Biology and Host Specificity of Gall-Inducing Acythopeus

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Biology and host specificity of gall-inducing Acythopeus burkhartorum (Coleoptera: Curculionidae), a biological-control agent for the invasive weed Coccinia grandis (Cucurbitaceae) in Guam and Saipan

Anantanarayanan Raman, Zerlene T. Cruz, Rangaswamy Muniappan & Gadi V. P. Reddy

An introduced cucurbit Coccinia grandis (Linnaeus) Voigt has grown into problem proportions in Hawaii and the Pacific islands of Guam and Saipan. The biology of Acythopeus burkhartorum O’Brien, 1998, a potential biological-control agent of C. grandis, has been described. By inducing the gall – a sink for nutrients – and by deriving nutrition, A. burkhartorum places C. grandis under stress. Especially during the late larval stage, the weevil displays an unusual behaviour of shredding dry gall tissues with its mandibles to ‘prepare’ the ‘pupal case’ and using the shredded sclerenchyma fibres to fill the open and cut ends of the pupal case. This ability to ‘create’ such a pupal case is unique among weevils. Because A. burkhartorum is able to sever tender shoots of C. grandis at points where galls are induced, we consider that this weevil will be highly relevant in C. grandis management. Although non- gall-inducing species of baridine weevils have a wide host range, the known gall- inducing species are specific to their respective hosts, similar to the majority of gall-inducing insects. A. burkhartorum prefer consistently either petioles or stems, behave identically by tunnelling through soft tissues within the host organs, and induce galls. Because of the potential of A. burkhartorum in biological control of C. grandis, we tested its specificity against C. grandis and Zehneria guamensis (an endemic cucurbit of Guam and Mariana Islands), following ‘choice’ and ‘no choice’ test modes. No feeding hole or gall development occurred on Z. guamensis indicating a categorical response that A. burkhartorum is specific to C. grandis. This result encouraged field release of A. burkhartorum in Guam and Saipan. Anantanarayanan Raman*, Charles Sturt University, PO Box 883, Orange, NSW 2800, Australia. [email protected] Z.T. Cruz, R. Muniappan & G.V.P. Reddy, Agricultural Experiment Station, University of Guam, Mangilao, Guam 96923, USA

Introduction on plants belonging to Asteraceae and Caryophyl- Within Curculionoidea, high numbers of gall induc- laceae (Anderson 1962; Kaplin 1981; Dieckmann ers exist only in Ceurthorhynchinae, Baridinae, and 1988). Currently only five species of gall-inducing Curculioninae (Korotyaev et al. 2005). Several spe- Baridinae are known, including Acythopeus bur- cies of Curculioninae are well-known gall inducers khartorum O’Brien, 1998 (Table 1). Gall-inducing

