Biology and Ecology of Delia planipalpis (Stein) (Diptera: Anthomyiidae), an Emerging Pest of Broccoli in Mexico.

Delia planipalpis (Stein) (Diptera: Anthomyiidae) is a pest of crucifers, such as broccoli, radish, cauliflower, turnip and cabbage. It has been recently described in Mexico as a significant emerging pest of broccoli. Due the lack of knowledge of this pest, the present study aimed to determine its life cycle, female sexual maturation, copulation, oviposition behavior and adult longevity. The identity of the fly in Mexico was confirmed genetically by sequencing the cytochrome oxidase subunit 1 gene (COI). The mean development time of D. planipalpis was 32-33 days on radish at 24 °C under laboratory conditions. Females became sexually mature 1-2 days after emergence, and the highest incidence of matings was recorded on the second day (60%). Under choice conditions, D. planipalpis females preferred to oviposit on radish plants, rather than broccoli plants, possibly due to the use of radish for rearing the laboratory colony. Oviposition and the mean number of eggs laid varied among the broccoli varieties, with the highest oviposition observed on the Tlaloc variety. Repeated attempts to rear the laboratory colony on broccoli plants failed. Radish-reared insects of both sexes lived longer when individualized in the adult stage (14.5-22.5 days) than when adult flies were maintained in groups (10-11 days). This study contributes to the understanding of D. planipalpis biology and provides information that can be used to establish future control strategies against this pest.

Drosophila suzukii Management in Latin America: Current Status and Perspectives.

Spotted-wing drosophila, Drosophila suzukii Matsumura, was first established in Latin America in Mexico in 2011. The vinegar fly has since been detected in 296 municipalities in Argentina, Brazil, Chile, Mexico, and Uruguay. Drosophila suzukii is polyphagous and is found on 64 host plants in 25 families in Latin America, with most hosts also exotic species. In Latin America, D. suzukii is attacked by 14 species of parasitoid wasps in the families Diapriidae, Figitidae, and Pteromalidae, which are promising native parasitoids for control of the pest. This article analyzes results from studies on monitoring, biological, chemical, and cultural control, and sterile insect techniques to provide a basis for the development of area-wide and sustainable D. suzukii management programs in Latin America. The review examines how D. suzukii has been managed in Latin America and how research conducted in this region can contribute to management of the species in other parts of the world.

Addition of Ammonium Acetate to Protein-Borax Baited Traps Does Not Improve Attraction of Anastrepha obliqua or Anastrepha serpentina (Diptera: Tephritidae).

Ammonia is considered a key component in the attraction of tephritid flies to protein-based lures. The addition of ammonium acetate to improve hydrolyzed protein-borax mixtures used to monitor tephritids has not been evaluated, although it has improved attraction to toxic baits. The presence of ammonium acetate crystals in a ventilated tube inside a trap containing hydrolyzed protein + borax, did not improve the capture of Anastrepha obliqua or Anastrepha serpentina flies in field experiments when compared with hydrolyzed protein + borax alone. In contrast, the addition of 1% ammonium acetate into the drowning solution of a hydrolyzed protein + borax mixture resulted in significantly reduced captures of both pests. Laboratory tests indicated that the emission of ammonia gas was increased 1.6-4.5-fold from traps that included ammonium acetate. These results confirm the hypothesis that a higher release of ammonia does not improve the attraction of tephritids when protein-derived odors are also present.

Does Ammonia Released from Protein-Based Attractants Modulate the Capture of Anastrepha obliqua (Diptera: Tephritidae)?

