ARTHROPOD BIOLOGY Symbiotic Relationship Between Hypothenemus hampei (Coleoptera: Scolytidae) and Fusarium solani (Moniliales: Tuberculariaceae) JUAN A. MORALES-RAMOS,1 M. GUADALUPE ROJAS,1 HELGA SITTERTZ-BHATKAR,2 3 AND GUADALUPE SALDAN˜ A Ann. Entomol. Soc. Am. 93(3): 541Ð547 (2000) ABSTRACT We found evidence of a symbiotic relationship between Fusarium solani (Martius) and Hypothenemus hampei (Ferrari). Females of H. hampei colonizing coffee beans infested with F. solani produced signiÞcantly more progeny than those colonizing sterile beans. Beans infested by F. solani had signiÞcant amounts of ergosterol, which was not present in sterile beans. Fecundity and survival of H. hampei was positively correlated with the content of ergosterol in their diet. Other evidence of symbiosis includes the presence of F. solani spores caught in cuticular protuberances, known as asperites, on the pronotal area of female H. hampei. The asperites are ßat and arranged in a radial pattern presenting a pronounced inclination directed to the center of this radial pattern which originates at a protuberance on the pronotal area. The asperites appear to increase the chances of fungal deposition on the pronotum when female beetles bore into infested coffee beans. The existence of a mutualistic association between H. hampei and F. solani and the role of the pronotal structures as primitive mycangia are discussed. KEY WORDS Hypothenemus hampei, mycangia, symbiosis, coffee berry borer, ambrosia, ergosterol THE COFFEE BERRY borer, Hypothenemus hampei (Fer- the signiÞcance of this association to the biology of H. rari) originated in Angola, from where it spread to the hampei has not been established. These are the only 2 rest of Africa by the late 1920s (Corbett 1933). It was reports of species of Cryphalini associated with am- introduced into the Brazilian State of Sao Pablo in 1924 brosia fungi. (Corbett 1933) from where it spread to all South More than half of the species of Scolytidae are American coffee producing countries, reaching Gua- xylomycetophagous (feeding on fungi inside tunnels temala in 1971 and Mexico in 1978 (Baker 1984). H. excavated into sound wood), including members of hampei is able to colonize and reproduce in 12 differ- the tribes Xyloterini, Xyleborini, Hylesinini, Scolytini, ent species of the genus Coffea (Johanneson and Scolytoplatypodini, and Corthylini (Wood 1982). Mansingh 1984). Beetles included within this category are known as Hypothenemus hampei, a member of the Scolytidae, ambrosia beetles (Batra 1963, Francke-Grossman belongs to the tribe Cryphalini. The members of this 1963). A mutualistic relationship exists between the tribe have been described as phloephagous (feeding ambrosia beetles and some species of fungi (ambrosia on phloem tissues of inner bark) or myelophagous fungi) (Batra 1966, 1972) based on a nutritional de- (feeding on the pit of small stems) (Wood 1982). pendence of the beetles on the ambrosia fungi (Kok Earlier reports on the biology of the genus Hypothen- 1979). Most ambrosia beetles, speciÞcally those from emus described these beetles as myelophagous, some- the tribe Xyleborini, are unable to complete develop- times attacking fruits and seeds (Wood 1982), but no ment and reproduce in the absence of their symbiotic fungal associations were reported. Beaver (1986) re- fungi (Norris 1965, 1979; Norris and Baker 1967; Barras ported a symbiotic association between Hypothenemus 1973; French and Roeper 1975). curtipennis (Schedl) and an undetermined species of The best example is the symbiosis between Xyle- fungus based on the presence of mycangia in the borus ferrugineus (F.) and F. solani. The immature prothorax of this scolytid of Western Samoa. Recently, stages of X. ferrugineus are unable to complete devel- an association between H. hampei and Fusarium solani opment in the absence of F. solani (Norris and Baker (Martius) (Moniliales: Tuberculariaceae) has been 1967, Norris and Chu 1971, Kingsolver and Norris reported from populations of 2 different continents 1977). Ergosterol is essential for development and (America and Africa) (Rojas et al. 1999). However, reproduction of X. ferrugineus (Norris et al. 1969, Chu et al. 1970). This sterol is absent in the wood, but F. 1 Formosan Subterranean Termite Research Unit, USDAÐARS solani provides it in sufÞcient quantities (Kok et al. SRRC, New Orleans, LA 70124. 1970). This fungus also provides X. ferrugineus with 2 Electron Microscopy Center, Texas A&M University, College Station, TX 77843Ð2257. essential fatty acids and phospholipids (Kok 1979). 