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Downloaded from Brill.com09/25/2021 12:54:19PM via free access Raman et al.: Biology of Acythopeus burkhartorum (Curculionidae) 183 species of Baridinae show no noteworthy affinity 2003). Gall-inducing Coleoptera are particularly either to particular plant families or to any related useful agents in weed management (Muniappan & clusters of plant families, a trait, which is evident in McFadyen 2005), mainly because these insects are the non-gall-inducing species of Baridinae (Pakaluk highly specific to their host plants (Raman 1996; 1994; Korotyaev et al. 2001; Marvaldi 2003). Raman et al. 2005), and they induce modifications While describing A. burkhartorum and A. cocciniae in their host plants – from fracturing of vascular O’Brien, 1998 from Kenya, O’Brien & Pakaluk strands and distorting nutrient transport (Raman (1998) implicated them to be potential biological- & Dhileepan 1999; Raman et al. 2006) to reduced control agents of Coccinia grandis (= C. indica Wight photosynthetic efficiency, stomatal conductance, & Arnold), a native plant of the tropical and sub- and water potential (Florentine et al. 2005). In such tropical Ethiopian region. C. grandis was introduced a context, A. burkhartorum demonstrates traits of a by human action into the Hawaiian Islands and in the reliable biological-control agent, because it is both recent past its populations have grown into problem a coleopteran and a gall inducer (Fig. 1). With proportions. In the last two decades, C. grandis has C. grandis turning into an invasive plant in the emerged as an invasive weed in the islands of Guam Hawaiian Islands, the Hawaii Department of Ag- and Saipan, where it is a problem plant both in man- riculture searched for potential natural enemies for aged gardens and natural areas (Pacific Island Eco- C. grandis. Acythopeus burkhartorum was one of the systems at Risk 2005). Although used extensively as three organisms obtained from eastern African re- a vegetable (e.g., Wasantvisut & Viriyapanich 2003) gion and field-released in Hawaii in 1999. and a medicinal plant (e.g., Patel & Srinivasan 1997) Adults of A. burkhartorum live up to 24 months in south-east and south Asian countries (e.g., India, feeding on the leaves of C. grandis. Eggs inserted Sri Lanka, Thailand), the Global Invasive Species into tender shoots (petioles or tendrils or stems) of Database (2005) lists C. grandis as a noxious weed. C. grandis hatch in 7–10 days. Five larval stages de- Coccinia grandis grows overwhelmingly in Hawaii, veloping in 25±1 days have been reported in Hawaii Guam, and nearby islands, and competes intensely (Murai et al. 1998). Mid-portions of galls, in the with the native vegetation. It acts as a host for melon shape of barrels, adequate enough to enclose the pu- fly (Bactrocera cucurbitae Coquillett, 1899; Diptera: pae, sever from their respective plant axes and drop Tephritidae) and as a reservoir for several other insect to the soil. Pupation occurs within the ‘dry’ severed pests and microbial pathogens (e.g., ring-spot virus) gall portions, which act as pupal cases. Adults emerge of cucurbitaceous crops. in 25–30 days (Murai et al. 1998; personal observa- Among diverse plant-feeding insects that are being tions R. Muniappan, G.V.P. Reddy, Z. Cruz). explored currently for use in weed biological-con- Because only limited information is currently trol campaigns, Coleoptera are gaining relevance, available on gall-inducing baridine weevils and especially because during their developmental stages A. burkhartorum is considered a potential candidate they tunnel through axial organs of host plants, and for the biological control of C. grandis in the Pacific their mandibulate larvae chew and consume large Islands (Murai et al. 1998), in the present paper quantity of tissues of their herbaceous host plants we provide details of the biology of A. burkharto- besides fracturing vascular strands (May 1984; Paka- rum in the context of its gall-inducing behaviour. luk 1994; Bailey et al. 2002; Korotyaev & Gültekin This paper also includes details of gall growth and a

Figs 1–8. Acythopeus burkhartorum on Coccinia grandis. – 1, Swollen stem (mature gall) inhabited by fourth stage larva (Bar=1 cm); 2, egg (oblong outline with a distinct dark-brown solidified secretion at the proximal end; nu- cleus within the egg visible clearly below) deposited on petiole base (longitudinal-sectional view) (Bar=100 μm); 3, mature ‘paired’ galls induced both on basal tendril and adjacent petiole; vertically slit to show larval chambers (Bar = 1 cm); 4, downward descent path (arrow) of a neonate larva. [note that the exposed gall is that of a second- stage larva]; LC – larval chamber (Bar=1 mm); 5, gall of a first-stage larva on the petiole (Bar=1 cm); 6, nutritive tissue of first-stage larva (cross sectional view); LC – larval chamber; arrows – damaged callus cells; intact callus cells bordering the larval chamber include dense cytoplasm and prominent nuclei (Bar=50 μm); 7, nutritive tissue of second-stage larva (cross-sectional view) with radial files of regenerating pith parenchyma cells; LC – larval chamber; arrow – callus cells damaged by larval feeding; cells away from the larval chamber include inclusions testing posi- tively for starch (Bar=100 μm); 8, gall (cross-sectional view) of late third/early fourth larval stage showing regenerated parenchyma filling the damaged pith; arrows – polyphenolic substances (tested with diazotized-sulphanilic acid reaction) occluding the parenchyma cells; ph – phloem showing dilated elements; scl – inner cortical sclerenchyma; chl – outer cortical chlorenchyma (Bar=100 μm).

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Table 1. Gall-inducing Baridinae, host plants, and distribution.

