Biological control of the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae) by Phymastichus coffea (Hymenoptera: Eulophidae) in Colombia

The potential of the eulophid parasitoid Phymastichus coffea LaSalle to control coffee berry borer Hypothenemus hampei (Ferrari) populations under field conditions in Colombia was evaluated. Parasitoid adults were released one, five and nine 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 parasitism levels were assessed ten days after each P. coffea release. Parasitism of H. hampei by P. coffea was significantly affected by the age of the berries at the time of infestation, and by the position of the beetle inside the berries. Highest levels of parasitism were recorded in 150-day-old berries (75-85%) and in 90-day-old berries (75%) when P. coffea were released one day after the artificial infestation with H. hampei. In 150-day-old berries, highest levels of parasitism were recorded for H. hampei found in the outer layer of the endosperm followed by beetles penetrating the exocarp. Increasing the time of P. coffea releases after the artificial infestations with H. hampei led to decreased levels of parasitism in beetles attacking 90- and 150-day-old coffee berries. Low levels of parasitism were recorded in H. hampei females infesting older coffee berries because most of the beetles had already constructed galleries deep in the endosperm of the berries, i.e. out of reach of the parasitoid. The potential of P. coffea for biological control of coffee berry borer in Colombia is discussed.     

A review of the biology and control of the coffee berry borer, Hypothenemus hampei (Coleoptera: Scolytidae)

The coffee berry borer, Hypothenemus hampei Ferrari, is a serious problem for the majority of the world's coffee growers and has proved to be one of the most intractable of present day pests. Despite a great deal of research, control still depends largely on the application of the organochlorine insecticide endosulfan, which is damaging to the environment, or a series of cultural and biological control methods which give variable and unpredictable results. This review summarizes the most important aspects of the biology and ecology of H. hampei and its control and identifies weak points in the knowledge about this pest. Emphasis is placed upon an analysis of the non-chemical control methods available and suggestions are offered for novel ecological and environmental factors worthy of further research, in the search for viable and sustainable control methods.     

Increased pathogenicity against coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae) by Metarhizium anisopliae expressing the scorpion toxin (AaIT) gene

Coffee berry borer (CBB) is the Worlds most devastating coffee pest causing an estimated US$500 million worth of losses annually through damage and control costs. Beauveria bassiana and Metarhizium anisopliae have been employed to control this pest but their low virulence (slow kill and large inoculums) is an important factor constraining their use. M. anisopliae (AaIT-Ma549) has been modified to express the scorpion toxin (AaIT) in insect hemolymph and this greatly increased pathogenicity against Manduca sexta and Aedes aegypti. Here, we demonstrate that AaIT-Ma549 was also dramatically more virulent against CBB, and we provide a much more comprehensive analysis of infection processes and post-mortality development than in the previous research. We evaluated several spore concentrations (10¹ through 10⁷ spores/ml) of both the wild type and recombinant strain. At concentrations of 10¹, 10² and 10³ spores/ml, the recombinant strain significantly increased mortality of CBB by 32.2%, 56.6% and 24.6%, respectively. The medial lethal concentration (LC₅₀) was reduced 15.7-fold and the average survival time (AST) was reduced by 20.1% to 2.98 ± 0.1 days with 10⁷ spores/ml. This is the first occasion that an entomopathogenic fungus has been found to kill CBB in less than 3 days. However, AaIT-Ma549 produces significantly fewer spores on cadavers than the parental strain.     

Interactions among bethylid parasitoid species attacking the coffee berry borer,Hypothenemus hampei (Coleoptera: Scolytidae)

The question of whether biological control is most likely achieved by deploying single or multiple species of biological control agents is much debated. While utilizing several natural enemies may enhance control, there is also the potential for disruptive inter-specific interactions. Such interactions may be studied in the laboratory by focusing on the details of the interactions themselves and attempting to infer population level consequences from their sum, or by focusing more directly on the overall effects on natural enemy populations: we term these approaches ‘reductionist’ and ‘holistic.’ Here we conduct a holistic laboratory study on interactions between three species of parasitoid wasps that are parasitoids of the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae): Cephalonomia stephanoderis Betrem, C. hyalinipennis Ashmead and Prorops nasuta Waterston (all Hymenoptera: Bethylidae). We find evidence for both intra- and inter-specific resource competition. Interactions between C. stephanoderis and P. nasuta, both indigenous to Africa, appear to be approximately symmetrical, while C. hyalinipennis, naturally found in the coffee plantations of Chiapas, Mexico, may exert a disruptive influence. C. hyalinipennis also has a low population growth rate. We now consider it to be a detrimental invader of the Mexican coffee agro-ecosystem that should not be encouraged by augmentative release or introduced into other regions. Overall, the most successful species, in terms of both emergence and female production, was P. nasuta. We compare these results with those from prior reductionist and holistic studies, and with observations on patterns of establishment of these bethylid species in the field. Given that it is increasingly clear that disruptive inter-specific interactions are generally common when multiple species are deployed in biological control, screening of potential agents should consider such interactions alongside the more ‘traditional’ focus on host specificity.     

