Characterization of a Costa Rican granulovirus strain highly pathogenic against its indigenous hosts,Phthorimaea operculella and Tecia solanivora

A granulovirus isolate collected from diseased Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) larvae in Costa Rican potato [Solanum tuberosum L. (Solanaceae)] crops was characterized at the molecular and biological level. Restriction endonuclease analysis identified this isolate as a novel P. operculella granulovirus (PhopGV) (Baculoviridae: Betabaculovirus) strain and was designated as PhopGV‐CR1. In addition, PCR amplification of four specific variable genomic regions yielded multiple amplicons for two open reading frames, revealing the presence of different genotypic variants within the virus population. Biologically, PhopGV‐CR1 was highly pathogenic for its two indigenous hosts, although significant differences of up to four‐fold were detected against P. operculella [LD₅₀ = 17.9 occlusion bodies (OBs) mm^?2] and Tecia solanivora (Povolny) (Lepidoptera: Gelechiidae) (LD₅₀ = 69.1 OBs mm^?2). The two P. operculella colonies, from Costa Rica and France, were equally susceptible to PhopGV‐CR1. Serial passage of PhopGV‐CR1 over four generations in T. solanivora increased its pathogenicity by five‐fold in three generations, suggesting an ongoing adaptation to its alternate host.     

Costa Rican soils contain highly insecticidal granulovirus strains against Phthorimaea operculella and Tecia solanivora

The aim of this work was to isolate and characterize novel Phthorimaea operculella granulovirus (PhopGV) strains from Costa Rican soils. Three novel strains, named PhopGV‐CR3, PhopGV‐CR4 and PhopGV‐CR5, were isolated from three locations in Costa Rica, Alvarado, Zarcero and Abangares, respectively, by means of soaking potato tubers with diluted soil samples. An additional strain, PhopGV‐CR2, was identified from diseased larvae from a Tecia solanivora laboratory culture. Restriction fragment length polymorphisms obtained for each isolate with six restriction endonucleases (RENs) allowed their identification as four distinct PhopGV strains. Both REN and Polymerase chain reaction analyses indicated the existence of an array of genotypic variants present in all isolates. Bioassays in P. operculella and T. solanivora showed that PhopGV‐CR3 was well adapted to the two coexisting hosts with high levels of pathogenicity against both pest species. The mean lethal dose values of this strain were 2.8 OBs/mm² for P. operculella and less than 0.5 OBs/mm² for T. solanivora. We conclude that PhopGV‐CR3 shows great promise for soil application against these pests in Costa Rican potato crops.     

Reproductive plasticity in <Emphasis Type="Italic">Petrolisthes armatus</Emphasis> (Anomura,Porcellanidae): a comparison between a Pacific and an Atlantic population

The porcellanid crab Petrolisthes armatus, with a known geographic distribution covering a wide range of latitudes, was selected to conduct a comparative study on egg production and reproductive output between two populations from Pacific and Atlantic coasts. Ovigerous females were collected between September and November 2005 in Punta Morales, Pacific coast of Costa Rica (n = 137) and from March 2005 to July 2006 in Araçá region, São Paulo, Brazil (n = 46). The mean size of females was statistically different between both populations with larger females from Brazil than Costa Rica (7.9 ± 1.31 and 6.6 ± 0.95 mm CW, respectively). Likewise, egg production was significantly different in both populations and started in Pacific Costa Rica at a smaller female size than in Brazil. Specimens from Pacific Costa Rica produced roughly three times more eggs than equally sized females from Brazil, while newly extruded embryos were larger in Brazil than in Costa Rica (0.045 and 0.039 mm³, respectively). Egg volume increased during embryogenesis by 112.8 and 164.5% in Costa Rican and Brazilian populations, respectively. The egg water content increased steadily in both populations; however, eggs produced in Brazil contained always more water than those from the same developmental stage in Costa Rica. Average reproductive output (based on dry weight) was substantially lower in Brazil (0.031) than in Costa Rica (0.065). Our results confirm an impressive intraspecific plasticity of reproductive features in an intertidal decapod. The observed phenotypical variability might be related to local environmental conditions as well as to the location of the studied population concerning its geographic range of distribution.     