Weevil Plant organ Host Host family Distribution Reference bearing the gall

Acythopeus burkhartorum Stem Coccinia grandis Cucurbitaceae Kenya O’Brien & O’Brien, 1998 (Linnaeus) Voigt Pakaluk 1998 Baris cordiae Marshall, Petiole Cordia obliqua Boraginaceae Peninsular Krishnamurthy et al. 1923 Willdenov India 1977; Mani 2000 Hollisiella crabro Stem Fissistigma Annonaceae Vietnam Korotyaev et al. (Faust, 1888) polyanthum 2005 (Hooker, f. & Thomson.) Merrill Thanius biennis Prena Stem Psychotria marginata Rubiaceae Costa Rica Prena & & Nishida, 2005 Swartz Nishida 2005 Ulobaris loricata Root Species Chenopodiaceae Middle and Korotyaev et (Boheman, 1836) indeterminate Central Asia al. 2005

host-specificity test of A. burkhartorum conducted vations. A hand lens (20× magnification) was used against Zehneria guamensis (Merry.) F. R. Forsberg to locate oviposition scars and egg locations, which (Cucurbitaceae), an endemic plant in Guam and were marked with a soft-tipped, permanent marker nearby islands, and that of the introduction and pen. Sequence of gall development was followed, us- establishment of A. burkhartorum in Guam and ing increase in stem girth as an indicator (i.e., gall Saipan. growth), which was further supplemented by pen markings of egg locations. Age of the gall was de- termined by the inhabiting stage of the immature Materials and methods stage (larval stages 1–5, prepupa, pupa), which was noted by slitting the gall in the vertical median axis Plant and insect material with a sharp razor blade, taking care not to dam- Several shoot cuttings of C. grandis obtained from age the inhabitant. Several galls were slit to observe the natural areas that have been infested by C. grandis larval behaviour and gall development: stem tissue in the island of Guam (13°15'–13°30'N; 144°40'– with oviposition scars: n=9; galls housing different 144°55'E) were propagated in plastic pots (45 cm developmental stages: n=39 (1st larval stage – 4, 2nd diameter) with commercial grade garden-potting larval stage – 10, 3rd larval stage – 14, 4th larval stage mix. These pots were maintained in the greenhouse – 7, 5th larval stage – 2, prepupal stage – 2), and of the Agricultural Experiment Station (AES) at the galls including the pupal stage: n=23. Morphometric University of Guam (UoG) (Mangilao, Guam) in data of the dissected galls and the inhabiting larval controlled environmental conditions (L:D – 12:12, stages were obtained using a calibrated ocular scale mean day temperature 32°C, mean night tempera- fitted in the stereo-binocular dissection microscope ture 17°C, and relative humidity 65–80%). Fifty (Leica™ Zoom 2000, Leica, Wetzlar, Germany) and vigorously regenerating plants were quarantined to by measuring at the widest point. Mean and stand- insect-proof cages (65×60×60 cm) and maintained ard deviation were calculated using summarized con- in the greenhouse with the environmental condi- tent of variates in Genstat® for Windows® (2005). tions described earlier. Morphometric parameters (length and width) of galls (in different growth stages) and inhabiting Gall induction larval stages were analyzed with simple-linear regres- Fifty adults (equal number of males and females) of sion with Genstat® for Windows® (2005); length A. burkhartorum reared on C. grandis in the quar- and width parameters of the larvae were compared antine laboratory at the AES were released on the as explanatory variates of length and width of galls, regenerated shoot cuttings of C. grandis at different respectively. growth stages and maintained in the insect-proof cages in January 2005. Microscopy Whenever mating pairs were observed, the plant Galls of C. grandis with oviposition scars and those that harboured them was separated for closer obser- housing different stages of A. burkhartorum were