Tephritid fly responses to food-based attractants involve a complex range of food-derived semiochemicals, including ammonia. We performed laboratory and field experiments to compare the attraction of Anastrepha obliqua (Macquart) to ammonia with the attraction to commercial food attractants and torula yeast at a range of pHs. A positive correlation was established between the concentration of ammonia in solution (1.5-150 mM ammonium solution) and gaseous ammonia released by bottle-type traps. This resulted in an asymptotic response in captures of A. obliqua flies in traps that released 99-295 µg ammonia/h. Pairwise comparisons in laboratory cages revealed that traps baited with 150 mM ammonia solution captured similar numbers of A. obliqua as traps baited with Biolure 2C, CeraTrap, and hydrolyzed protein products (Captor, Winner, and Flyral) plus borax, despite the low quantities of ammonia (11-56 µg/h) released from these attractants. Subsequent choice experiment captures in traps containing ammonia solution were similar or higher than those of commercial attractants, with the exception of Winner + borax, but were not correlated with the ammonia released from attractants. Captures of flies in traps containing ammonia solution were increased by the addition of 1% torula yeast or torula yeast alkalized with sodium hydroxide or borax despite differences in the quantities of ammonia released. Fly captures generally increased with increasing alkalization of torula yeast (pH 7.5-9.5). In the field, torula yeast in ammonia solution captured similar numbers of A. obliqua flies as Captor + borax when traps were evaluated after 24 h but not after a 7-day trapping period. Traps baited with ammonia solution or Winner + borax were significantly less attractive than Captor + borax in both field experiments. We conclude that A. obliqua flies are attracted to ammonia solutions of increasing concentration, up to 150 mM, in the absence of other stimuli, whereas attraction to commercial attractants or alkalized torula yeast is not correlated with the release of ammonia.

Agricultural-Grade Apple Cider Vinegar Is Remarkably Attractive to Drosophila suzukii (Diptera: Drosophiliadae) in Mexico.

Due to its availability and low cost, apple cider vinegar (ACV) is a frequently used as an attractant for monitoring the invasive spotted wing drosophila, Drosophila suzukii. In laboratory cage experiments, the attraction of ACV alone was compared with ACV in mixtures with different concentrations of acetic acid, propionic acid, different hydrolyzed proteins, synthetic fruit flavors (strawberry, blackberry and apple) and the addition of fruit nectars (grape, pineapple and apple). The addition of 5% apple nectar to ACV significantly increased fly captures, whereas other combinations were similar to or less attractive than ACV alone. Apple flavored vinegar was not attractive to flies. Captures did not vary significantly among the brands of ACV commonly sold in Mexico, except for one poorly-performing brand, but cup traps baited with an agricultural-grade ACV unfit for human consumption captured approximately two-fold more flies than the commercial attractants Suzukii Trap, Suzukii Trap Max Captures or ACV alone in cage experiments. Field trials performed in polytunnels planted with raspberry crops in Mexico resulted in two-fold to ten-fold higher numbers of D. suzukii captured by the agricultural-grade ACV compared to Droskidrink (a mixture of ACV, red wine and sugar), Suzukii Trap, Suzukii Trap Max Captures or edible grade ACV alone. The species selectivity of the agricultural grade ACV was similar to that of other attractants tested. Agricultural-grade ACV also captured higher numbers of female than male flies in field trials. We conclude that the remarkably high attractiveness and low cost of agricultural-grade ACV makes it a useful tool for monitoring D. suzukii populations in berry crops.

Yeast Species, Strains, and Growth Media Mediate Attraction of Drosophila suzukii (Diptera: Drosophilidae).

Specific ecological interactions between insects and microbes have potential in the development of targeted pest monitoring or control techniques for the spotted wing drosophilid, Drosophila suzukii (Matsumura), an exotic invasive pest of soft fruit. To evaluate D. suzukii attraction to yeast species from preferred types of fruit, three yeasts were isolated from blackberry fruit and two yeasts from raspberry fruit and used to bait simple plastic bottle traps. Saccharomyces cerevisiae and Hanseniaspora uvarum were identified from blackberries, whereas a different H. uvarum strain was identified from raspberry. Yeast identification was based on sequence analysis of the D1/D2 domain of the large subunit 26S rRNA gene. Commercial baker's yeast (S. cerevisiae) was similar or more effective for the capture of D. suzukii males and females than yeasts isolated from blackberry or raspberry when grown in sucrose. However, when grown in corn syrup, a strain of S. cerevisiae from blackberry captured the highest number of females and a strain of H. uvarum isolated from raspberry captured high numbers of males and females. Species of Candida, Hanseniaspora, and Pichia from a laboratory yeast collection did not outperform baker's yeast in pairwise tests when grown in sucrose solution or yeast-peptone-dextrose medium. The raspberry strain of H. uvarum grown in corn syrup outperformed S. cerevisiae grown in sucrose, in terms of captures in baited traps under laboratory conditions. We conclude that yeast species, strain, and growth medium can have a marked influence on D. suzukii attraction to baited traps, a finding that could assist in the development of yeast-related monitoring or control techniques targeted at this pest.