Lepanthes variabilis (Orchidaceae: Pleurothallidinae), a new endemic species from Costa Rica

Lepanthes variabilis, a new species from Costa Rica, is described and illustrated. It is endemic to the northern part of the Talamanca mountain range, from 2750–2850 m. It is compared to the similar L. minutilabia, which also occurs in Costa Rica.     

Antifungal Activity of Nanocapsule Suspensions Containing Tea Tree Oil on the Growth of Trichophyton rubrum

The aim of this study was to evaluate, for the first time, the antifungal efficacy of nanocapsules and nanoemulsions containing Melaleuca alternifolia essential oil (tea tree oil) in an onychomycosis model. The antifungal activity of nanostructured formulations was evaluated against Trichophyton rubrum in two different in vitro models of dermatophyte nail infection. First, nail powder was infected with T. rubrum in a 96-well plate and then treated with the formulations. After 7 and 14 days, cell viability was verified. The plate counts for the samples were 2.37, 1.45 and 1.0 log CFU mL^?1 (emulsion, nanoemulsion containing tea tree oil and nanocapsules containing tea tree oil, respectively). A second model employed nails fragments which were infected with the microorganism and treated with the formulations. The diameter of fungal colony was measured. The areas obtained were 2.88 ± 2.08 mm², 14.59 ± 2.01 mm², 40.98 ± 2.76 mm² and 38.72 ± 1.22 mm² for the nanocapsules containing tea tree oil, nanoemulsion containing tea tree oil, emulsion and untreated nail, respectively. Nail infection models demonstrated the ability of the formulations to reduce T. rubrum growth, with the inclusion of oil in nanocapsules being most efficient.     

Growth, production and carbon sequestration of silvopastoral systems with native timber species in the dry lowlands of Costa Rica

The multiple environmental issues of loss of forest cover due to cattle farming combined with pasture degradation leading to low levels of production, consequent extensification and therefore to more deforestation, are serious concerns in Costa Rica. To test the feasibility of countering these by combining a more productive pasture system with indigenous tree species, a silvopastoral experiment was established on a farm in the seasonally dry lowlands of Cañas, Guanacaste Province. A rapidly growing pasture species (Brachiaria brizantha) was tested against a traditional pasture dominated by Hyparrhenia rufa. Three indigenous tree species were established: Pithecellobium saman, Diphysa robinioides and Dalbergia retusa. Plots were grazed by cattle for 4 or 5 days with one to 2 month intervals between grazing episodes. After 51 months, D. robinioides was the fastest growing species, and P. saman the slowest, while B. brizantha produced three times the above ground and twice the below ground biomass as H. rufa, and trees had no effect upon grass yield. Contrary to competition theory, there was no effect of pasture species upon the two faster growing tree species. The carbon in above and below ground phytomass varied between 3.5 and 12.5 Mg C ha^?1 in treeless pasture controls and silvopastoral systems, respectively, and total soil organic carbon (TSOC) in the upper 0.6 m averaged 110 Mg ha^?1. B. brizantha appeared to stimulate tree root production, which in turn was highly correlated with TSOC, resulting in annual increments in TSOC of up to 9.9 Mg ha^?1 year^?1. These early results indicate the promising potential of this silvopastoral system for combining cattle production, and increasing tree cover and carbon sequestration.     

Mechanical damage to pollen aids nutrient acquisition in Heliconius butterflies (Nymphalidae)

Neotropical Heliconius and Laparus butterflies actively collect pollen onto the proboscis and extract nutrients from it. This study investigates the impact of the processing behaviour on the condition of the pollen grains. Pollen samples (n = 72) were collected from proboscides of various Heliconius species and Laparus doris in surrounding habitats of the Tropical Research Station La Gamba (Costa Rica). Examination using a light microscope revealed that pollen loads contained 74.88 ± 53.67% of damaged Psychotria pollen, 72.04 ± 23.4% of damaged Psiguria/Gurania pollen, and 21.35 ± 14.5% of damaged Lantana pollen (numbers represent median ± first quartile). Damaged pollen grains showed deformed contours, inhomogeneous and/or leaking contents, or they were empty. Experiments with Heliconius and Laparus doris from a natural population in Costa Rica demonstrated that 200 min of pollen processing behaviour significantly increased the percentage of damaged pollen of Psychotria compared to pollen from anthers (P = 0.015, Z = ?2.44, Mann–Whitney U-test). Examination of pollen loads from green house reared Heliconius butterflies resulted in significantly greater amounts of damaged Psiguria pollen after 200 min of processing behaviour compared to pollen from flowers (P < 0.001, Z = ?4.583, Mann–Whitney U-test). These results indicate that pollen processing functions as extra oral digestion whereby pollen grains are ruptured to make the content available for ingestion.     