Mating Behavior of the Coffee Berry Borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae)

The mating behavior of the coffee berry borer, Hypothenemus hampei (Ferrari), was observed under laboratory conditions. Pairs of adult virgin male and female beetles were placed in the wells of a polystyrene microtiter plate, one pair per well. The mating activity of each pair was recorded for 24?h. The mating behavior of the coffee berry borer was similar to that of other Scolytinae and was clearly divided into precopulatory, copulatory and postcopulatory phases. The beetles started to mate within a few hours of emergence. Repeated mating occurred during the 24-hour period and increased in frequency with age. However, we cannot address multiple matings in H. hampei, since we did not simulate the female-biased sex ratio of this species and the experimental design did not allow females to avoid additional mating attempts by males. In addition, further studies are necessary that focus on the effectiveness of sperm transmission and direct and indirect effects of multiple matings on the H. hampei females and their offspring.     

Sequences and transcriptional analysis of Coffea arabica var. Caturra and Coffea liberica plant responses to coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae) attack

The coffee berry borer (CBB) is the most prevalent pest of coffee plantations. Within the Coffea genus, C. arabica is susceptible to CBB and C. liberica shows a lower susceptibility. Two EST libraries were constructed from the total RNA of C. arabica and C. liberica fruits artificially infested with CBBs for 24 h. Using 6000 clones sequenced per library, a unigene database was generated, obtaining 3634 singletons and 1454 contigs. For each contig, the proportion of sequences present in both species was determined and a differential gene expression between the species was detected. C. arabica displayed a higher relative expression of proteins involved in general stress responses, whereas C. liberica showed the induction of a higher number of insect defense proteins. In order to validate the results, quantifications through real-time PCR were done. A hevein-like protein, an isoprene synthase, a salicylic acid carboxyl methyltransferase and a patatin-like protein gene were highly upregulated in C. liberica at 24 and/or 48 h after insect infestation compared to C. arabica. The identification of metabolic pathways induced by this pest insect provides tools to take advantage of the genetic resources available for the control of CBB.     

Bacillus thuringiensis serovar israelensis is highly toxic to the coffee berry borer, Hypothenemus hampei Ferr. (Coleoptera: Scolytidae)

A native collection of Bacillus thuringiensis strains was screened, once a reliable bioassay technique to assess the toxicity against the coffee berry borer (CBB) first-instar larvae was developed. A first round of bioassays with 170 strains indicated that the great majority of them showed no or very little insecticidal activity and that very few showed significant levels of toxicity. Interestingly, only those strains that had previously been associated with mosquitocidal activity were also toxic to CBB. Qualitative bioassays (using one high dose) were carried out only with those native mosquitocidal strains, corroborating their significant toxicity towards the CBB first-instar larvae. Most of these strains belong to serovar israelensis. In a second approach, strains from the Institut Pasteur type collection, whose mosquitocidal activity had been previously demonstrated, were also subjected to bioassays. Only those strains that showed a comparable protein content in their parasporal crystals to the israelensis type strain also showed high levels of toxicity towards CBB. Finally, an accurate LC(50) was estimated, using purified parasporal crystals from B. thuringiensis serovar israelensis type strain, at 219.5 ng cm(-2) of diet. All the statistical requirements for a reliable estimator were fulfilled. This is the first report of B. thuringiensis serovar israelensis being active against a coleopteran species.     

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.     

Alimentary canal and reproductive tract of Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae, Scolytinae)

The alimentary canal and the reproductive tract of males and females of Hypothenemus hampei Ferrari are described. The alimentary canal of H. hampei showed the crop with several spine-like structures and the midgut with few gastric caeca. We evidenced for the first time that adult females need to feed on coffee in order to produce viable eggs before and during oviposition period. The presence of air bubbles inside the anterior midgut may be due to starvation rather than the response of the environment during flying as previously reported. Two ovaries and the same structures and arrangements common to individuals of the Curculionidae beetles composed the female reproductive system. The male reproductive tract showed a significant difference with respect to other Curculionidae and even other Scolytinae species as it showed a sclerotized aedeagus with several pore-like structures in the terminal portion where sperm is released during mating.     

