Evaluation of Beauveria bassiana (Ascomycota: Hypocreales) as a control of the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae) emerging from fallen,infested coffee berries on the ground

The effects of Beauveria bassiana strains on coffee berry borers (CBB), which emerge from infested berries left on soil, and its impact on the infestation of coffee berries on tree branches were evaluated at two Experimental Stations (Naranjal-Caldas and Paraguaicito-Quindio) in the Colombian coffee zone. Using a completely randomized design with 10 repetitions, 50 coffee berries artificially infested with CBB were placed on the base of a coffee tree. Four treatments including B. bassiana strain Bb9205, a mixture of Cenicafé strains (Bb9001, Bb9024 and Bb9119), a commercial formulation of B. bassiana and a control (water) were sprayed with 1×10⁹ conidia per tree. After 30 days, all fungal strains lowered the infestation levels of the coffee berries on the trees at both locations. The mixture of Cenicafé strains decreased the tree infestation between 50 and 30% at both locations. In the berries dissected from each treated tree, insect mortality was about 40% at both locations compared to 15% in the control. B. bassiana strains also decreased the insect population inside the newly infested berries on the trees by 55–75%. The mixture of Cenicafé strains was the most effective for decreasing insect populations. B. bassiana significantly decreased CBB populations that emerged from fallen, infested, coffee berries and reduced future insect generations.     

Evaluation of potential entomopathogenic fungus,Beauveria bassiana,for controlling the coffee berry borer Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) in Taiwan

Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae), a coffee berry borer (CBB), is a serious pest of coffee fruits and causes economic losses in the coffee industry. With the concerns of chemical control, an alternative eco-friendly management strategy such as microbial control has emerged as the times require. Herein, a total of thirty-three fungi were isolated from CBB cadavers. Two entomopathogenic fungal isolates, NCHU-271 and NCHU-272, which showed fast insect-killing activity, were subjected to molecular identification and named “Beauveria bassiana-NCHU-271” (Bb-NCHU-271) and “Beauveria bassiana -NCHU-272” (Bb-NCHU-272). Moreover, a virulence test of Bb-NCHU-271 and Bb-NCHU-272 against CBBs was also performed. Both Bb-NCHU-271 and Bb-NCHU-272 caused 100% mortality at 8 days post inoculation (d.p.i.) and a LT₅₀ of Bb-NCHU-271 by spraying 10⁸ conidia/ml was less than Bb-NCHU-272. Therefore, histological evidence of Bb-NCHU-271 infected CBBs was provided to proven the infection process of B. bassiana in CBBs. Furthermore, an infestation test was performed to evaluate the differences in CBB control efficacy between the treatments of “Borers exposed to EPF first” or “Berries exposed to EPF first”. The results revealed that the “Borers exposed to EPF first” of Bb-NCHU-272 caused significantly lower survival rates and higher mycosis rates than those of Bb-NCHU-271. Only slight control effects of Bb-NCHU-271 and Bb-NCHU-272 were also observed in the “Berries exposed to EPF” treatment. In conclusion, Bb-NCHU-271 and −272 showed controlling ability to the CBBs either before or after CBB infestation into coffee berries, while application of EPFs before the CBBs bored into coffee fruits could enhance the control effect.     

Influence of two formulation types and moisture levels on the storage stability and insecticidal activity of Beauveria bassiana

The formulation of mycopesticides may require a physical separation of conidia from the substrate and subsequent drying. In the present study, Beauveria bassiana conidia produced by solid-state fermentation were harvested either through a dry or washing protocol. Washed conidia were used to design a water-dispersible granule (WG) formulation, whereas sieved conidia were mixed with an emulsifiable oil to achieve an oil-based formulation (OD). Potential harmful effects caused by the formulation type on the storage stability and insecticidal activity against Hypothenemus hampei were assessed. As expected, the time for initial conidial germination to drop 50% (GT₅₀) in all treatments was deeply influenced by storage temperatures, which varied from over 180 days at 4 °C to less than 90 days at 35°C. In all four tested temperatures, GT₅₀s for unformulated dry conidia were significantly higher than for those formulated as WG, and the latter was similar to conidia formulated as OD in the two highest temperatures. Residual water content in the OD formulation (1,600 vs. 340?ppm) had a negative influence on conidial survival under storage, whereas WG granules immediately dried after the washing protocol showed conidial germination similar to granules exposed to a slower dehydration regime. Mortality of H. hampei adults exposed to different concentrations of B. bassiana formulated as WG was slightly lower (10–15%) than either the OD or the unformulated conidia. In brief, we have demonstrated that formulation type and their moisture level can affect the storage stability and insecticidal activity of B. bassiana conidia toward the coffee berry borer. Of particular importance, we have shown that drying oils prior to formulation could improve the storage of mycopesticides, an approach that may find industrial applications.     