Downloaded from Brill.com09/25/2021 12:54:19PM via free access Raman et al.: Biology of Acythopeus burkhartorum (Curculionidae) 185 fixed in formal-acetic alcohol (FAA: 70% ethanol to ca. 100 cm height bearing multiple branches and – 90 ml, 40% formalin – 5 ml, glacial acetic acid were used in tests by maintaining them in the in- – 5 ml) for 72 h; each developmental stage of galls sect-proof cages. ‘Choice’ experiments, including (oviposition scars, galls housing different immature growing plants of both C. grandis and Z. guamensis stages) was stored in separate glass vials in 70% etha- in the same cage, and ‘no choice’ experiments, in- nol. FAA-fixed materials were cut at 5–7 μm in a cluding growing plants of either C. grandis or Z. gua- rotary microtome (Leica™ 2245 [semi-motorized], mensis in one cage, were conducted by releasing the Leica, Wetzlar, Germany). Most of the sections ob- 20 mixed-sex pairs of adult weevils into each cage. tained were contrasted with 1% toluidine blue (dis- All plants were adequately and regularly watered and solved in 1% aqueous borax solution) for use in maintained. Observations were made every day on bright-field microscopy; a few, randomly chosen sec- the number of feeding holes cut by adult weevils on tions of galls of known age, were prepared unstained leaves and galls induced on either petioles or tendrils for phase-contrast microscopy. All the stained (and or stems. contrasted in weak acid-alcohol solution) and the unstained sections were mounted in 5% aqueous Field release glycerine on glass slides. From fresh galls, hand-cut After conducting host specificity tests, an environ- sections were obtained for histochemical tests for mental-assessment statement was prepared and starch (Periodic acid – Schiff’s reagent test; Feder & submitted to Animal and Plant Health Inspection O’Brien 1968), lipids (Sudan black B reaction; Go- Service (APHIS) of US Department of Agriculture mori 1952), and polyphenols (diazotized-sulphanilic (USDA 2004). After securing permits from APHIS acid reaction; Gahan 1984) All bright-field and to field release this weevil in Guam and Saipan, adult phase-contrast microscopic observations and photo- weevils were released on C. grandis vines in selected graphs were made in a Nikon™ Elipse 80 – i micro- areas on Guam and Saipan. scope fitted with a digital DS-L1 camera control unit (Nikon Corporation, Tokyo, Japan). Samples of vertically slit galls with the larvae were photographed in Results a stereo-binocular microscope (Leica™ Zoom 2000, Leica, Wetzlar, Germany). Oviposition Adult females oviposit frequently in either tender Host-specificity test petioles or tendrils and less frequently in tender Adults of A. burkhartorum obtained from the Hawaii stems. The frequency of oviposition into petioles is Department of Agriculture were reared on potted usually higher than into tendrils and stems. In a sam- C. grandis plants maintained in the insect-proof ple of mature galls (n=98), the ratio of galls on peti- cages in the quarantine laboratory of AES follow- oles, tendril bases, and young stems was 3:1:1. Fe- ing the same procedures described under ‘Plant and males dig pits with their rostrum and deposit one egg insect materials’. Because Murai et al. (1998) have in each pit. After oviposition, the female covers the tested the host range of A. burkhartorum using differ- egg (400×220 μm) with a ‘secretion’, which solidifies ent plant species belonging to Violales (Caricaceae, upon exposure to air and turns brownish-black; no Cucurbitaceae, Flacourtiaceae, Passifloraceae, Viol- noteworthy change, either subcellular or growth, oc- aceae, and Turneraceae), following centrifugal phy- curs in the cells surrounding the egg (Fig. 2). Usually logenetic method (Wapshere 1974), we tested the one egg occurs at one node, which later results in a specificity of A. burkhartorum using its established host single gall at that node. However, paired galls at the C. grandis and an endemic species of Cucurbita- same node arising from two eggs deposited in the ceae in Guam and Mariana Islands, viz., Zehneria petiole and tendril have also been observed (three in guamensis, following ‘choice’ and ‘no choice’ test a population of 98) (Fig. 3). modes. We focussed on testing the susceptibility of Z. guamensis, mainly because of the concern on Gall growth and larval activity the possibility of A. burkhartorum radiating on to From the peripheral region where oviposition oc- Z. guamensis in Guam and neighbouring islands in curred, neonate larvae move into the soft central 2002. core of the plant organ; in a few instances, neonate Individuals of Z. guamensis were raised in the green- larvae move upwards especially when the eggs had house from seeds (propagation methods similar to been deposited at the bases of petioles or tendrils. the methods followed for raising shoot cuttings of The larvae move by chewing and feeding on the C. grandis) obtained from the limestone forests in parenchymatous cells (Fig. 4). In three to four days Guam. Both C. grandis and Z. guamensis were raised after the emergence of the first larval stage, the