The Effect of Seasonal Humidity on Survival and Duration of Dormancy on Diverging Mexican Rhagoletis pomonella (Diptera: Tephritidae) Populations Inhabiting Different Environments.

Phytophagous insects synchronize emergence with plant phenology by engaging in dormancy during periods of host scarcity and environmental stress. Regulation of dormancy is achieved through response to seasonal cues. While temperature and photoperiod are important cues in temperate latitudes, seasonal humidity, such as the onset of rains, can be a reliable cue to for synchronization of emergence and affects survival of overwintering insects. We compared response of Mexican Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) populations inhabiting subtropical environments differing in humidity patterns, to seasonal humidity regimes. Both populations emerged as adults in high proportions and suffered lower mortality under humidity regimes ending with a humid summer, but the effect was more pronounced for the Eje Volcanico Trans Mexicano (EVTM) population, which inhabits a dryer environment and undergoes longer dormancy. While there were no differences among pupae from the Sierra Madre Oriental (SMO) in percent of non-emerged pupae surviving and engaging in prolonged dormancy after a year, EVTM pupae exposed to an initial humid period engaged in prolonged dormancy in higher proportions than those exposed to other regimes. Seasonal humidity had little effect on the duration of dormancy, but EVTM pupae exposed to consecutive dry periods took longer to emerge than those exposed to other regimes. Our results suggest that rather than being used as a token stimulus, humidity affected survival of overwintering R. pomonella, especially at the end of dormancy when energy reserves are depleted and there is an increase in metabolic rate that renders EVTM pupae more susceptible to desiccation.

The Effect of Winter Length on Duration of Dormancy and Survival of Rhagoletis completa (Diptera: Tephritidae) and Associated Parasitoids From Northeastern Mexico.

The walnut husk fly Rhagoletis completa (Cresson), native to the Midwestern United States and Mexico, is invasive in California and Europe. It is one of the most important pests of walnuts in areas gathering 30% of the world production. Knowledge of life-history regulation is important for the design of management strategies. Research on dormancy has been performed on invasive populations, and not on populations at the southern extreme of its native range. Here, we examined the effect of winter length on fly and parasitoid emergence, survival, and duration of dormancy. Percent emergence was higher for chill periods at 5°C ranging from 8 to 20 wk. No or insufficient chill resulted in low emergence and a significant proportion of individuals in prolonged dormancy (>1 yr). Duration of dormancy was longer for pupae at constant temperatures and a 4-wk chill period than longer winter durations. Dormancy was longer for Mexican than that reported for U.S. populations, suggesting the existence of a latitudinal cline where populations at southern latitudes have evolved slower metabolic rates. Three parasitoid species were found associated with R. completa (Aganaspis alujai (Wharton and Ovruski) (Hymenoptera: Figitidae), Diachasmimorpha juglandis Muesebeck, and Diachasmimorpha mellea Gahan) (Hymenoptera: Braconidae). Results suggest that rearing of R. completa is possible by subjecting pupae to chill periods between 8 and 20 wk. Overwintering mortality of flies and A. alujai could be further reduced above 5°C. Our findings can contribute for the accurate development of predictive models on invasion potential, development, fly and parasitoid rearing, and biological control.