Group hunting by workers of two Neotropical swarm-founding paper wasps, Parachartergus apicalis and Agelaia sp.

Here we report field observations of group hunting by two Neotropical species of paper wasps, Parachartergus apicalis in Costa Rica and Agelaia cf. angulata in Peru. In both cases, multiple workers simultaneously attacked live caterpillar prey. We describe the wasps’ behavior and their interactions with the relatively large-bodied (>80 mm length) caterpillars, and we discuss the implications of these observations for paper wasp behavior and ecology.     

Development and Optimisation of Spironolactone Nanoparticles for Enhanced Dissolution Rates and Stability

Stable solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) formulations to enhance the dissolution rates of poorly soluble drug spironolactone (SP) were being developed. Probe ultra-sonication method was used to prepare SLNs and NLCs. All NLCs contained stearic acid (solid lipid carrier) and oleic acid (liquid lipid content), whereas, SLNs were prepared and optimised by using the solid lipid only. The particles were characterised in terms of particle size analysis, thermal behaviour, morphology, stability and in vitro release. The zeta sizer data revealed that the increase in the concentration of oleic acid in the formulations reduced the mean particle size and the zeta potential. The increase in concentration of oleic acid from 0 to 30% (w/w) resulted in a higher entrapment efficiency. All nanoparticles were almost spherically shaped with an average particle size of about ～170 nm. The DSC traces revealed that the presence of oleic acid in the NLC formulations resulted in a shift in the melting endotherms to a higher temperature. This could be attributed to a good long-term stability of the nanoparticles. The stability results showed that the particle size remained smaller in NLC compared to that of SLN formulations after 6 months at various temperatures. The dissolution study showed about a 5.1- to 7.2-fold increase in the release of the drug in 2 h compared to the raw drug. Comparing all nanoparticle formulations indicated that the NLC composition with a ratio of 70:30 (solid:liquid lipid) is the most suitable formulation with desired drug dissolution rates, entrapment efficiency and physical stability.     

Genetic diversity analysis of the endangered slipper orchid Phragmipedium longifolium in Costa Rica

Phragmipedium longifolium is an endangered terrestrial orchid. In Costa Rica, these plants are found growing in small and isolated patches, some of them consisting of just four to six individuals. Information about the genetic variability within and among populations is very important for the conservation of this endangered species. A total of 160 samples were collected in six locations and analyzed with amplified fragment length polymorphism technique. The genetic diversity of P. longifolium in Costa Rica (Hw = 0.1711) is high and differentiation among sampled locations is moderate (φ_pt = 0.2013) in comparison with results of studies in some other terrestrial orchid species using the same technique. The percentage of polymorphic loci was, on average, 51.5. The analysis of molecular variance (AMOVA) indicated the main genetic variation was within sampled locations (80%), even though the variation among locations was also significant. In situ conservation is recommended because, in addition to protecting habitat and avoiding fragmentation, mycorrhizal fungi and pollinators are also protected. Close proximity between populations is required to maintain high genetic variability through a gene flow continuum. It is suggested that conservation of patches with higher genetic variability be prioritized as many are located in unprotected areas. A germplasm bank for ex situ conservation has been established in the living collection at Lankester Botanical Garden using the plants collected in this study. Finally, a search for new locations of this species is also suggested.     