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Downloaded from Brill.com09/25/2021 12:54:19PM via free access Raman et al.: Biology of Acythopeus burkhartorum (Curculionidae) 187 host-plant organ housing the larva, which could val chamber, induced dryness, shredding of the scaf- be either petiole or tendril or stem, shows a mod- folding hard tissue (e.g., sclerenchyma) induce sever- est swelling as the visible expression of the gall ing at both ends of this ‘portion’ of the gall (viz., the (Fig. 5). Larval feeding on the pith parenchyma larval chamber) into a barrel-shaped pupal case with cells, coupled with its tunnelling behaviour (either its ends closed with shredded fibres (Fig. 15, 16). upwards or downwards in the plant organ) induces The severed pupal case drops to soil and the adult the parenchyma cells to regenerate callus cells es- emerges from it after 25–30 days. pecially along the perimeter of the larval chamber (Fig. 6). The inhabiting larva derives its nutrition Measured parameters such as the gall length and from these callus cells and the supplementing ra- width, and larval length and width show a gradual dial files of parenchyma cells, which include dense and statistically significant increase during gall de- cytoplasm and prominent nuclei. Histochemical velopment (ANOVA: p<0.001 in all instances) localization of starch and lipids indicated that the (Fig. 17). Maximum gall growth (both in terms of regenerated parenchymatous callus cells lining the length and width) occurs during the development larval chamber include lipidic inclusions, whereas of the first and second larval stages. Gall length, in parenchyma cells away from the larva include starch particular, shows a drop, especially during the in- (Fig. 7). With maturation of the larva and intense feed- habitation time of the fifth larval stage, whereas gall ing, the gall grows in length and in width, essentially by width values remain at 4±1 mm throughout the gall increment in the radially arranged pith-parenchyma growth period, including the inhabitation time of cells. When the larva is in its late third larval stage of the fifth larval stage. A comparison of gall length vs. development, feeding extends up to the outer cortex larval length presents a significant positive relation- of the host organ. In doing so, the larva consumes ship (p<0.001) (Fig. 18), whereas that of gall width vascular tissues as well; however, regenerative callus vs. larval width presents a less significant, negative parenchyma fills the bulk of the gall. As the larva ma- relationship (p<0.015) (Fig. 19). tures to the fourth larval stage, gall cells accumulate polyphenolic materials in the callus parenchyma cells Host-specificity test (Fig. 8). The number of days lapsed from the time of release As the larva turns into fifth stage, several notable of weevils into cages, the time taken by them to com- changes occur in the gall rapidly and abruptly. The mence feeding, and the time that lapsed before galls fifth-stage larva deposits frass at both extremities of became visible on the tested plants (C. grandis and the larval chamber (Fig. 9). Deposition of frass (prob- Z. guamensis) are provided in Table 2. No feeding ably because of its ‘toxic’ nature) at either end of the hole or gall development occurred on Z. guamensis larval chamber triggers localized degeneration of gall either in the ‘choice’ or in the ‘no choice’ tests. tissue at those points (Fig. 10). Because the vascular strands are fractured by larval feeding, the gall dries. Field release At this stage, the inhabiting mature larva shreds the A total of 321 adults of A. burkhartorum was field cortical sclerenchyma using its mandibles and thus released in various locations in Guam (Ordat Pump prepares a part of the gall as a ‘pupal case’ (Figs. 11, Station, Public Health, Tai area, Cross Island Road 12, 13). Matching with the dimensions of the larval next to Baza Gardens, and UoG house #35) on chamber (10–15 mm in length), the prepupa fills the 8 October 2004. Further 202 adults of A. burkhar- ends of the larval chamber (which subsequently be- torum were field released at different sites in Saipan comes the pupal case) with shredded sclerenchyma (Kagman, Ben Cepada’s Ranch and near Capital fibres (Fig. 14). Degenerating extremities of the lar- Hill) on 9 February 2005.

Figs 9–16. Acythopeus burkhartorum on Coccinia grandis – 9, gall (cross-sectional view) of fifth larval stage showing deposition of frass (f) at the one of the larval chamber (LC) terminals (Bar=100 μm); 10, gall of the fifth larval stage with a ring of degenerating tissue (arrows) preparing the ‘pupal case’ to be severed from the remainder of the gall (Bar=1 mm); 11, gall of the fifth larval stage (opened to expose the larva) showing the larva shredding the dry inner cortical sclerenchyma and outer cortical chlorenchyma (Bar=1 mm); 12, shredded tissue including sclerenchyma fibres, chlorenchyma cells, and parenchyma cells with polyphenolic (dark-brown) inclusions (Bar=100 μm); 13, ‘pupal case’ showing the ‘thin’ outer rim of fibrous and chlorenchymatous cortex. * – a portion of larval head. (Bar=100 μm); 14, pupal case severed from the gall (Bar=1 cm); 15, pupal case (slit with a scalpel) showing the pupa within (Bar=1 cm); 16, end of the pupal case showing the ‘stuffing’ of shredded dry tissue (Bar=0.5 cm);

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35 Larva length Gall length Larva width Gall width 30

25 ) m m (

20 t g m n e m l

l l

15 a G

0 Larva 1 Larva 2 Larva 3 Larva 4 Larva 5 Larval length (mm) 25 days

Fig. 17. Growth dynamics of A. burkhartorum galls Fig. 18. Fitted and observed simple linear relationships with respect to larval stages. between gall length and larval length parameters.