Raspberry as a Source for the Development of Drosophila suzukii Attractants: Laboratory and Commercial Polytunnel Trials.

Several commercial products and home-made attractants have been developed for monitoring and mass-trapping of the spotted wing drosophila, Drosophila suzukii. Growers in Mexico have adopted an attractant based on a fermenting mixture of raspberry pulp and sucrose, with anecdotally promising results. We compared the capture rates of traps baited with raspberry pulp + sucrose with captures from a range of alternative attractants. Raspberry pulp alone or with sucrose was more attractive than apple cider vinegar (ACV) or SuzukiiTrap and similar to baker's yeast + sucrose in laboratory cage studies. Synthetic raspberry aroma (0.1-10% concentration), in water or mixed with ACV, did not improve capture rates in the laboratory. Traps baited with raspberry + sucrose or ACV had similar captures of D. suzukii in raspberry or blackberry polytunnels in Michoacán, Mexico. Raspberry + sucrose baited traps captured significantly higher numbers of other drosophilid species, leading to higher total numbers of captured flies (all species), which may explain why Mexican growers favor the raspberry-based attractant. The commercial products SuzukiiTrap and Z-Kinol had lower captures than ACV in polytunnels, although SuzukiiTrap had the highest selectivity in captures of D. suzukii (81% of flies captured). A two-component trap (2C trap) baited with ACV + ethanol as the drowning solution and raspberry pulp + sucrose or baker's yeast + sucrose in a ventilated tube device was markedly more effective than the trap currently used by growers. We conclude that raspberry pulp + sucrose is as effective for the attraction of D. suzukii as ACV under commercial polytunnel conditions. The 2C trap performed better than the transparent cup trap currently used by berry producers in Mexico.

Sickly Sweet: Insecticidal Polyols Induce Lethal Regurgitation in Dipteran Pests.

Polyols are commonly used in food and medicines as sweeteners and preservatives but may also have insecticidal properties against some species of Diptera. Here we compared the insecticidal activity and feeding response of glycerol and propylene glycol (PG) on two tephritids: Anastrepha ludens and Anastrepha obliqua, and the drosophilid Drosophila suzukii. First, flies were exposed to solutions of 50% sucrose and the two polyols at concentrations of 1.67 M, 2.78 M and 4.18 M for 24 h and then observed at 24 h intervals for a period of three days. Both polyols elicited strong regurgitation behavior in the three flies and killed them. Regurgitation apparently also reduced flies' body weight, and this was particularly apparent in insects that fed on 4.18 M PG solutions. A high percentage of individuals exposed to PG solutions perished after 72 hours. The number of proboscis extensions, which is associated with feeding preference, was lower in the 4.18 M polyols + sucrose mixtures than in the 50% sucrose solution. Glycerol had a lower insecticidal effect in Anastrepha spp. and very little insecticidal effect in D. suzukii. Finally, elevated regurgitation and mortality was confirmed in A. ludens treated with 1.0⁻2.78 M of erythritol plus sucrose. Our results demonstrate that PG, and to a lower extent glycerol, have the potential for being used as a safer method of insect pest control. The hyper-regurgitation response may contribute to the insecticidal properties of these polyols in Diptera.

The Fruit Fly Lure CeraTrap: An Effective Tool for the Study of the Arboreal Ant Fauna (Hymenoptera: Formicidae).

Despite preliminary reports of ants trapped in food-baited fruit fly traps, little is known regarding the identity of the myrmecofauna that can be sampled using this technique. This study aimed to examine the inventory completeness, activity and species occurrence of canopy ant assemblages collected in baited traps used for monitoring fruit flies in different fruit orchards in central Veracruz, Mexico. The trap models used in the sampling were Multilure, McPhail glass, and 500 ml blue polyethylene bottles. Three commercial fruit fly food attractants (CeraTrap, Captor + Borax, and BioLure) and two grape juice products (Jumex grape juice and Tang) were used as baits for sampling. In total 3,626 ant workers belonging to 54 species, 19 genera, 10 tribes, and 5 subfamilies were collected. Among the five food attractants used in this study, CeraTrap recorded a markedly higher inventory completeness, ant activity and species occurrence per trap. This study reports for the first time the use of CeraTrap, as a promising and effective food attractant for collecting the foraging ants in the canopy of agroecosystems, which may be applicable to other habitats such as natural forests, mangroves, or agricultural settings such as coffee plantations.

