkalbessie Game Farmand Kapama Game Reserve in the Mixed Lowveld Bushveld vegetation zone, andHans Merensky Nature Reserve in the Mopane Bushveld zone (Van Rooyen andBredenkamp 1996). The density of Commiphora across these localities varied fromhigh (Fig. 3) to occasional (Fig. 4).

Polyclada sp. was observed and photographed on its host plant, S. birrea(Anacardiaceae), in Okalongo village, Omusati region, and Ondobe village,Ohangwena region, northern Namibia (Figs. 14–19) (February 1998 and January2001 by AL). This region is located in the Acacia tree-shrub savanna vegetationtype (Mendelsohn et al. 2002). Sclerocarya density (Fig. 14) may have beeninfluenced and extended by indigenous people who harvest marula fruits (Fig. 15)

THE COLEOPTERISTS BULLETIN 61(2), 2007 299

to make alcoholic brews and AmarulaH liqueur, and to extract valuable cookingoil from the dried kernels.

Insect vouchers are deposited in the South Africa National Collection ofInsects (SANC), Republic of South Africa, American Museum of NaturalHistory (AMNH), NY, U.S.A., and Essig Museum, University of California-Berkeley (EMEC), CA, U.S.A. Plant vouchers are deposited in the herbaria ofthe South African National Biodiversity Institute (SANBI) and the New YorkBotanical Garden (NYBG). Host plants and beetles were identified by theauthors through feeding association and with collections of immature stages from

Figs. 1–4. 1) San man digging up Diamphidia cocoons, N of Botlapatlou, 23u45910.90S,25u28926.70E, Botswana, April (photograph, E. C. Grobbelaar); 2) applying Diamphidiapupal hemolymph as poison on arrows (photograph, E. C. Grobbelaar); 3) Commiphoraforest (Burseraceae), Tembe Elephant Reserve, Kwa-Zulu Natal, South Africa, January(photograph, C. Chaboo); 4) typical Commiphora habitat, Ndumo Game Reserve, NdumoGame Reserve, Kwa-Zulu Natal, South Africa, January (photograph, E. Grobbelaar).


Figs. 5–11. Diamphidia femoralis Gerstaecker on Commiphora host, South Africa. 5)egg case with hard, dark smoothened fecal cover (photograph, C. Chaboo); 6) early instarscovered with semi-solid fecal pellets (photograph, K. Ober); 7) mature larvae with fecalstrands hanging from anus (photograph, E. Grobbelaar); 8) mature larvae covered withsemi-solid fecal pellets (photograph, E. Grobbelaar); 9) pre-pupa entering soil for pupation;10) pupation cocoon (broken from adult emergence), L ca. 1 cm; 11) adult (photograph,K. Ober).


the Iziko Museum of Cape Town (SAMC), Museum of Comparative Zoology(MCZ), South African National Collection of Insects (SANC), TransvaalMuseum (TMSA), and the United States National Museum (USNM). Outdatedlocality names follow Broadley and Minshull (1986).

Live Diamphidia beetles were maintained on host plants at SANC forobservations of oothecal and fecal egg cover production by females and offecal retention by larvae. Field and laboratory photography were conductedas follows: by EG (SANC) with an Olympus OM4 camera and Zuiko Auto-S50 mm F1.4 lens, as well as a Telescopic Auto Extension Tube 65–116 with ZuikoAuto-1:1 Macro 80 mm F4 lens and T10 Ring Flash 1; by AL with digitaland film cameras; and by CSC (AMNH) with the MicropticsTM system (www.microptics-usa.com, accessed 2006).

Results

The life cycle of galerucines generally is comprised of the egg, four larval stadia,pupa and adult (Jolivet and Verma 2002). Larvae are root miners, leaf miners,external root feeders, and free-living phyllophagous feeders (Doguet 1994; Smith1986; Jolivet and Hawkeswood 1995). The life cycles of Diamphidia and Polycladaare similar, including oviposition on host plant stems (Figs. 5, 17), free-livingleaf-chewing larvae (Figs. 6–8, 12, 18), and underground pupation (Figs. 9–10).

