Unusual Behaviour — Unusual Morphology: Mutualistic Relationships between Ants (Hymenoptera: Formicidae) and Diaphorina enderleini (Hemiptera: Psylloidea), Associated with Vernonia amygdalina (Asteraceae)

ABSTRACT Diaphorina enderleini, known from Sudan, Tanzania and Yemen, is reported for the first time from Cameroon, Ethiopia and Kenya. Observations and records from Cameroon and Ethiopia show that it develops on Vernonia amygdalina. The species is diagnosed and the last instar larva is described and illustrated. The larvae lack a functional circumanal ring and do not produce wax, a feature unusual for psyllids. The lack of wax deprives them of the protection common in various free-living psyllids. Consequently the psyllid is involved in an unusual mutualistic interaction with the ants Pheidole megacephala and Crematogaster striatula. This association is described and discussed.


INTRODUCTION
Most Sternorrhyncha and Fulgoromorpha are phloem feeders. Phloem sap provides a highly unbalanced diet, resulting in the excretion of large amounts of honeydew. This carbohydrate-rich liquid provides a valuable food resource for various insects such as ants (Way 1963;Gullan 2001). Ants actively collect honeydew and often live in close for both partners (Buckley 1987;Delabie 2001;Davidson et al. 2003). The ants obtain high quality food and the hemipterans receive protection from predators and parasitoids (Novak 1994). Ant attendance is well known from aphids (Kaplan & Eubanks 2005;Idechiil et al. 2007;Grover et al. 2008;Nielsen et al. 2010) and scale insects (Heckroth et al. 1998;Johnson et al. 2001;Ueda et al. 2008;Ben-Dov 2010) but seems less relevant in psyllids (Novak 1994). Based on observations conducted in Cameroon, we report ant attendance of the widely distributed Afrotropical psyllid Diaphorina enderleini Klimaszewski, 1964, developing on Vernonia species.
Psyllids or jumping plant-lice are one of the four superfamilies of Sternorrhyncha comprising to date some 3850 described species worldwide. The group is particularly species-rich in the tropics. Psyllids are generally monophagous or narrowly oligophagous with respect to their angiosperm hosts (Hodkinson 1974). Often, related psyllid species develop on related host-plant taxa. A major exception to this high host conservatism is the large Old World genus Diaphorina Löw, 1880, which recruits hosts from at least 19 families in 10 plant orders of dicotyledonous angiosperms (Hollis 1987;Burckhardt & van Harten 2006).
Diaphorina enderleini was originally described by Enderlein (1910), as Gonanoplicus guttulatus, based on material collected from Tanzania. Klimaszewski (1964) transferred G. guttulatus to Diaphorina and replaced the name for reasons of homonymy with D. enderleini. Loginova (1978) described Diaphorina siluncula from Sudan. Burckhardt D. siluncula with D. enderleini, provided a detailed morphological description of the adults and reported the species from Yemen. The psyllid is currently known from Tanzania, Sudan and Yemen in the Afrotropical Region (Burckhardt & van Harten 2006). So far nothing has been known about the larvae and host plants of D. enderleini apart from the very brief and not diagnostic description by Enderlein (1910).
Vernonia amygdalina Delile (Asteraceae) is a perennial shrub native to tropical Africa and the southern tip of the Arabian Peninsula. It is a multipurpose plant used as a medicinal plant throughout its distribution range (CVPSL 2003;Banjo et al. 2006;Nzigidahera 2008), or as a vegetable in West Africa (Anonymous 2005;Asawalam & Hassanali 2006). V. amygdalina naturally colonises secondary forests and cultivated land. For this reason it can be regarded as a non-timber forest product (NTFP). It is also planted in gardens and as fences around houses. For cultivation it is generally propagated by planting shoots (Shippers 2000), and thus can be regarded as a crop. Some studies highlight its insecticidal or insect repellent properties (Asawalam & Hassanali 2006;Kabeh & Jalingo 2007). These properties, if proved, may be due to the presence of lactone, oxalic acid and hydrocyanic acid (HCN) found in its leaf dust (Kabeh & Jalingo 2007). Other studies have tested its fodder potentials (Bonsi et al. 1995;El Hassan et al. 2000;Mekonnen et al. 2009;Tabuti & Lye 2009). In Cameroon V. amygdalina is known as Ndolè, an essential ingredient of the popular meal offered by all local restaurants. production in urban and periurban areas (Anonymous 2005). It is even sold outside Cameroon, as in Nigeria, Equatorial Guinea or Gabon, and sometimes in Europe for niche markets (e.g. Africans in European countries) (Bosch et al. 2009).
Several insects, including Hemiptera (Sternorrhyncha, Cicadomoprpha, Fulgoromorpha and Heteroptera), Coleoptera (Coccinellidae and Cucurlionidae), Lepidoptera and Orthoptera, are reported feeding on leaves of this important crop (Banjo et al. 2006;Alene et al. unpubl. data) and D. enderleini constitutes a potential pest. The present paper aims and to describe the relationships between the psyllids and four associated ant species.