Altitude and coffee production systems influence extent of infestation and bean damage by the coffee berry borer

The coffee berry borer (CBB), Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae), is one of the major insect pests of coffee worldwide. The present study was designed to assess the level of infestation of coffee berries at different developmental stages across different altitudes and coffee management systems. The experiment was carried out at three locations in southwestern Ethiopia under two coffee management systems and four coffee berry development stages with three replications. Results of the study showed significantly highest proportion of damaged berries (37.5%), number of holes per berry (10.88) and number of adult CBB per berry (7.55) on dried leftover berries at low-altitude study sites. On the other hand, the lowest mean percent damaged berries, number of holes per berry and number of adults were recorded at mid- and high-altitude study sites. The study also showed that, CBB caused significantly highest damage in plantation coffee management system than garden coffee. Results of this study highlight proper harvesting at red ripe stage in order to minimise incidence of CBB. It is also important to design integrated management strategies to mitigate CBB damage especially in lowland plantation coffee production systems.     

Abundance of Coffee berry borer in feral,abandoned and managed coffee on Hawaii Island

Coffee berry borer (Hypothenemus hampei Ferrari), the most damaging insect pest of coffee worldwide, was first detected on Hawaii Island in 2010. Poorly managed, abandoned and feral coffee sites on the island have since been thought to harbour coffee berry borer (CBB) populations, which then negatively impact neighbouring coffee farms. In the present study, we sought to quantify CBB abundance in these sites, which vary in management intensity and vegetation structure and diversity. We collected data on trap catch as a measure of CBB flight activity, fruit production and fruit infestation by CBB in eight well-managed farms and sites that were either poorly managed, abandoned or feral (wild) coffee. Sites were sampled bi-weekly over a period of 2 years from 2016 to 2017. We found that CBB flight activity was significantly higher in poorly managed sites relative to abandoned and feral sites, but was not significantly different from well-managed sites. Coffee production in well-managed farms was significantly higher than in abandoned and feral sites, but was not significantly different from poorly managed farms. CBB infestation in poorly managed sites was significantly higher than that observed in well-managed, abandoned and feral sites. We estimated an average load of 11–25 CBB per branch at poorly managed sites, compared to 3–9 per branch at well-managed sites, 1–16 per branch at abandoned sites and 1–3 per branch at feral sites. Our findings suggest that poorly managed sites should be prioritized for implementation of CBB control measures as part of a landscape-level integrated pest management (IPM) programme.     

Where to sample? Ecological implications of sampling strata in determining abundance and impact of natural enemies of the coffee berry borer,Hypothenemus hampei

Several parasitoids of African origin have been introduced to coffee producing areas of the Americas and Asia as biological control agents of the coffee berry borer (CBB) Hypothenemus hampei (Coleoptera: Curculionidae). These parasitoids have become established in the field but their effect on the CBB has been limited. A two-year field study in Western Kenya has found Prorops nasuta (Hymenoptera: Bethylidae) to be the predominant parasitoid emerging from CBB-infested coffee berries collected on coffee trees or from the ground. P. nasuta comprises more than 75% of the total natural enemies collected. The density of P. nasuta was 90% higher in the berries collected from the ground than from the trees. Its hyperparasitoid, Aphanogmus sp. (Hymenoptera: Ceraphronidae), also emerged from both type of berries. Across the two seasons, the average P. nasuta density per berry was 18–35 times higher than that of Aphanogmus sp. Throughout the two years sampled, significantly higher numbers of P. nasuta and Aphanogmus sp. occurred between February and March, which coincides with the beginning of the rainy season. Higher numbers of live CBB females were recorded in berries collected from the trees. Nevertheless, mortality of adult CBB was considerably higher from January to March and started to decrease from April onwards. The possibly negative effects of cultural control practices in Latin America which include the removal of berries fallen to the ground on biological control of CBB are discussed, and the use of screened collection devices for these berries which would permit the release of parasitoids but prevent escape of the pest is proposed.     