Metaparasitylenchus hypothenemi n. sp. (Nematoda: Allantonematidae), a parasite of the coffee berry borer, Hypothenemus hampei (Curculionidae: Scolytinae)

Metaparasitylenchus hypothenemi n. sp. (Nematoda: Allantonematidae) is described from the coffee berry borer, Hypothenemus hampei (Ferrari) (Curculionidae: Scolytinae), in Chiapas, Mexico. This species differs from other members of the genus by its small size, annulated cuticle, lateral fields with 3 ridges, free-living stages with an excretory pore located between the pharyngeal gland orifices, a distinct stylet with basal swellings in free-living females, a postvulval uterine extension, a thin stylet lacking basal swellings in males, 2 separate spicules, a gubernaculum, and a peloderan bursa. Parasitic females are white, with a straight or slightly curved body and are ovoviviparous. Third-stage juveniles emerge from parasitized beetles and molt twice before reaching the adult stage. Because the coffee berry borer is the most important pest of coffee throughout the world and this parasite partially or completely sterilizes female beetles, it is worthy of further investigation as a potential biological control agent.     

An alpha-amylase inhibitor gene from Phaseolus coccineus encodes a protein with potential for control of coffee berry borer (Hypothenemus hampei)

Plant alpha-amylase inhibitors are proteins found in several plants, and play a key role in natural defenses. In this study, a gene encoding an alpha-amylase inhibitor, named alphaAI-Pc1, was isolated from cotyledons of Phaseolus coccineus. This inhibitor has an enhanced primary structure to P. vulgaris alpha-amylase inhibitors (alpha-AI1 and alpha-AI2). The alphaAI-Pc1 gene, constructed with the PHA-L phytohemaglutinin promoter, was introduced into tobacco plants, with its expression in regenerated (T0) and progeny (T1) transformant plants monitored by PCR amplification, enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis, respectively. Seed protein extracts from selected transformants reacted positively with a polyclonal antibody raised against alphaAI-1, while no reaction was observed with untransformed tobacco plants. Immunological assays showed that the alphaAI-Pc1 gene product represented up to 0.05% of total soluble proteins in T0 plants seeds. Furthermore, recombinant alphaAI-Pc1 expressed in tobacco plants was able to inhibit 65% of digestive H. hampei alpha-amylases. The data herein suggest that the protein encoded by the alphaAI-Pc1 gene has potential to be introduced into coffee plants in order to increase their resistance to the coffee berry borer.     

Natural occurrence of Beauveria bassiana in Hypothenemus hampei (Coleoptera: Curculionidae) populations in unsprayed coffee fields

Three unsprayed coffee farms (farm 1, 2 and 3) were studied for the natural occurrence of the insect pathogenic fungus Beauveria bassiana in Hypothenemus hampei populations throughout the rainy season of 2004 (July-November) and 2005 (July-December). B. bassiana infections were found during most sampling dates in both years, on all three farms. The B. bassiana infection levels were higher in 2005 than in 2004 with mean prevalence of 12.1% and 2.7%, respectively. No consistent significant differences in infection level between farms were found in any of the years. B. bassiana infection levels fluctuated widely throughout the season, and peaked at 13.5% on farm 3 in 2004 and at 44.0% on farm 1 in 2005. The H. hampei population was significantly higher in 2004 than in 2005, with 6.9% of the berries infested in 2004 and only 0.7% in 2005. In both years, the H. hampei infestation level was significantly higher on farm 2. No consistent significant differences in H. hampei infestation levels were found between sampling dates on any of the farms. H. hampei infestation levels fluctuated throughout both seasons, and peaked at 15.3% on farm 2 in 2004 and 2.2% on farm 2 in 2005. No consistent density dependent correlation between H. hampei infestation level and B. bassiana infection level was found. Correlations between climatic conditions and B. bassiana or H. hampei were not found.     

