Ecology and Evolution of Insect-Fungus Mutualisms.

The evolution of a mutualism requires reciprocal interactions whereby one species provides a service that the other species cannot perform or performs less efficiently. Services exchanged in insect-fungus mutualisms include nutrition, protection, and dispersal. In ectosymbioses, which are the focus of this review, fungi can be consumed by insects or can degrade plant polymers or defensive compounds, thereby making a substrate available to insects. They can also protect against environmental factors and produce compounds antagonistic to microbial competitors. Insects disperse fungi and can also provide fungal growth substrates and protection. Insect-fungus mutualisms can transition from facultative to obligate, whereby each partner is no longer viable on its own. Obligate dependency has (a) resulted in the evolution of morphological adaptations in insects and fungi, (b) driven the evolution of social behaviors in some groups of insects, and (c) led to the loss of sexuality in some fungal mutualists.

Caradiophyodidae, a New Family of Micro-Wasps (Hymenoptera: Platygastroidea) Based on the Description of Caradiophyodus saradae gen. et sp. nov. in Mid-Cretaceous Burmese Amber.

The female micro-wasp in mid-Cretaceous Burmese amber is described as a new genus and species in the extinct family Caradiophyodidae fam. nov. (Hymenoptera: Platygastroidea). Features of the specimen are its small body size (1.3 mm), no elbows, elongated, 15-segmented antennae, a deep cleft in the top of its head, a tarsal formula of 5-5-5, a reduced venation with a small pterostigma but no uncus in the forewing, no anal lobe in the hind wing, and a possible coiled ovipositor in the metasoma. Large unidentified expanded structures, considered to be possible seeds, plant secretions, or host eggs, are attached to each antenna.

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.

New record for the coffee berry borer, Hypothenemus hampei, in Hawaii.

The coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) is endemic to Africa and is the most devastating pest of coffee worldwide. The female bores a hole in the coffee berry and deposits her eggs inside. Upon hatching, larvae feed on the seeds, thus reducing both quality and yields of the marketable product. The coffee berry borer was found in the district of Kona on the island of Hawaii in August 2010 and appears to be restricted to that area.

Cultivation and Genome Sequencing of Bacteria Isolated From the Coffee Berry Borer (Hypothenemus hampei), With Emphasis on the Role of Caffeine Degradation.

The coffee berry borer, the most economically important insect pest of coffee worldwide, is the only insect capable of feeding and reproducing solely on the coffee seed, a food source containing the purine alkaloid caffeine. Twenty-one bacterial species associated with coffee berry borers from Hawai'i, Mexico, or a laboratory colony in Maryland (Acinetobacter sp. S40, S54, S55, Bacillus aryabhattai, Delftia lacustris, Erwinia sp. S38, S43, S63, Klebsiella oxytoca, Ochrobactrum sp. S45, S46, Pantoea sp. S61, Pseudomonas aeruginosa, P. parafulva, and Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75) were found to have at least one of five caffeine N-demethylation genes (ndmA, ndmB, ndmC, ndmD, ndmE), with Pseudomonas spp. S31, S32, S37, S60 and P. parafulva having the full complement of these genes. Some of the bacteria carrying the ndm genes were detected in eggs, suggesting possible vertical transmission, while presence of caffeine-degrading bacteria in frass, e.g., P. parafulva (ndmABCDE) and Bacillus aryabhattai (ndmA) could result in horizontal transmission to all insect life stages. Thirty-five bacterial species associated with the insect (Acinetobacter sp. S40, S54, S55, B. aryabhattai, B. cereus group, Bacillus sp. S29, S70, S71, S72, S73, D. lacustris, Erwinia sp. S38, S43, S59, S63, K. oxytoca, Kosakonia cowanii, Ochrobactrum sp. S45, S46, Paenibacillus sp. S28, Pantoea sp. S61, S62, P. aeruginosa, P. parafulva, Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75, Stenotrophomonas sp. S39, S41, S48, S49) might contribute to caffeine breakdown using the C-8 oxidation pathway, based on presence of genes required for this pathway. It is possible that caffeine-degrading bacteria associated with the coffee berry borer originated as epiphytes and endophytes in the coffee plant microbiota.

Molecular diagnosis of a previously unreported predator-prey association in coffee: Karnyothrips flavipes Jones (Thysanoptera: Phlaeothripidae) predation on the coffee berry borer.