Exploiting the genetic diversity of Beauveria bassiana for improving the biological control of the coffee berry borer through the use of strain mixtures

Beauveria bassiana is an entomopathogen widely used to control the coffee berry borer in Colombia, as part of an Integrated Pest Management strategy. Traditionally, the development of fungal insect pathogens as biocontrol agents in crop pests has been oriented towards the selection and formulation of elite clonal strains. Instead, we explored the potential application of genetic diversity in B. bassiana by determining the effect of strain mixtures on coffee berry borer mortality compared to clonal isolates. Genomic DNA from 11 strains was characterized using internal transcribed spacers and β-tubulin sequences as well as amplified fragment length polymorphism markers. Cluster analysis produced three genetic groups and confirmed the low but significant intraspecific genetic diversity present among the strains. Single strain virulence towards the coffee berry borer under laboratory conditions, using 1×10⁶ conidia ml⁻¹, ranged between 89.9 and 57.5%. All the inoculations with mixtures resulted in coinfection events. Combinations of genetically similar strains showed no significant differences when their virulences were compared. However, mixtures of genetically different strains led to both antagonism and synergism. The lowest virulence percentage (57%) was obtained by putting together the most virulent strain of each group, contrary to the highest virulence percentage (93%) that resulted from mixing the three least virulent strains. The results indicate the promising potential of designing strain mixtures as an alternative for the biocontrol of Hypothenemus hampei and other pests and provide tools for the understanding of the ecological dynamics of entomopathogen populations under natural conditions.     

A Keystone Ant Species Provides Robust Biological Control of the Coffee Berry Borer Under Varying Pest Densities

Species’ functional traits are an important part of the ecological complexity that determines the provisioning of ecosystem services. In biological pest control, predator response to pest density variation is a dynamic trait that impacts the provision of this service in agroecosystems. When pest populations fluctuate, farmers relying on biocontrol services need to know how natural enemies respond to these changes. Here we test the effect of variation in coffee berry borer (CBB) density on the biocontrol efficiency of a keystone ant species (Azteca sericeasur) in a coffee agroecosystem. We performed exclosure experiments to measure the infestation rate of CBB released on coffee branches in the presence and absence of ants at four different CBB density levels. We measured infestation rate as the number of CBB bored into fruits after 24 hours, quantified biocontrol efficiency (BCE) as the proportion of infesting CBB removed by ants, and estimated functional response from ant attack rates, measured as the difference in CBB infestation between branches. Infestation rates of CBB on branches with ants were significantly lower (71%-82%) than on those without ants across all density levels. Additionally, biocontrol efficiency was generally high and did not significantly vary across pest density treatments. Furthermore, ant attack rates increased linearly with increasing CBB density, suggesting a Type I functional response. These results demonstrate that ants can provide robust biological control of CBB, despite variation in pest density, and that the response of predators to pest density variation is an important factor in the provision of biocontrol services. Considering how natural enemies respond to changes in pest densities will allow for more accurate biocontrol predictions and better-informed management of this ecosystem service in agroecosystems.     

The biology of Phymastichus coffea LaSalle (Hymenoptera: Eulophidae) under field conditions

The coffee berry borer (CBB) Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae) was accidentally introduced into México in 1978, and rapidly became the main pest of coffee. As an exotic pest, its management has been mainly based on biological control methods through the introduction of parasitoids from Africa. In this context, at the beginning of the present decade, the parasitoid Phymastichus coffea LaSalle (Hymenoptera: Eulophidae) was imported to Mexico. Since then, several studies have been carried out as part of the post introduction evaluation of this parasitoid. In this paper, information concerning the parasitism and life-cycle of P. coffea in coffee farms is presented with the objective of providing information that elucidates its role as a biological control agent. P. coffea showed highly significant preferences for allocation of two eggs per host, usually one female and one male. Both offspring are able to develop and reach the adult stage successfully. Lifespan of adults is 2–3 days only. The degree of parasitism by P. coffea was more than 95% at the three altitudes tested, when releases consisted of a ratio of 10 CBB:1 parasitoid. The median survivorship of CBB parasitized by this wasp was 13, 15 and 19 days at the low, medium and high altitude coffee zones, respectively. The parasitism by P. coffea was higher when parasitoid releases were carried out simultaneously with the CBB, and decreased with the time between host and parasitoid releases. We showed that using P. coffea at a density of 1 parasitoid per 10 hosts resulted in a 3- to 5.6-fold decrease in CBB damage to the coffee seeds when compared to the control. The importance and value of these results are discussed in terms of the use of P. coffea as a biological control agent of the CBB in Latin America.     