Discussion

Host relations of gall-inducing Baridinae Although the Baridinae are a large subfamily of the

Curculionidae, little is known about host associations )

and life histories of both gall-inducing and non-gall- m m (

inducing species of Baridinae. Available information h t d does not indicate any definite pattern of host-plant i w

l l

use by gall-inducing species on either related plant a families or related plant genera (Prena 2001, 2003). G The known species of gall-inducing Baridinae (Ta- ble 1) feed on diverse and unrelated host plants belonging to large clades of Angiosperms (sensu Angiosperm Phylogeny Group 2003): Acythopeus burkhartorum feeds only on some Cucurbitaceae, belonging to ‘Rosids: Eurosids I’ [Core Eudicots]; Larval width (mm) Hollisiella crabro feeds on one species of Annonaceae, belonging to the basal angiosperm clade Magnoliids; Fig. 19. Fitted and observed simple linear relationships between gall width and larval width parameters. Baris cordiae and Thanius biennis feed on species of Boraginaceae and Rubiaceae, belonging to ‘Asterids: Euasterids I’ [Core Eudicots]; Ulobaris loricata feeds on one species of Chenopodiaceae, belonging to fidelity to their host plants (Raman 1996, Abra- Caryophyllales [Core Eudicots]. Nonetheless, what hamson et al. 1998, Raman et al. 2005). In such a is consistent at least among B. cordiae, T. biennis, and context, the role of A. burkhartorum in biological- A. burkhartorum is that they prefer either petioles control campaigns against C. grandis gains in rel- (that are similar to stems in primary structure) or evance. stems, behave identically by tunnelling through soft tissues within the host organs, and induce galls. Al- Unique characteristics of A. burkhartorum though non-gall-inducing species of baridine weevils The interaction between growth stages of A. burkhar- feed on more than one host plant (Korotyaev et al. torum and C. grandis exhibits high levels of synchro- 2001), the known gall-inducing species are specific nization. By depositing one egg per node on tender to their respective hosts showing a strong level of shoots, gravid females of A. burkhartorum display

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Table 2. Host specificity tests for Acythopeus burkhartorum on Coccinia grandis and Zehneria guamensis.

‘Choice’ test ‘No choice’ test C. grandis Z. guamensis C. grandis Z. guamensis

Adult feeding holes/leaf 63.8±10.4 0.0 32.5±13.1 0.0 Days taken to initiate galls 13 0.0 13 0.0 Days lapsed to start feeding 1 No feeding 1 No feeding