MATERIAL AND METHODS
Material of D. enderleini from Cameroon collected on V. amygdalina was examined from the localities listed in Table 1. The specimens are preserved dry, slide-mounted, or stored in 70 % ethanol, and are deposited in the following institutions: Laboratory of Zoology, University of Yaounde 1, Cameroon (LZUY1); Muséum d'histoire naturelle, Genève, Switzerland (MHNG); Naturhistorisches Museum Basel, Switzerland (NHMB). Additional material examined comes from Ethiopia (Vernonia amygdalina, NHMB), as well as Kenya and Yemen (both without host data, MHNG).
The drawings and measurements were made from slide-mounted specimens. The morphological terminology follows Hollis (1976Hollis ( , 1984 and Ossiannilsson (1992

Description:
Adult the concave dorsal margin of the female proctiger. Fifth instar larva (Figs 1-5). Colouration. Sclerites dark brown; membrane beige. Eye reddish pink. Antenna yellow basally, dark brown in apical quarter. Wing pads with dark brown pattern. Morphology. Body almost as long as wide (Fig. 1). Antenna (Fig. 2) short, 3-segmented, 1.5× longer than the other. Dorsal sclerites large and forewing pads well sclerotised; dorsal and ventral sclerites covered in short setae. Forewing pad large and massive, with well-developed humeral lobe, outer margin (Fig. 3) bearing short slender lanceolate setae, posterior apex angled; dorsal surface covered in very short setae. Hindwing pad broad, apex almost rounded, margin and dorsal surface as in forewing pad. Tarsal arolium (Fig.  4) almost twice as long as claws, widened apically with a very short petiole. Caudal plate (Fig. 5) about 1.5× wider than long, rounded posteriorly, weakly indented at apex. Abdominal margin with slender lanceolate setae slightly longer than those on forewing pad margin, setae becoming longer towards apex. Abdominal venter sparsely covered in fairly long setae. Anus terminal, circumanal ring vestigial. Measurements (in mm) and ratios (11 specimens  Among these ants, a very unusual behaviour of P. megacephala and C. striatula was observed. While female psyllids are laying eggs on the lower leaf surface, workers of P. megacephala quickly pile up detritus combined with soil around and partly above the psyllids, resulting in a shelter-like structure (Figs 6, 7). Those of C. striatula build carton shelters (Fig. 8). During this procedure the adult female psyllids hardly move away from their eggs and larvae. The psyllid larvae are arranged in tight rows along the leaf veins, especially the principal one, and sometimes at the base of very young branches. The larvae are mostly sedentary and only move, slowly and steadily, when disturbed. They do not produce wax, as can be expected from the almost complete lack of the circumanal ring. Females and larvae excrete large drops of honeydew, which are immediately recovered by ants.
In C. acvapimensis and M. opaciventris the workers did not build any structure around the psyllids but they were observed taking honeydew from them (Figs 9, 10).

DAMAGE TO THE HOST
Adults and larvae of Diaphorina enderleini feed by sucking the plant sap. Feeding by the larvae is especially injurious due to their large number and their immobility (they feed at the same spot for a long time). A noticeable symptom is the appearance of translucent aureoles on the leaves, apparently resulting from the toxicity of the saliva injected by the psyllids.

DISCUSSION AND CONCLUSION
The larvae of Diaphorina enderleini share a series of morphological characters with other Diaphorina spp., such as the three-segmented antenna with four rhinaria on segment 3, the presence of a humeral lobe on the forewing pad and the marginal lanceolate setae on the wing pads and caudal plate. However, while a circumanal ring of wax-producing pores is well-developed in the larvae of the other known species of Diaphorina (Burckhardt 1985(Burckhardt , 1986Burckhardt & Mifsud 1998;Burckhardt & van Harten 2006), it is almost completely lacking in D. enderleini, and is not functional. In fact, the larvae do not produce wax. This habit probably exposes them to environmental constraints and natural enemies.
Among psyllids, different strategies for the protection of larvae are realised. In several free-living psyllids, such as Mesohomotoma and Psylla spp., the larvae are completely covered in abundant wax produced by wax glands which are connected to the In Diclidoplebia xuani Messi, 1998, andDiclidoplebia harrisoni Osisanya, 1969, the larvae covered in wax induce leaf roll galls in which they live Aléné et al. 2008). Many larvae of the Australian Spondyliaspidini form lerps, i.e. protecting shields mostly composed of starch under which they live (Hollis 2004). The New World Euphalerus spp. associated with Lonchocarpus spp. live either in galls or under lerps (Hollis & Martin 1997), whereas many other taxa live in closed galls, such as the Afrotropical Phytolyma spp. on Moraceae (Hollis 1973), some Afrotropical Pseudopharcopteron spp. (Malenovský et al. 2007;Malenovský & Burckhardt 2009) or members of the South American Calophya rubra (Blanchard, 1852) group on Schinus spp. (Burckhardt & Basset 2000).
As the larvae of D. enderleini neither induce gall-like structures nor produce wax, they need an extrinsic protection which may be provided by the attendance of ants. The latter expect to be recompensed by honeydew excreted by the psyllids. Two ant species, P. megacephala and C. striatula, appear as good partners in this interaction since they build shelters to protect the psyllids from environmental constraints and natural enemies.
The shelter is probably also a means for the ants to protect the source of honeydew from competitors such as other hymenopterans and dipterans. Similar relationships are widely recorded in several groups of Sternorrhyncha and Auchenorrhyncha (Delabie 2001;Naskrecki & Nishid 2007;Verheggen et al. 2009) and a few groups of Heteroptera (Pfeiffer & Linsenmair 2000;Gibernau & Dejean 2001). There are very few records of this type of trophobiosis in psyllids (Novak 1994).