Effect of physiological status on olfactory and visual responses of female Hypothenemus hampei during host plant colonization

The response of Hypothenemus hampei (Ferr.) (Coleoptera, Scolytidae) (coffee berry borer, CBB) females to olfactory stimuli was demonstrated to be related with the physiological status; especially with age, whether they were mated or not, and whether they have already oviposited. A special behaviour, the abandonment of the native berry, was also found to play a major role. In contrast, response to visual stimuli (false coffee berries) was found to be independent of the physiological status of CBB females. Variations in behavioural responses to visual and olfactory stimuli are discussed in relation to colonization strategy.     

Identification of attractant and repellent plants to coffee berry borer,Hypothenemus hampei

Colombia is one of the world's largest producers of coffee [Coffea arabica L. (Rubiaceae)]. The coffee berry borer (CBB), Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae), is the main pest of coffee. This insect is controlled through an integrated pest management program that includes cultural, biological, and chemical control strategies. Despite research seeking CBB attractants and repellents, these potential management tools have not been successfully incorporated into control programs. This work proposes the use of plant functional diversity for CBB management, for which a number of plants related to coffee and weeds were selected. CBB preference to these plants was determined by olfactometry and volatile compounds emitted by them were identified. Field trials were performed to test CBB preference under field conditions. These trials determined the olfactory preference of CBB to coffee berries accompanied by material of the plants Crotalaria micans Link (Fabaceae), Lantana camara L. (Verbenaceae), Nicotiana tabacum L. (Solanaceae), Artemisia vulgaris L., Calendula officinalis L., Stevia rebaudiana (Bertoni) Bertoni, and Emilia sonchifolia (L.) DC. (all four Asteraceae). Under laboratory conditions N. tabacum, L. camara, and C. officinalis were identified as repellents for CBB in olfactometer assays, whereas E. sonchifolia functioned as attractant. Controlled field trials corroborated CBB repellency of N. tabacum and L. camara; both release volatile sesquiterpenes. Selected candidate attractants included E. sonchifolia plants, for showing attraction in the laboratory. The potential use of these plants in agroecological management of coffee plantations is discussed.     

Sterile males of Ceratitis capitata (Diptera: Tephritidae) as disseminators of Beauveria bassiana conidia for IPM strategies

Sterile Mediterranean fruit fly, Ceratitis capitata (Wied.), males were evaluated as vectors to spread Beauveria bassiana (Bals) conidia to wild C. capitata populations under field conditions. The inoculated sterile males were released by air, using the chilled adult technique over 7000 ha of coffee growing in Chimaltenango, Guatemala, Central America. The impact of releases was determined using dry traps baited with a food attractant. The effects of these releases on Apis mellifera, Linnaeus (honey bee), Hypothenemus hampei, Ferrari (coffee berry borer) and the parasitic mite Varroa destructor (Oudeman) were also evaluated. Inoculated sterile males were able to transmit fungal spores to 44% of the wild C. capitata flies captured in traps, which likely were infected through intra- and intersexual interactions during leks, mating or mating attempts. There was no transmission of the fungal spores to non-target insect species such as coffee berry borer, honey bees or varroa. We conclude that sterile males of Mediterranean fruit fly inoculated with B. bassiana can act as effective vectors of conidia to wild populations, constituting a safe, environmentally friendly and selective alternative for suppressing the medfly under a Sterile Insect Technique-based IPM approach.     