The coffee berry borer, Hypothenemus hampei, is the most important pest of coffee throughout the world, causing losses estimated at US $500 million/year. The thrips Karnyothrips flavipes was observed for the first time feeding on immature stages of H. hampei in April 2008 from samples collected in the Kisii area of Western Kenya. Since the trophic interactions between H. hampei and K. flavipes are carried out entirely within the coffee berry, and because thrips feed by liquid ingestion, we used molecular gut-content analysis to confirm the potential role of K. flavipes as a predator of H. hampei in an organic coffee production system. Species-specific COI primers designed for H. hampei were shown to have a high degree of specificity for H. hampei DNA and did not produce any PCR product from DNA templates of the other insects associated with the coffee agroecosystems. In total, 3,327 K. flavipes emerged from 17,792 H. hampei-infested berries collected from the field between April and September 2008. Throughout the season, 8.3% of K. flavipes tested positive for H. hampei DNA, although at times this figure approached 50%. Prey availability was significantly correlated with prey consumption, thus indicating the potential impact on H. hampei populations.

Genus Hamigera, six new species and multilocus DNA sequence based phylogeny.

Genus Hamigera was erected for Talaro-myces species that make asci singly instead of in chains. Initially it contained two species, H. avellanea and H. striata. We describe six new species in the genus, H. fusca, H. inflata, H. insecticola, H. pallida, H. paravellanea and H. terricola. Merimbla ingelheimensis is a distinct anamorphic species in the Hamigera clade. None of our DNA sequence data (BT2, calmodulin, ITS, 1su rDNA, RPB2, Tsr1 and Mcm7) supported the placement of H. striata in the same clade as H. avellanea, thus we accepted Talaromyces striatus. In addition to Hamigera species we examined the phylogenetic disposition of Warcupiella spinulosa, Penicillium megasporum, Penicillium arenicola and Merimbla humicoloides. Despite nominal similarity of some of these species to Merimbla, none of these species are part of the Hamigera clade and M. humicoloides is placed in Penicillium to have a monophyletic genus Hamigera.

Entomopathogenic fungi (Hypocreales: Ophiocordycipitaceae) infecting bark lice (Psocoptera) in Dominican and Baltic amber.

Ophiocordyceps dominicanus Poinar & Vega sp. nov. in Dominican amber and Polycephalomyces baltica Poinar & Vega sp. nov. (Hypocreales: Ophiocordycipitaceae) in Baltic amber are described as entomopathogenic fungi of bark lice (Psocoptera). The specimens possess several features unknown in extant synnematous entomopathogenic fungi such as a tubular dark synnema with a straight, pointed tip bearing spores over the entire surface in O. dominicanus, and a globular yellowish synnema developing on the tip of the host's antenna in P. baltica. These are the only known fossil entomopathogenic fungi of bark lice, making them unique not only for their characters but also in respect to their selection of developmental sites on their bark lice hosts.

A mid-Cretaceous female scale insect (Hemiptera: Sternorrhyncha: Coccomorpha) in Burmese amber.

A new genus and species of scale insect (Hemiptera: Coccomorpha) is described from a female specimen in mid-Cretaceous Burmese (Myanmar) amber. Fossil female scales are rare and the present species, described as Paleolepidotus macrocolus gen. et sp. n., has such an unusual assortment of morphological features that it could not be assigned to any particular extant or extinct family. The small, ferruginous specimen exhibits a series of long wax pencils that extend around the body, including the head. The antennae and legs are quite long compared to other extant and extinct scale fossils. Of special interest are the protruding eyes, and a conical-triangular rostrum arising from between the forelegs; the claws with bifid apices are also unique. The ovisac contains immature stages.

Draft Genome Sequence of Pseudomonas sp. Strain CES, Containing the Entire Alkylxanthine Gene Cluster for Caffeine Breakdown.

Pseudomonas strain CES was isolated from caffeine-enriched soil and found to possess the N-demethylation pathway for caffeine breakdown. We report the nucleotide sequence of the draft genome with 5,827,822 bp, 62.6% G+C content, and 5,427 protein-coding regions.

Differential Metabolic Responses Caused by the Most Important Insect Pest of Coffee Worldwide, the Coffee Berry Borer (Hypothenemus hampei).

The world's coffee supply is threatened by the coffee berry borer, Hypothenemus hampei, the most destructive pest affecting coffee production and quality. This study hypothesized that coffee berry borer infestation induces distinct metabolic responses in the green coffee seeds of Coffea arabica and Coffea canephora (robusta). A targeted metabolomics approach was conducted using liquid chromatography tandem mass spectrometry to quantify intracellular metabolites in infested and uninfested arabica and robusta green seeds. In parallel, the seed biomass content and composition were assessed for the same conditions. Coffee berry borer attack induced increases in the levels of chlorogenic acids in arabica seeds, whereas organic acids and sugar alcohols were more abundant in infested robusta seeds. Most importantly, a set of compounds was identified as biomarkers differentiating the metabolic response of these taxa to the coffee berry borer.

Accurate Differentiation of Green Beans of Arabica and Robusta Coffee Using Nanofluidic Array of Single Nucleotide Polymorphism (SNP) Markers.