Figs. 12–13. Diamphidia nigroornata Stal on Commiphora host, Mkuzi Game Reserve,South Africa, January (photographs, E. Grobbelaar). 12) late instar larva covered withsemi-solid fecal pellets; 13) adult.


These characteristics are consistent with those described for Blepharida (Becerra2004a, b).

Diamphidia femoralis is known from Burundi, Ethiopia, Mozambique,Rwanda, South Africa and Tanzania (Weise 1924). Diamphidia nigroornata waspreviously known from KwaZulu-Natal, South Africa (Heikertinger and Csiki1940) but our observations extend its range into Botswana.

Oviposition was observed in the three species. Females deposited clusterscontaining elongate eggs oriented vertically, and then covered these with a thickcoating of olive-green feces. Feces were deposited in similar-sized bands that wereoriented longitudinally. The texture was very rubbery and extremely sticky.Mature cases were hard, blackish, and not sticky (Figs. 5, 17). Under laboratoryobservations of D. nigroornata, this greenish coat hardened and blackened andwas no longer sticky within one hour. The initial banded pattern was notapparent in the egg case of D. femoralis (Fig. 5) but were distinct in Polyclada

Figs. 14–19. Polyclada species, the Omusati and Ohangwena regions, northernNamibia, January–February. 14) host plant, Sclerocarya birrea (Anarcardiaceae), marulatree (photograph, M. Hyde); 15) local women harvest ripe fruits for brews (photograph, K.Fleissner); 16) egg case; 17) egg case showing overlapping rows of dried feces; 18) gregariouslarvae; 19) adult. (photographs, A. Larsen)


where each ridge retained its integrity, hardening with a flange that was slightlyelevated over the following ridge (Fig. 17).

All observed instars retained their feces. Larval feces of Diamphidia femoralis(Figs. 6–8) and D. nigroornata (Fig. 12) were retained in two patterns. Larvaesometimes also had long, somewhat dry, fecal filaments hanging from the anus(Fig. 7); these were easily broken and appeared to be similar to those describedfor Blepharida sacra (Weise) (Furth 2004). In the Polyclada larvae, feces were inform of an anal string (Fig. 18). In different instars of the same Diamphidia series,larvae were completely or partially covered with thick, semi-solid, short fecalpellets. These pellet forms were distinct or blended with neighboring pellets. Priorto pupation, the mature larva discarded its fecal coat by passively rubbing againstthe host plant and by no longer retaining additional feces. Eventually it moveddown the plant and entered the soil for pupation. Pupation cocoons ofDiamphidia (Fig. 10) and Polyclada are composed of soil particles glued togetherand appear not to include fecal materials.

Discussion

In the three species observed, D. femoralis, D. nigroornata and Polyclada sp.,females lay small groups of eggs that they then completely cover with feces. Asthis fecal coat dries, hardens and darkens, the covering displays distinctivepatterns. In both Diamphidia species, the cover texture is somewhat smooth, butin Polyclada, the cover retains the pattern of fecal deposition with rows thatoverlap. Each row is slightly flared with ragged edges.

Larvae remain clustered, perhaps as a consequence of hatching together. Olderinstars are solitary, and migrated to the soil alone. Museum collections compriseadults, larvae and some broken cocoons, all collected from September to Aprilbut most frequently in December–January. Pupation occurs within a soil cocoon(Fig. 10). The San (Bushmen) collect cocoons throughout the year. The life cycleis prolonged during the pupal stage which can vary from 2–3 years (Maulik 1931).As far as we know this may be the longest galerucine life cycle yet reported, andcontrasts with that of Blepharida where the entire life cycle is completed in a singleseason (Becerra 2004a).