Distribution of the Entomopathogenic Fungus Beauveria bassiana in Rice Ecosystems and Its Effect on Soil Enzymes

Fungal entomopathogens, especially Beauveria bassiana, are often studied within the context of their use in biological pest control; however, there is limited knowledge of their distributions in host plants and soil ecosystem. We examined the distribution of B. bassiana and its influence on rice plants and paddy soils. B. bassiana could only be detected on the foliar surfaces of rice plants within 15 days under Bb-4 (7.5 × 10⁴ conidia/mL) and Bb-7 (7.5 × 10⁷ conidia/mL) treatments. The endophytic colonization of B. bassiana could not be found in stems, roots, or seeds of rice plants under Bb-4 and Bb-7 treatments. The fungus was found only in the leaves of rice plants under Bb-4 and Bb-7 treatments at 15 days after inoculation. Moreover, B. bassiana was absent from paddy soils under Bb-4 and Bb-7 treatments at all times. Enzyme activity (urease and phosphatase) in the paddy soils of Bb-4 and Bb-7 treatments showed no significant difference from the control. It is possible that B. bassiana was not able to colonize paddy soil. Detailed understanding of distribution and ecological interactions of B. bassiana is helpful for understanding and predicting the effects of fungal entomopathogens on host populations, and the interactions among fungal entomopathogens and other organisms in the community.     

Testing what we know about coffee volatiles affecting behaviour of Hypothenemus hampei

Coffee berry borer (CBB), Hypothenemus hampei (Ferrari) (Coleoptera: Scolytinae), is a major pest of coffee worldwide. CBB females use berry volatiles while searching for hosts. Coffee volatiles can be used for monitoring CBB populations. The main objective of this study was to test under laboratory and field conditions bioactive compounds for H. hampei. In the laboratory, CBB females were attracted to the methanol-ethanol blend (MEB), but not to methylcyclohexane, ethylbenzene, nonane, methyl salicylate, verbenone, α-pinene, farnesene, and frontalin compared to clean air. In contrast, CBB females preferred clean air over linalool. Females were attracted to most of the tested compounds combined with the MEB over clean air, except farnesene, α-pinene, and linalool. Females preferred clean air to α-pinene or linalool, and they showed no preference for clean air or farnesene. Females also preferred the MEB over linalool, verbenone, α-pinene, or farnesene when combined with MEB. The combination of tested compounds and the MEB did not increase the capture of CBB in the field. Interestingly, α-pinene and linalool alone, or combined, inhibited the capture of CBB females and non-target insects by trapping bait with the MEB. Thus, α-pinene or linalool can be used in a push-pull strategy considering the low impact over non-target insects.     

Field superparasitism by Phymastichus coffea, a parasitoid of adult coffee berry borer, Hypothenemus hampei

Superparasitism by Phymastichus coffea LaSalle (Hymenoptera: Eulophidae), a parasitoid of adults of the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae), was recorded under field conditions in a coffee plantation in Colombia. Parasitoid adults were released 1, 5, and 9 days after artificial infestations of 90‐, 150‐, and 210‐day‐old coffee berries with H. hampei females. The position of the beetle inside the berry and the number of P. coffea larvae per female host were assessed 10 days after each parasitoid release. Under laboratory conditions, P. coffea usually lays two eggs per host, one female and one male. In our studies, we often recorded more than six P. coffea larvae in an individual host and mean numbers of larvae per host ranged from two to 4.45. Superparasitism by P. coffea under field conditions was influenced by the age of the coffee berries, which is the most important factor determining the speed of penetration by H. hampei, and therefore the time the beetles are exposed to a P. coffea attack. The number of parasitoid larvae in each H. hampei female gradually decreased with the age of the berry, and also linearly decreased with the time of parasitoid release. Age‐dependent effects of coffee berries that alter the ratio of available hosts to searching parasitoids by providing refuges to the herbivore, largely determine the extent of superparasitism of H. hampei by P. coffea under fields conditions in Colombia.     

Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)

The entomopathogenic fungus Beauveria bassiana was established in coffee seedlings after fungal spore suspensions were applied as foliar sprays, stem injections, or soil drenches. Direct injection yielded the highest post-inoculation recovery of endophytic B. bassiana. Establishment, based on percent recovery of B. bassiana, decreased as time post-inoculation increased in all treatments. Several other endophytes were isolated from the seedlings and could have negatively influenced establishment of B. bassiana. The recovery of B. bassiana from sites distant from the point of inoculation indicates that the fungus has the potential to move throughout the plant.     