Green (unroasted) coffee is one of the most traded agricultural commodities in the world. The Arabica (Coffea arabica L.) and Robusta (Coffea canephora Pierre ex A. Froehner) species are the two main types of coffees for commercial production. In general, Arabica coffee is known to have better quality in terms of sensory characteristics; thus, it has a higher market value than Robusta coffee. Accurate differentiation of green beans of the two species is, therefore, of commercial interest in the coffee industry. Using the newly developed single nucleotide polymorphism (SNP) markers, we analyzed a total of 80 single green bean samples, representing 20 Arabica cultivars and four Robusta accessions. Reliable SNP fingerprints were generated for all tested samples. Unambiguous differentiation between Robusta and Arabica coffees was achieved using multivariate analysis and assignment test. The SNP marker panel and the genotyping protocol are sufficiently robust to detect admixture of green coffee in a high-throughput fashion. Moreover, the multilocus SNP approach can differentiate every single bean within Robusta and 55% of Arabica samples. This advantage, together with the single-bean sensitivity, suggests a significant potential for practical application of this technology in the coffee industry.

Early growth phase and caffeine content response to recent and projected increases in atmospheric carbon dioxide in coffee (Coffea arabica and C. canephora).

While [CO2] effects on growth and secondary chemistry are well characterized for annual plant species, little is known about perennials. Among perennials, production of Coffea arabica and C. canephora (robusta) have enormous economic importance worldwide. Three Arabica cultivars (Bourbon, Catimor, Typica) and robusta coffee were grown from germination to ca. 12 months at four CO2 concentrations: 300, 400, 500 or 600 ppm. There were significant increases in all leaf area and biomass markers in response to [CO2] with significant [CO2] by taxa differences beginning at 122-124 days after sowing (DAS). At 366-368 DAS, CO2 by cultivar variation in growth and biomass response among Arabica cultivars was not significant; however, significant trends in leaf area, branch number and total above-ground biomass were observed between Arabica and robusta. For caffeine concentration, there were significant differences in [CO2] response between Arabica and robusta. A reduction in caffeine in coffee leaves and seeds might result in decreased ability against deterrence, and consequently, an increase in pest pressure. We suggest that the interspecific differences observed (robusta vs. Arabica) may be due to differences in ploidy level (2n = 22 vs. 2n = 4x = 44). Differential quantitative and qualitative responses during early growth and development of Arabica and robusta may have already occurred with recent [CO2] increases, and such differences may be exacerbated, with production and quality consequences, as [CO2] continues to increase.

Poisonous setae on a Baltic amber caterpillar.

The knobbed setae on a small caterpillar in 45-55 million years old [Eocene] Baltic amber were studied and characterized as urticating, with evidence of liquid release implying the production of poisons. It is presumed that the caterpillar had been disturbed just prior to falling into the resin, as some of its setae showed defensive responses. The swollen tips of the setae are equipped with "trip hairs" and when disturbed, the tips release liquid deposits, some of which contain rod-like bodies. These setal responses to a disturbance are the first report of poisonous setal defense mechanisms in a fossil insect.

A mid-Cretaceous trichomycete, Priscadvena corymbosa gen. et sp. nov., in Burmese amber.

Priscadvena corymbosa gen. et sp. nov., is described from thalli and sporangia emerging from the oral cavity of a click beetle (Coleoptera: Elateridae) in mid-Cretaceous Burmese amber. The fossil contains several features unknown in extant Trichomycetes including a click beetle (Coleoptera: Elateridae) host, spiny, aerial thalli with the entire thallus bearing numerous small uninucleate globular spores and stalks attached to the oral cavity of its host. Based on these features, P. corymbosa gen. et sp. nov. is placed in a new family, Priscadvenaceae fam. nov., and new order, Priscadvenales ord. nov. The new morphological and behavioral features of the fossil add to the diversity of the trichomycetes as currently defined.

Anatomical study of the coffee berry borer (Hypothenemus hampei) using micro-computed tomography.

Traditionally, the study of anatomy in insects has been based on dissection techniques. Micro-computed tomography (micro-CT) is an X-ray based technique that allows visualization of the internal anatomy of insects in situ and does not require dissections. We report on the use of micro-CT scans to study, in detail, the internal structures and organs of the coffee berry borer (Hypothenemus hampei), the most damaging insect pest of coffee worldwide. Detailed images and videos allowed us to make the first description of the aedeagus and the first report of differences between the sexes based on internal anatomy (flight musculature, midgut shape, hindgut convolutions, brain shape and size) and external morphology (lateral outline of the pronotum and number of abdominal tergites). This study is the first complete micro-CT reconstruction of the anatomy of an insect and is also the smallest insect to have been evaluated in this way. High quality rendered images, and additional supplementary videos and 3D models are suitable for use with mobile devices and are useful tools for future research and as teaching aids.