Experimental hybridization and reproductive isolation between two sympatric species of tephritid fruit flies in the Anastrepha fraterculus species group.

Among tephritid fruit flies, hybridization has been found to produce local adaptation and speciation, and in the case of pest species, induce behavioral and ecological alterations that can adversely impact efficient pest management. The fraterculus species group within Anastrepha (Diptera: Tephritidae), is a rapidly radiating aggregate, which includes cryptic species complexes, numerous sister species, and several pest species. Molecular studies have highlighted the possibility of introgression between A. fraterculus and A. obliqua. Reproductive isolation has been studied among morphotypes of the A. fraterculus species complex as a tool for species delimitation. Here we examined the existence and strength of prezygotic and postzygotic isolation between sympatric populations of two closely related species within the highly derived fraterculus group (A. fraterculus and A. obliqua), coexisting in nature. Although adults of both species showed a strong tendency for assortative mating, a small proportion of hybrid pairings in both directions were observed. We also observed asymmetric postzygotic isolation, with one hybrid cross displaying a strong reduction in fecundity and F1 egg fertility. Survival was greater for the progeny of homotypic and hybrid crosses in the maternal host. There was a marked female biased sex ratio distortion for both F1 hybrid adults. Hybridization between A. fraterculus and A. obliqua in nature may be difficult but possible; these two species display stronger reproductive isolation than all pairs of species previously examined in the A. fraterculus species complex. Asymmetric postzygotic isolation is suggestive of Wolbachia mediated cytoplasmic incompatibilities that may be exploited in area-wide pest management.

Improved capture of Drosophila suzukii by a trap baited with two attractants in the same device.

The improvement of trap-lure combinations is an important part of integrated pest management programs that involve monitoring pests for timely insecticide applications, or for their use in control strategies such as mass trapping or bait stations. In this study improvements in the capture of Drosophila suzukii were not observed following the inclusion of different color stimuli with respect to a red-black stripe cup trap. This red-black stripe trap with a hemispherical dome-shaped lid had a significantly improved physical retention of flies compared to traps fitted with a flat lid. Retention was further improved when an additional tube device, which could be baited with a supplemental attractant, was introduced through the dome-shaped lid. Under laboratory conditions, this trap, in which apple cider vinegar + 10% ethanol was present as the drowning solution and the additional tube device was baited with a fermenting mixture of sugar and yeast, was significantly more effective in catching D. suzukii flies than other conventional attractants or a commercial lure. The capture rate of this trap-lure combination remained higher than that of a commercial lure, even after 20 days of use under laboratory conditions. In a guava orchard this trap was 15-fold more effective in catching D. suzukii flies than similar traps baited with apple cider vinegar alone, 4 to 7 fold more effective than similar traps baited with a commercial lure, and 1.7-fold more effective than a fermenting mixture of yeasts and wheat flour. In commercial blackberry orchards, this trap was 6-fold more effective in trapping D. suzukii flies than the clear trap baited with apple cider vinegar used by growers. The efficacy of this trap presents a promising line of future research for monitoring and control of D. suzukii and likely other drosophilid pests.

Baculovirus-Induced Climbing Behavior Favors Intraspecific Necrophagy and Efficient Disease Transmission in Spodoptera exigua.