Doxycycline interferes with quorum sensing-mediated virulence factors and biofilm formation in Gram-negative bacteria

Inhibition of quorum sensing (QS)-regulated virulence factors including biofilm is a recognized anti-pathogenic drug target. The search for safe and effective anti-QS agents is expected to be useful to combat diseases caused by multidrug-resistant bacteria. In this study, effect of a commonly used antibiotic, doxycycline on QS was evaluated using sensor strains of Chromobacterium violaceum (ATCC 12472 and CVO26) and Pseudomonas aeruginosa PAO1. Sub-MICs of doxycycline reduced QS-controlled violacein production in C. violaceum to a significant degree (70 %) and showed a significant reduction of LasB elastase (67.2 %), pyocyanin (69.1 %), chitinase (69.8 %) and protease (65 %) production and swarming motility (74 %) in P. aeruginosa PAO1 over untreated controls. Similar results were also recorded against a clinical strain of P. aeruginosa (PAF-79). Interestingly, doxycycline at respective sub-MICs (4 and 32 μg ml^?1) significantly reduced the biofilm-forming capability and exopolysaccharide production in both the strains of P. aeruginosa (PAO1 and PAF-79) over untreated controls. The results of this study highlight the multiple actions of doxycycline against QS-linked traits/virulence factors and its potential to attenuate virulence of P. aeruginosa.     

New genera and species of ericaceae (Vaccinieae) from Costa Rica and Panama

Two new monotypic genera,Didonica andUtleya, are described, withD. pendula from Panama andU. costaricensis from Costa Rica.Disterigma trimera (Panama),D. utleyorum (Costa Rica, Panama, Colombia, and Ecuador),Lateropora santafeensis (Panama),Lysiclesia panamensis (Panama),Macleania talamancensis (Costa Rica),Themistoclesia costaricensis (Costa Rica) andT. horquetensis (Panama),Vaccinium costaricense andV. orosiense (both from Costa Rica) and V.jefense (Panama) are all described as new. New combinations are provided for the PanamanianVaccinium floccosum (=Symphysia floccosa) and the West IndianVaccinium racemosum (=Symphysia racemosa). Keys are provided for the Central American species ofDisterigma andThemistoclesia, the species ofLateropora andLysiclesia, and the Costa Rican and Panamanian species ofVaccinium. Six new species are illustrated.     

Histopathology and effect on development of the PhopGV on larvae of the potato tubermoth, Phthorimaea operculella (Lepidoptera: Gelechiidae)

Larvae of the potato tubermoth (PTM), Phthorimaea operculella, feed on potato plants and tubers and are a major pest in the tropics and subtropics worldwide, causing up to 100% damage. The PTM granulovirus (PhopGV) provides significant potato protection, but little is known about its effect on larval development or its histopathology. Here we show that only 10% of larvae exited from PhopGV-treated tubers (1.4 × 10⁸ granule/ml), lagging significantly behind controls, and most of these died by 72 h after emergence. Histopathology studies showed the fat body and epidermis were the principal tissues infected. PhopGV morphogenesis was similar to other GVs, the exception being small vesicles between mature granules.     

A new species of Eimeria Schneider, 1875 (Apicomplexa: Eimeriidae) from the dusky rice rat Melanomys caliginosus (Tomes) (Mammalia: Rodentia) in Costa Rica

We collected faecal samples from 24 dusky rice rats, Melanomys caliginosus (Tomes) (Rodentia: Cricetidae: Sigmodontinae), in a Biological Reserve in Costa Rica, and found three (12.5%) to be infected with a species of Eimeria Schneider, 1875, which we describe here as new. Sporulated oöcysts of Eimeria caliginosa n. sp. are almost spheroidal and measure 16–21 × 17–20 (mean 19.6 × 18.2) μm; micropyle, oöcyst residuum and polar granule are absent. Sporocysts are ovoidal, 9–13 × 6–8 (mean 11.2 × 6.7) μm, with small Stieda and sub-Stieda bodies present, but a para-Stieda body is absent; the sporocyst residuum is a compact mass of c.11–15 granules, c.5 μm wide. Sporozoites are crescent-shaped, 5–8 × 2–3 (mean 6.8 × 2.4) μm. This is the third species of Eimeria described from the genus Melanomys Thomas.     

A new Costa Rican species of Drosophila visiting inflorescences of the hemi-epiphytic climber Monstera lentii (Araceae)

Drosophila monsterae sp. nov. is described from 11 males and 13 females collected from the inside of closed inflorescences of Monstera lentii (Araceae) at 1810 m altitude in the Forest Reserve of Cerro de La Carpintera, Canton La Unión, Province of Cartago, Costa Rica. Although flies have been seen wandering and copulating inside the floral chambers of closed inflorescences during the floral female phase, eggs or larvae have not yet been found either in the spathe or in the fleshy spadix. The new species is related to Drosophila tristani Sturtevant, 1921, from San José, Costa Rica, from which it differs mainly by having smaller slightly circular compound eyes, distinctly broader genae (cheek index ca. 2.4 vs 5 in D. tristani), and the inner capsule of spermathecae with an unusual folded duct at basal half of its very wide introvert. This is the eighth species to be included in the New World, essentially Neotropical, subgenus Phloridosa. Photomicrographs of male and female terminalia are also provided.     