3 BeneÞcial Insects Research Unit, USDAÐARS SARC, Weslaco, TX Most ambrosia beetles possess structures special- 78596. ized for the transport of the symbiotic fungi called 542 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 93, no. 3 mycangia or mycetangia (Batra 1963, Giese 1965). coffee beans were treated previously by 2 methods. Mycangia vary in complexity and position on the in- Treatment 1 consisted of 12.7 g of coffee beans with 6 sect. The most complex mycangia (prothoracic pleural ml of water autoclaved at 120ЊC for 20 min. Treatment and promesonotal) are sac-like organs with oil pro- 2 consisted of 12.5 g of coffee beans in 6 ml of distilled ducing glands to maintain the viability of the fungi water and 13 mg of sorbic acid autoclaved as above. (Francke-Grosmann 1963). The simplest structures After cooling, 750 ␮l of a Þlter sterilized vitamin so- consist of groups of setae or intrusions in the prothorax lution was added to both treatments to replace the that captures fungal spores (Francke-Grosmann 1967, vitamins lost during autoclaving. The composition of Furniss et al. 1987). the vitamin solution, in parts per million, was as fol- Mycangia within the genus Hypothenemus have lows: choline chloride 40,000, inositol 3,000, calcium been reported in H. curtipennis (Schedl) and this is the penthotanate 400, niacinamide 1,000, thiamine HCl 80, only member of the tribe Cryphalini reported to be riboßavin 200, pyridoxal 37, folic acid 750, biotin 12, symbiotically associated with fungi (Beaver 1986). cobalamine 4, and ascorbic acid 2,400. The 100 groups The mycangia of this species appear as depressions in of 5 beetles were equally divided into 2 treatments in each side of the pronotum with numerous hairs car- which treatment 2 received coffee beans treated with rying fungal hyphae and spores (Beaver 1986). sorbic acid to eliminate F. solani growth and treatment The existence of an association between H. hampei 1 received beans without inhibitors. and F. solani (Rojas et al. 1999) suggests that a similar The original experimental plan was to analyze the symbiotic relationship may exist between these 2 or- results of only 2 treatment groups, making treatment ganisms. The objectives of this research were to es- 1 the control. The sorbic acid was expected to elim- tablish the importance of F. solani in the diet of H. inate F. solani growth in treatment 2. Fungus growth hampei and its biology, to search for the presence of was only partially inhibited in treatment 2 as observed potential mycangia on the external cuticula of H. ham- by the presence of fungal mycelia on the coffee beans. pei, and to determine if fungal spores were present Because it was essential to compare a treatment with within these structures. the absence of F. solani, the experiment was modiÞed to take advantage of some groups lacking F. solani growth in treatment 1 1wk after beetle introduction. Materials and Methods The washing procedure described above eliminated F. Origin of Biological Materials. Adult beetles from 2 solani from all 5 beetles in 45% of the groups treated. populations of H. hampei were introduced to the quar- This was determined by the presence or absence of antine facilities of the USDA-APHIS Mission Biolog- fungal mycelia on the coffee beans as observed with ical Control Center in Mission, TX. One of the pop- a stereo microscope (45ϫ) 1 wk after the beetles were ulations was isolated from infested coffee berries introduced to the sterile beans. Previous observations collected from coffee plantations located near Tapa- conÞrmed that fungal growth is easily observed on the chula, Chiapas, Mexico. The 2nd colony was intro- coffee beans 2 d after inoculation. Treatment 1 was duced from Allada, Benin, Africa. This colony was divided into 2 subtreatments, the groups in which F. isolated from infested coffee berries collected from solani was not eliminated were treated as treatment 1A coffee plantations (from the ground and trees) near (control) and the rest as treatment 1B. However, as a Allada, Benin. consequence of this modiÞcation in the original ex- Effect of the Fungus on the Fecundity of Founder perimental design, the treatments (fungus present or Females. A total of 100 groups of 5 (totaling 500) H. absent) were of unequal size. Treatment 2 was not hampei females (from Benin, Africa) that exhibited subdivided and it was included in the analysis in its migrating behavior was selected from the main colony. original form. This modiÞcation improved the exper- Such females can be recognized because they move imental expectations because it allowed concerns away from the culture media and tend to take ßight about the effect of sorbic acid on beetle fecundity to readily. Migrating females were selected because most be addressed. of them have mated (Baker 1984). Under a laminar Observations were taken 2 wk and 3 wk after the ßow hood, the beetles were washed in a solution of introduction of the beetles to the sterile coffee beans. 0.1% Comite (Uniroyal, Middlebury, CT) for 30 s and During the 1st set of observations, 25 petri dishes from rinsed in sterile distilled water to eliminate associated each of the 2 original treatments were selected ran- mites. This operation was repeated 3 times. Next, the domly. The dishes were opened to extract the infested beetles were washed in a solution of 0.25% Benlate WP coffee beans. Beans from treatment 1 that showed (wettable powder, DuPont, Wilmington, DE) in a visible fungal growth were recorded as treatment 1A 0.9% NaCl solution for 30 s and rinsed in sterile dis- (control) and the ones that did not show fungal tilled water to eliminate secondary fungi.