Alpha-amylases of the coffee berry borer (Hypothenemus hampei) and their inhibition by two plant amylase inhibitors

The adult coffee berry borer (Hypothenemus hampei Ferrari [Coleoptera: Scolytidae]), a major insect pest of coffee, has two major digestive alpha-amylases that can be separated by isoelectric focusing. The alpha-amylase activity has a broad pH optimum between 4.0 and 7.0. Using pH indicators, the pH of the midgut was determined to be between 4.5 and 5.2. At pH 5.0, the coffee berry borer alpha-amylase activity is inhibited substantially (80%) by relatively low levels of the amylase inhibitor (alphaAI-1) from the common bean, Phaseolus vulgaris L., and much less so by the amylase inhibitor from Amaranthus. We used an in-gel zymogram assay to demonstrate that seed extracts can be screened to find suitable inhibitors of amylases. The prospect of using the genes that encode these inhibitors to make coffee resistant to the coffee berry borer via genetic engineering is discussed.     

Responses of coffee berry borer, Hypothenemus hampei (Ferrari)(Coleoptera: Scolytidae), to vertical distribution of methanol: ethanol traps

Captures of the coffee berry borer (CBB) Hypothenemus hampei (Ferrari) were assessed in traps in the field. IAPAR designed traps [plastic bottles (2 L) lured with methanol:ethanol (1:1) in a vessel] were placed either at 0.5, 1.0 and 1.5m high from the ground or simultaneously tested in the 2004 fructification season. Traps placed at the three heights trapped 5.5 times more CBB than the others, mostly at the traps placed at 0.5 m (75%). Treatments using the IAPAR designed trap placed at 1.2 m high; IAPAR trap with a white plastic plate above (IAPAR modified I) at 1.2 m high; IAPAR at 0.5 m high and two additional vessels at 1.0 and 1.5m high (IAPAR modified II) and T-163 trap [three red plastic cups (300 ml) and a red plastic plate as a cover] lured with M:E (1:1) at 1.2m height were compared in the vegetative (2005) and fructification (2006) periods. IAPAR modified II (dispenser vessels placed at 0.5, 1.0 and 1.5 m) trapped more beetles than the remaining types (2.72 times more beetles than IAPAR design); and IAPAR modified I traps trapped more beetles than T 163 and IAPAR traps in the vegetative period. In the reproductive period, IAPAR modified II trapped less beetles than IAPAR and IAPAR modified I. In 2007 vegetative season, IAPAR modified II trap were compared with IAPAR trap and trapped 2.8 times more beetles. The positive responses to a vertical distribution of the volatile attractants in the vegetative period of the planting allow the development of more efficient trapping systems for CBB.     

Progression in field infestation is linked with trapping of coffee berry borer, Hypothenemus hampei (Col., Scolytidae)

Phenology of the coffee plant and infestation by coffee berry borer Hypothenemus hampei Ferrari were studied in relation to trapping of adult females in kairomone-baited traps in a coffee plantation in New Caledonia. In a 0.4 ha coffee field, a group of 27 trees located along a transect beginning at an early infestation point was selected. The number of green, red and dry coffee berries, along with the number of larvae, adult males and females per berry was determined monthly from October 1993 to July 1994. Twelve, red multifunnel traps, each baited with a solution of methanol:ethanol (1:1 ratio, a mean solution release rate of 1 g/day) were placed within the coffee field, along the transect, within the selected trees, grouped in four zones named 1–4. Two additional traps were located outside the plantation.
The proportion of infested berries increased as berry maturity and harvest date approached, while the infestation rate decreased with distance from the epicentre. Over the 10 months of the study, beetle populations increased and spread from the original infestation point across the different zones, according to distance and availability of berries or appropriate physiological status. Traps near the epicentre caught the largest numbers of beetles. Linear relationship between trap catch and infestation level was demonstrated. Traps placed outside the field approached zero catch. Trap catch was highly influenced by rainfall events, and the highest captures coincided with rapidly declining berry numbers on trees. There are good prospects for management of this insect using traps.     

Isolation,morphological and molecular characterization of Bacillus thuringiensis strains against Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae)