Shortly prior to death, many species of Lepidoptera infected with nucleopolyhedrovirus climb upwards on the host plant. This results in improved dissemination of viral occlusion bodies over plant foliage and an increased probability of transmission to healthy conspecific larvae. Following applications of Spodoptera exigua multiple nucleopolyhedrovirus for control of Spodoptera exigua on greenhouse-grown sweet pepper crops, necrophagy was observed by healthy S. exigua larvae that fed on virus-killed conspecifics. We examined whether this risky behavior was induced by olfactory or phagostimulant compounds associated with infected cadavers. Laboratory choice tests and olfactometer studies, involving infected and non-infected cadavers placed on spinach leaf discs, revealed no evidence for greater attraction of healthy larvae to virus-killed over non-infected cadavers. Physical contact or feeding on infected cadavers resulted in a very high incidence of transmission (82-93% lethal disease). Observations on the behavior of S. exigua larvae on pepper plants revealed that infected insects died on the uppermost 10% of foliage and closer to the plant stem than healthy conspecifics of the same stage, which we considered clear evidence of baculovirus-induced climbing behavior. Healthy larvae that subsequently foraged on the plant were more frequently observed closer to the infected than the non-infected cadaver. Healthy larvae also encountered and fed on infected cadavers significantly more frequently and more rapidly than larvae that fed on non-infected cadavers. Intraspecific necrophagy on infected cadavers invariably resulted in virus transmission and death of the necrophagous insect. We conclude that, in addition to improving the dissemination of virus particles over plant foliage, baculovirus-induced climbing behavior increases the incidence of intraspecific necrophagy in S. exigua, which is the most efficient mechanism of transmission of this lethal pathogen.

Efficacy of commercial traps and food odor attractants for mass trapping of Anastrepha ludens (Diptera: Tephritidae).

One of the most important factors for the success of a mass trapping strategy to control a fruit fly involves the selection of an effective trap-lure combination. Because different species of fruit flies respond differently to the physical characteristics of a trap and to bait volatiles, the evaluation of commercial traps and lures that have proved useful against other tephtrids is necessary to determine their efficacy for mass trapping of Anastrepha ludens (Loew) (Diptera: Tephritidae). Under caged conditions, a commercial hemispherical trap with lateral holes (Maxitrap Plus) proved more attractive to A. ludens (both sexes) than five other commercial traps that were all baited with hydrolyzed protein. Among these traps, bottom invaginated traps and traps with invaginated lateral holes constructed with transparent cylinders had the best physical retention properties. When evaluated under field conditions, the lure was critical for the efficacy of the trap, and one of the traps that performed poorly in attraction and retention cage tests (MS2) resulted as one of the most effective traps when baited with CeraTrap lure. Considering the use of different trap models under field conditions, CeraTrap liquid bait was more effective in A. ludens capture than Biolure dry synthetic bait, but both lures were not replaced during the entire course of the experiment. The percentage of captured females was also slightly higher using CeraTrap lure (67.2%) than using Biolure baits (54.5-58.8%). In field tests, 75-81% of females were mated and no significant differences were observed among trap-lure combinations. Trap selectivity against nontarget adult lacewings also differed among trap-lure combinations.

Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo.

BACKGROUND: Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. RESULTS: Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in insect larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after insect processing (1 L extract represents about 125 g of insect biomass or about 375 insect larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. CONCLUSIONS: Our results open up the possibility of using insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that insects have been used as living biofactories to produce a VHH molecule.

Humoral and in vivo cellular immunity against the raw insect-derived recombinant Leishmania infantum antigens KMPII, TRYP, LACK, and papLe22 in dogs from an endemic area.

Leishmania infantum causes visceral leishmaniasis, a severe zoonotic and systemic disease that is fatal if left untreated. Identification of the antigens involved in Leishmania-specific protective immune response is a research priority for the development of effective control measures. For this purpose, we evaluated, in 27 dogs from an enzootic zone, specific humoral and cellular immune response by delayed-type hypersensitivity (DTH) skin test both against total L. infantum antigen and the raw Trichoplusia ni insect-derived kinetoplastid membrane protein-11 (rKMPII), tryparedoxin peroxidase (rTRYP), Leishmania homologue of receptors for activated C kinase (rLACK), and 22-kDa potentially aggravating protein of Leishmania (rpapLe22) antigens from this parasite. rTRYP induced the highest number of positive DTH responses (55% of leishmanin skin test [LST]-positive dogs), showing that TRYP antigen is an important T cell immunogen, and it could be a promising vaccine candidate against this disease. When TRYP-DTH and KMPII-DTH tests were evaluated in parallel, 82% of LST-positive dogs were detected, suggesting that both antigens could be considered as components of a standardized DTH immunodiagnostic tool for dogs.