resource-partitioning behaviour by either minimizing Although the biology and behaviour of A. burkharto- intra-specific competition within larval populations rum up to the pupal stage are similar to other weevils or eliminating it completely. Oviposition behaviour (e.g., see Gültekin 2004 for several citations), the of A. burkhartorum is different, when compared with ability of A. burkhartorum to ‘create’ a pupal case by other gall-inducing weevils, such as Conotrachelus al- severing a portion of the gall makes it exceptional, bocinereus Fiedler, 1940 (Curculionidae: Curculioni- since such an ability is unknown in any other Curcu- nae) living on Parthenium hysterophorus (Asteraceae) lionidae. However, the ability of Cleonidius poricollis (Florentine et al. 2002) and Ceurthorhynchus napi Mannerheim, 1843 (Curculionidae: Cleoninae) to Gyllenhal, 1837 (Curculionidae: Ceurthorhynchi- construct ‘sand tubes’ with root tissues of its host nae) living on Brassica napus var. oleifera Linneaus plant (Descurainia pinnata (Walter) Britton [Brassi- (Brassicaceae) (Le Papé & Bronner 1987), although caceae]) and particles of sand (O’Brien and Mar- all the three compared weevils deposit only one egg shall 1987) needs to be viewed in conjunction here. at each oviposition site. Conotrachelus albocinereus A. burkhartorum’s behaviour stands out in being and C. napi, while ovipositing on their respective able to utilize C. grandis for nutrition, and especially host plants, push eggs deep into mature stem tis- during late larval stages, when it is able to shred the sue, whereas, A. burkhartorum oviposits by sinking drying plant tissues to ‘prepare’ the pupal case, sever eggs superficially into tender shoot (e.g., petioles, the barrel-shaped pupal case from the gall, and use tendrils, stems). By inserting eggs deep into mature shredded sclerenchyma fibres to close the ends of the stems, C. albocinereus and C. napi minimize move- pupal case. ment for the neonate larvae within the cortical tissue of their respective host plants, because the egg is A. burkhartorum in the biological control of enclosed in a proteinaceous sheath and the egg and C. grandis the secretion together induce gall growth (Le Papé Gall-inducing Coleoptera and Lepidoptera are fa- & Bronner 1987, Florentine et al. 2002). On the voured presently in weed management campaigns, contrary, the egg of A. burkhartorum is not enclosed because these arthropods either are monophagous or in a proteinaceous sheath and the egg, per se, does have a narrow host range (Muniappan & McFadyen not trigger any gall growth. The neonate larvae of 2005). Their capability to re-canalize the host-plant’s A. burkhartorum gnaw their way into the soft cor- developmental processes and metabolic activity, to tical parenchyma until they reach the point where place their host plants in stress by draining nutrients they settle eventually and the gnawing initiates gall and other key metabolic products to the inducing growth in C. grandis. We speculate that the mode larva, and to induce anoxic conditions within the of insertion of eggs – either superficially or deeply – host plant render them as candidates of choice in depends on the softness of tender shoots or hardness weed-biological control programmes (Florentine of mature shoots of the chosen host-plant site and in et al. 2001, 2002, 2005, Raman et al., 2006). The high probability this behaviour is an adaptive strat- usefulness of A. burkhartorum in the management of egy of the species concerned. During oviposition, C. grandis is simple, yet elegant; it demonstrates its C. albocinereus covers the sites of egg deposition with usefulness in the management of C. grandis by sever- chewed plant materials, whereas, A. burkhartorum ing parts tender stems, tendrils, and petioles (viz., by and C. napi (Le Papé & Bronner 1987) cover egg creating the pupal case). sites with a ‘secretion’, which solidifies upon expo- A key criterion used in the choice of biological- sure to air. Covering of the egg site with a solidifying control agents for weeds is that the agents will not secretion is currently known also in two non-gall- become pest organisms subsequent to introduction inducing species of Baridinae (Korotyaev & Gültekin in new areas (Littlefield & Buckingham 2004). In 2003) and the possible reason for such behaviour is assessing the broad-spectrum host specificity of to protect the egg. A. burkhartorum, Murai et al. (1998) have tested

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several species of plants; in our ‘choice’ and ‘no- Feder, N. & T.P. O’Brien, 1968. Plant microtechnique: choice’ host-specificity test, we used one endemic some principles and new methods. – American Journal species of Cucurbitaceae (Z. guamensis), which has of Botany 55: 123–142. yielded a categorical response that A. burkhartorum Florentine, S.K., A. Raman & K. Dhileepan, 2001. Gall- is specific to C. grandis. In the context of such a de- inducing insects and biological control of Parthenium finitive outcome, an environmental assessment was hysterophorus L. (Asteraceae). – Plant Protection Quar- prepared, which was approved by APHIS, USDA; terly 16: 1–7. consequently field-release of A. burkhartorum on the Florentine, S.K., A. Raman & K. Dhileepan, 2002. islands of Guam and Saipan was authorized. Data Responses of the weed Parthenium hysterophorus pertaining to establishment in the field at the release (Asteraceae) to the stem gall-inducing weevil Conotra- sites are being accrued; predation of the pupae of chelus albocinereus (Coleoptera: Curculionidae). – En- A. burkhartorum – in spite of being confined to tomologia Generalis 26: 195–206. the ‘pupal case’ – by ants appears to be a hurdle in Florentine, S.K., A. Raman & K. Dhileepan, 2005. Effects of gall induction by Epiblema strenuana on gas the successful establishment of A. burkhartorum in exchange, nutrients, and energetics in Parthenium hys- Guam and Saipan. Monitoring of horizontal spread terophorus. – BioControl 50: 787–801. from the released sites is ongoing. Gahan, P.B., 1984. Plant histochemistry and cytochem- istry – An introduction. Academic Press, New York. 301 pp. Acknowledgements GenStat® for Windows®, 2005. GenStat Release 8.1. 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