Fecal retention in Chrysomelidae has been discussed as a protection againstsolar radiation, desiccation, parasitoids and predators (Olmstead and Denno1992; Hilker 1992; Olmstead 1994, 1996; Vencl and Morton 1999; Muller andHilker 2003, 2004). The oothecal and larval fecal coats in Diamphidia andPolyclada may fulfill these roles and may also provide protection by crypsisbecause egg mass coverings blend with the host plant stems (Figs. 5, 17). Larvae(Figs. 8–9), pupae, and adults of D. femoralis (Fig. 11) are brightly coloredorange-red, and the adults are particularly so, suggesting a case of aposematiccoloration. This appears contradictory given that the larvae are partially orcompletely obscured by green to black-brown feces and the pupae areunderground. While diamphotoxin was reported only in pupae, it is stillambiguous as to whether larvae and adults are also toxic.

Our finding of fecal retention in Diamphidia and Polyclada species similar tothat found in Blepharida supports a historical hypothesis of a close relationshipamong these three genera (Seeno and Wilcox 1982). The currently availablehigher-level phylogenies for Galerucinae (Lingafelter and Konstantinov 2000;Kim et al. 2003; Duckett et al. 2004; Gillespie et al. 2003, in press) have sampledsome Blepharida species but not Polyclada or Diamphidia. In her phylogeneticanalysis of Blepharida and allies, Becerra (1993, 2004a, b) resolved three sampled


Diamphidia, D. femoralis, D. nigroornata and D. vittatipennis Baly, in a mono-phyletic lineage sister to Blepharida and allied genera. To explore the evolutionaryfecal ecology of Galerucinae, Polyclada and additional genera at the root nodeshould be analyzed in an expanded dataset. Fecal retention is widespread inBlepharida where 25 species are known with it (Evans et al. 2000). If this behaviororiginated once within Galerucinae, then fieldwork may unveil additional genericand species records. Since some Blepharida larvae do not retain feces,phylogenetic analysis will reveal the number and temporal sequence of gainsand losses of retention.

Preliminary studies indicate that specialized morphological machinery enablesfecal retention in Chrysomelidae. Shield-retaining cassidine larvae have abdom-inal segment X sub-segmented to form a mobile telescoped anus and tergum IXwith urogomphal processes (Chaboo and Nguyen 2004; Chaboo in press). InCryptocephalinae, the maternal rectal-abdominal apparatus constructs a compli-cated fecal case, and the larvae have a modified flattened head, inflated recurvedabdomen and long legs associated with case construction and retention (Erber1968, 1969, 1988; Chaboo et al. in press). In Criocerinae, where larvae makesimilar wet and semi-solid fecal pellets as do the galerucines discussed here,a specialized neuro-muscular anatomy has been hypothesized for moving fecesalong the larva’s body towards the head (Riley 1869, 1874). We predict that larvaeof both Criocerinae and the three fecal-retaining galerucine genera may exhibita convergently similar suite of anal, setal, and muscular features correlated withfecal constructions, retention, repair and manipulation.

Acknowledgments

We are grateful to the following for facilitating this study: research andcollecting permits: Adrian Armstrong (KZN Parks Authority, South Africa),Vincent Egan (Limpopo Parks Authority, South Africa), Clarke and JennyScholtz (University of Pretoria, South Africa); field assistance: Cornel du Toit,Vincent van der Merwe, Karen Ober and Justin Schmidt; photographs: E. C.Grobbelaar, M. Hyde, K. Fleissner and K. Ober; specimen loans: M. Cochrane(SAMC), P. Perkins (MCZ), and D. Furth (USNM); funding for CSC by UC-Berkeley Hellman Fund (Kipling Will), AMNH Cullman Fund (Joel Cracraft andGeorge Barrowclough) and the Systematic Association (UK); for EG by the PlantProtection Research Institute (SANC) (Gerhard Prinsloo); and for AL byDanChurchAid (Denmark). Comments and Spanish translation by FernandoMerino was helpful.

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(Received 30 August 2006; accepted 2 January 2007. Publication date 26 July 2007.)


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