The coffee berry borer Hypothenemus hampei Ferrari, 1876 (Coleoptera: Curculionidae: Scolytinae) is considered the most serious pest of the coffee crop and is controlled primarily with the use of chemical insecticides. An alternative to this control method is the use of the entomopathogenic bacterium, Bacillus thuringiensis Berliner, 1911. Therefore, the objective of this work was to select strains of B. thuringiensis virulent against H. hampei and characterize them by morphological and molecular methods to identify possible genes for the production of genetically modified plants. To achieve this objective, 34 strains of B. thuringiensis underwent a selective bioassay to evaluate their toxicity to H. hampei first-instar larvae. Among the strains tested, 11 and the standard B. thuringiensis subspecies israelensis (IPS-82) caused mortality above 90%. Then, the median lethal concentration (LC₅₀) was estimated for these strains followed by characterization using morphological, biochemical and molecular methods. The lowest LC₅₀ was obtained for strain BR58, although this concentration did not differ significantly from that of the standard strain IPS-82 or from that of strains BR137, BR80 and BR67. The molecular characterization detected cry4A, cry4B, cry10, cry11 and cyt1 genes in 10 of the most virulent strains (BR58, BR137, BR80, BR81, BR147, BR135, BR146, BR138, BR139, BR140). Strain BR67 differed completely from the others and amplified only the cry3 gene. This strain was more virulent than BR135, BR146, BR138, BR139 and BR140, but it did not differ from BR58, BR137, BR80, BR81 and BR147. The protein profile revealed proteins of 28, 65, 70 and 130 kDa, and the morphological analysis identified spherical crystalline inclusions in all strains. The results showed that the 11 strains studied have potential for use as a gene source for insertion into coffee plants for the control H. hampei, especially the cry3, cry4A, cry4B, cry10, cry11 and cyt1 genes, that were repeated in the most virulent isolates.     

Diversity of Bacillus thuringiensis strains isolated from coffee plantations infested with the coffee berry borer Hypothenemus hampei

The coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) was first reported infecting Costa Rican coffee plantations in the year 2000. Due to the impact that this plague has in the economy of the country, we were interested in seeking new alternatives for the biological control of H. hampei, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 202 B. thuringiensis isolates obtained from Costa Rican coffee plantations infested with H. hampei were analyzed through crystal morphology of the crystal inclusions and SDS-PAGE of 6-endotoxins, while 105 strains were further evaluated by PCR for the presence cry, cyt and vip genes. Most of the Bt strains showed diverse crystal morphologies: pleomorphic (35%), oval (37%), bipyramidal (3%), bipyramidal and oval (12%), bipyramidal, oval and pleomorphic (10%) and bipyramidal, oval and cubic (3%). The SDS-PAGE analyses of the crystal preparations showed five strains with delta-endotoxin from 20 to 40 kDa, six from 40 to 50 kDa, seven from 50 to 60 kDa, 19 from 60 to 70 kDa, 29 from 70 to 100 kDa and 39 from 100-145 kDa. PCR analyses demonstrated that the collection showed diverse cry genes profiles having several genes per strain: 78 strains contained the vip3 gene, 82 the cry2 gene, 45 the cry1 and 29 strains harbored cry3-cry7 genes. A total of 13 strains did not amplified with any of the cry primers used: cry1, cry2, cry3-7, cry5, cry11, cry12 and cry14. Forty-three different genetic profiles were found, mainly due to the combination of cry1A genes with other cry and vip genes. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against H. hampei and other insect pests of agricultural importance.     

Increasing coffee berry borer (Coleoptera: Curculionidae: Scolytinae) female density in artificial diet decreases fecundity

Three experiments were conducted to determine the influence of number of coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae), females (one, two, or five) reared in artificial diet on fecundity and subsequent development of larvae, pupae, and adults. Our results demonstrated that increasing female density from one to two or five individuals did not result in the expected two- or five-fold increase in progeny, despite ample food resources available. Instead, decreased fecundity was observed with increasing density for all experiments. The mechanism reducing fecundity was not identified, but possibly, volatiles are being produced (e.g., host-marking pheromones). The decrease in fecundity may explain why infestations of only one colonizing female per berry are the norm in the field.     

Optimization of coffee berry borer, Hypothenemus hampei Ferrari (Col., Scolytidae), mass trapping with an attractant mixture

Mass trapping is a technique currently being considered to control the coffee berry borer (CBB), Hypothenemus hampei Ferrari (Col., Scolytidae), as part of the integrated management of its populations. However, the research undertaken in this field has variable and sometimes contradictory results. The purpose of this study carried out with a single type of trap (experimental model '1B') was to evaluate the technique and define parameters for its efficient use. The first result obtained was detection of the excellent attractant properties of the ethanol–methanol mixture, and its efficiency related to the proportion of the two alcohols. Coffee borers were not attracted to traps with caffeine, green coffee powder, freshly pulped and hulled coffee berries and ground CBB. It was shown that the ethanol–methanol mixture (1 : 1) release rate varied without affecting the capture rate, and that the red colour of the trap substantially increased CBB attraction. In terms of trap set-up, it was found that capture rates were three times better with a trap height at 1.2 m, than that for a position near ground level. In addition, the best density of traps for achieving efficient mass trapping was 22 units per hectare. These results show the importance of developing an attractant using pure compounds, and of improving the trap and the trapping technique in line with CBB behaviour.