Review and redescription of species in the Oecetis avara group,with the description of 15 new species (Trichoptera,Leptoceridae)

The O. avara group of Oecetis is formally defined to include 4 described species, O. avara (Banks), O. disjuncta (Banks), O. elata Denning & Sykora, and O. metlacenis Bueno-Soria, and 15 new species. Oecetis marquesi Bueno-Soria, previously considered a member of the O. avara group, is treated as incertae sedis to species group, but is also redescribed and treated in the current work. New species described here (with their respective distributions) include: O. acciptrina (Costa Rica, Panama, Ecuador), O. agosta (Mexico), O. angularis (Guatemala to Ecuador), O. apache (SW USA), O. campana (Ecuador), O. constricta (Mexico to Ecuador, Venezuela, and Trinidad), O. houghtoni (North America), O. maritza (Costa Rica), O. mexicana (Mexico to Ecuador), O. patula (Guatemala, Nicaragua), O. protrusa (Mexico to Ecuador), O. sordida (Mexico, USA, Canada), O. tumida (Costa Rica), O. uncata (Costa Rica), and O. verrucula (Mexico to Costa Rica). A key to the species is also provided.     

Comparison of aerial conidia and blastospores from two entomopathogenic fungi against Diaphorina citri (Hemiptera: Liviidae) under laboratory and greenhouse conditions

This study compared the insecticidal activity of liquid culture-produced blastospores and solid substrate-produced aerial conidia of Beauveria bassiana GHA and Isaria fumosorosea ARSEF3581 strains against Diaphorina citri adults. Insects exposed to 10⁷ propagules/ml in a spray residue contact leaf bioassay died within 6 days at 25°C, with no significant differences between fungal treatments. At higher concentrations (10⁸ propagules/ml), I. fumosorosea conidia killed psyllids faster compared to its blastospore formulation, i.e. 4 versus 5 days, respectively. In greenhouse tests, the same treatments applied to infested citrus plants (2?×?10⁶ spores/ml) all significantly reduced the number of nymphs compared with the untreated controls over 3 weeks; however, only I. fumosorosea blastospores significantly reduced the number of F1 adult psyllids when compared with controls. Similar results were observed in the follow-up greenhouse test, where I. fumosorosea blastospores were the most effective treatment overall, reducing D. citri populations by about 60% after 21 days; by contrast, imidacloprid killed almost 100% of psyllids within a week in both tests. Fewer psyllids exhibited mycosis in the greenhouse (i.e. ≈20 versus?≥?87% in the laboratory). This is the first report comparing both conidial and blastospore formulations of B. bassiana and I. fumosorosea for the control of a psyllid pest. Field testing is required to determine how successful different spore formulations might be under various environmental conditions.     

Immobilized anaerobic fermentation for bio-fuel production by Clostridium co-culture

Clostridium thermocellum/Clostridium thermolacticum co-culture fermentation has been shown to be a promising way of producing ethanol from several carbohydrates. In this research, immobilization techniques using sodium alginate and alkali pretreatment were successfully applied on this co-culture to improve the bio-ethanol fermentation performance during consolidated bio-processing (CBP). The ethanol yield obtained increased by over 60 % (as a percentage of the theoretical maximum) as compared to free cell fermentation. For cellobiose under optimized conditions, the ethanol yields were approaching about 85 % of the theoretical efficiency. To examine the feasibility of this immobilization co-culture on lignocellulosic biomass conversion, untreated and pretreated aspen biomasses were also used for fermentation experiments. The immobilized co-culture shows clear benefits in bio-ethanol production in the CBP process using pretreated aspen. With a 3-h, 9 % NaOH pretreatment, the aspen powder fermentation yields approached 78 % of the maximum theoretical efficiency, which is almost twice the yield of the untreated aspen fermentation.