Whorl and pollen-shed stage application of Beauveria bassiana for suppression of adult western corn rootworm

The corn rootworm complex is the most damaging insect pest of corn (Zea mays L.). This study was conducted to determine the efficacy of whorl and pollen-shed stage applications of a granular formulation of Beauveria bassiana (Balsamo) Vuillemin for control of adult western corn rootworm (Diabrotica virgifera virgifera Le Conte). The effect of application time (whorl-stage, pollen-shed) and plant surface exposed (leaves and leaf collars; silks; leaves, leaf collars, and silks) on level of beetle fungal infection were investigated. In addition, the number of colony forming units of B. bassiana in the corn leaf collar area was quantified. In the three years (1998–2000) of the study, application of B. bassiana at whorl-stage did not significantly increase beetle fungal infection. Beauveria bassiana applied to plants at pollen-shed in 1998 resulted in a significant increase in beetle infection with 51% of beetles from treated plants infected and 6.0% from control plants. Similar applications at pollen-shed in 1999 and 2000 resulted in very low infection levels. Beauveria bassiana application at pollen-shed stage significantly increased the number of colony forming units per leaf collar during all years of the study. Beetle infection with B. bassiana did not differ consistently among plant surface to which beetles were exposed for either application. Increased fungal load in leaf collars was not correlated with increased levels of adult infection. Increased rates of B. bassiana and application when beetles are present on the plants are likely needed to significantly increase infection rates.     

Infection of the Coffee Berry Borer Hypothenemus hampei (Coleoptera: Scolytidae) by Brazilian Isolates of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes)

The virulence of four fungal isolates (three Beauveria bassiana and one Metarhizium anisopliae ) against adult female coffee berry borers (CBB) was investigated. The most virulent isolate from initial bioassays, B. bassiana LPP1, with a LT 50 of 3.4 days, was further investigated by application to berries prior to infestation and to berries already infested with CBB. At the highest concentration applied to berries (1 ×10 7 conidia mL -1 ), CBB mortality was 83% (berries inoculated prior to infestation) and 62% (berries inoculated after infestation).     

Evaluation of Beauveria bassiana applications against adult house fly, Musca domestica, in commercial caged-layer poultry facilities in New York state

Applications of a commercially produced Beauveria bassiana product, balEnce, were compared with pyrethrin treatments for the control of adult house flies in New York high-rise, caged-layer poultry facilities. An integrated fly management program, which included the release of house fly pupal hymenopteran parasitoids, was used at all facilities. Adult house fly populations were lower in B. bassiana-treated facilities during the spray and post-spray periods, as recorded on spot cards. Concurrently, the numbers of house fly larvae recovered in B. bassiana-treated facilities were less than one-half that of the pyrethrin-treated facilities. House fly pupal parasitism levels were low, but similar under both treatment regimes. The numbers of adult and larval Carcinops pumilio, a predatory beetle, recovered from B. bassiana-treated facilities were 43 and 66% greater than from the pyrethrin-treated facilities, respectively.     

Multiple cryptic genetic units in Hypothenemus hampei (Coleoptera: Scolytinae): evidence from microsatellite and mitochondrial DNA sequence data

Hypothenemus hampei is the most important insect pest of coffee and has spread to most coffee‐growing countries worldwide. There have been very few studies and none have addressed the population genetics of the beetle using microsatellite markers. In the present study, 683 individuals collected from 37 locations in 18 countries worldwide were screened at nine polymorphic microsatellite loci. Sixty‐five out the 683 and six additional individuals were analyzed on a 400‐bp fragment of the mitochondrial cytochrome oxidase I gene. Bayesian clustering analysis and phylogenetic approaches were used to infer the genetic structure of H. hampei over the sampling that encompassed almost all its range. Microsatellite markers made it possible to achieve sufficiently significant power for the delineation of five morphocryptic evolutionary units. Supported by 27 new COI haplotypes, an unexpected considerably high level of genetic differentiation and genetic divergence was revealed between five geographically delineated clusters. Both markers and approaches showed that the clusters included specimens from (1) Ethiopia, (2) Kenya and Uganda, (3) Brazil, (4) Central America excluding Jamaica, and (5) all samples from Asia, West Africa, and Jamaica. These findings clearly suggest the existence of a ‘species complex in H. hampei’. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 113–129.