Insecticidal properties and microbial contaminants in a Spodoptera exigua multiple nucleopolyhedrovirus (Baculoviridae) formulation stored at different temperatures.

The Spodoptera exigua (Hübner) multiple nucleopolyhedrovirus (SeMNPV) is currently being tested as a biological insecticide for use in greenhouse crops in southern Spain. We performed a study in which semipurified SeMNPV occlusion bodies (OBs) were formulated in phosphate-buffered saline, pH 6.5, with 5% (vol:vol) glycerol and 0.15% (wt:vol) sorbic acid, and they were stored at -20, 4, or 25 degrees C during 18 mo. Initial aerobic counts (+/-SE) averaged 1.4 (+/-0.17) x 10(7) colony-forming units/ml after 17-h incubation at 37 degrees C. Aerobic counts of microorganisms that contaminated OB formulations stored at 250C decreased markedly over the period of the study, whereas only small decreases were observed in counts from OBs stored at 4 or -20 degrees C. The principal microbial contaminants of OB suspensions were Enterococcus spp., Enterobacteriaceae, and yeasts. Potential human pathogens (Salmonella, Shigella, and Vibrio species) were not detected, and populations of Staphylococcus aureus and Bacillus cereus were extremely low. Compared with newly formulated OBs, the estimated LD50 values of OBs stored at 25 degrees C increased by >16,666-fold over the 18 mo of storage, whereas LD50 values were not greatly affected by storage at 4 or -20 degrees C. Significant changes over time in OB concentrations were only observed in the 25 degrees C treatment. Complete degradation of viral DNA was observed at 25 degrees C but not in refrigerated or frozen OBs. We conclude that OB formulation with bacteriostatic or antioxidant additives, together with storage and distribution in refrigerated conditions, will likely result in an SeMNPV biopesticide shelf life that exceeds 18 mo.

Effect of tinopal LPW on the insecticidal properties and genetic stability of the nucleopolyhedrovirus of Spodoptera exigua (Lepidoptera: Noctuidae).

The influence of an optical brightener, Tinopal LPW, on the activity of a purified genotype of the nucleopolyhedrovirus (SeMNPV) of the beet armyworm, Spodoptera exigua (Hübner), was determined in second to fifth instar (L2-L5) S. exigua. When mixed with viral occlusion bodies (OB) 1% Tinopal LPW significantly reduced the median lethal dose (LD50) of the virus in all instars compared with insects treated with SeMNPV alone. Levels of enhancement, as determined by LD50 values, ranged from 2.6- to 580-fold, depending on the instar. The greatest enhancement occurred on the two later instars, L4 (70-fold) and L5 (580-fold), which show a much higher resistance to SeMNPV infection than earlier instars. The median time to death (MTD) values were not significantly different in any instar among larvae treated with SeMNPV + Tinopal LPW and those treated with SeMNPV alone. Larval development in SeMNPV + Tinopal LPW treated larvae was retarded, in second and fourth instars, compared with controls or larvae treated with SeMNPV alone. The OB yields from SeMNPV treated larvae were almost 1.6-fold greater in second instars (9.3 x 10(6) OBs/larvae), and 1.9-fold greater in fourth instars (1.9 x 10(8) OBs/larvae), than those obtained in larvae treated with SeMNPV + Tinopal LPW. The addition of 1% Tinopal LPW to the virus suspension did not alter the genotypic composition of viral progeny during four successive passages of the virus.