(Table 1) Hepatic EROD activity and and retinoid concentrations in the liver, and plasma thyroid hormones of northern fulmars (Fulmarus glacialis) from Bjørnøya

We investigated whether the hepatic cytochrome P450 1A activity (measured as 7-ethoxyresorufin-O-deethylase (EROD)) and plasma thyroid hormone and liver retinoid concentrations were explained by liver and blood levels of halogenated organic contaminants (HOCs) in free-ranging breeding northern fulmars (Fulmarus glacialis) from Bjornoya in the Norwegian Arctic. Hepatic EROD activity and liver levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) were positively correlated, suggesting that hepatic EROD activity is a good indicator for dioxin and dioxin-like HOC exposure in breeding northern fulmars. There were not found other strong relationships between HOC concentrations and hepatic EROD activity, plasma thyroid or liver retinoid concentrations in the breeding northern fulmars. It is suggested that the HOC levels found in the breeding northern fulmars sampled on Bjornoya were too low to affect plasma concentrations of thyroid hormones and liver levels of retinol and retinyl palmitate, and that hepatic EROD activity is a poor indicator of polychlorinated biphenyl (PCB) and pesticide exposure.

Spatio-temporal dynamics of viruses are differentially affected by parasitoids depending on the mode of transmission by their insect vectors.

Relationships between agents in multitrophic systems are complex and very specific. Insect-transmitted plant viruses are completely dependent on the behaviour and distribution patterns of their vectors. The presence of natural enemies may directly affect aphid behaviour and spread of plant viruses, as the escape response of aphids might cause a potential risk for virus dispersal. The spatio-temporal dynamics of Cucumber mosaic virus (CMV) and Cucurbit aphid-borne yellows virus (CABYV), transmitted by Aphis gossypii in a non-persistent and persistent manner, respectively, were evaluated at short and long term in the presence and absence of the aphid parasitoid, Aphidius colemani. SADIE methodology was used to study the distribution patterns of both the virus and its vector, and their degree of association. Results suggested that parasitoids promoted aphid dispersion at short term, which enhanced CMV spread, though consequences of parasitism suggest potential benefits for disease control at long term. Furthermore, A. colemani significantly limited the spread and incidence of the persistent virus CABYV at long term. The impact of aphid parasitoids on the dispersal of plant viruses with different transmission modes is discussed.

Insect growth regulators as potential insecticides to control olive fruit fly (Bactrocera oleae Rossi): insect toxicity bioassays and molecular docking approach

Olive fruit fly, Bactrocera oleae (Rossi), is a key pest in olive orchards, causing serious economic damage. To date, the pest has already developed resistance to the insecticides commonly applied to control it. Thus, in searching for new products for an accurate resistance management programme, targeting the ecdysone receptor (EcR)might provide alternative compounds for use in such programmes. RESULTS: Residual contact and oral exposure in the laboratory of B. oleae adults to the dibenzoylhydrazine-based compounds methoxyfenozide, tebufenozide and RH-5849 showed different results. Methoxyfenozide and tebufenozide did not provoke anynegative effectsontheadults,but RH-5849 killed98-100%of the treated insects15 days after treatment. Theligand-binding domain (LBD) of the EcR of B. oleae (BoEcR-LBD) was sequenced, and a homology protein model was constructed. Owing to a restricted extent of the ligand-binding cavity of the BoEcR-LBD, docking experiments with the three tested insecticides showed a severe steric clash in the case of methoxyfenozide and tebufenozide, while this was not the case with RH-5849. CONCLUSION: IGR molecules similar to the RH-5849 molecule, and different from methoxyfenozide and tebufenozide, might have potential in controlling this pest.

Integration of physical, chemical and biological tactics against insect pests and virus diseases in horticultural crops

Actualmente, la gestión de sistemas de Manejo Integrado de Plagas (MIP) en cultivos hortícolas tiene por objetivo priorizar los métodos de control no químicos en detrimento del consumo de plaguicidas, según recoge la directiva europea 2009/128/CE ‘Uso Sostenible de Plaguicidas’ (OJEC, 2009). El uso de agentes de biocontrol como alternativa a la aplicación de insecticidas es un elemento clave de los sistemas MIP por sus innegables ventajas ambientales que se utiliza ampliamente en nuestro país (Jacas y Urbaneja, 2008). En la región de Almería, donde se concentra el 65% de cultivo en invernadero de nuestro país (47.367 ha), MIP es la principal estrategia en pimiento (MAGRAMA, 2014), y comienza a serlo en otros cultivos como tomate o pepino. El cultivo de pepino, con 8.902 ha (MAGRAMA, 2013), tiene un protocolo semejante al pimiento (Robledo et al., 2009), donde la única especie de pulgón importante es Aphis gossypii Glover. Sin embargo, pese al continuo incremento de la superficie de cultivo agrícola bajo sistemas MIP, los daños originados por virosis siguen siendo notables. Algunos de los insectos presentes en los cultivos de hortícolas son importantes vectores de virus, como los pulgones, las moscas blancas o los trips, cuyo control resulta problemático debido a su elevada capacidad para transmitir virus vegetales incluso a una baja densidad de plaga (Holt et al., 2008; Jacas y Urbaneja, 2008). Las relaciones que se establecen entre los distintos agentes de un ecosistema son complejas y muy específicas. Se ha comprobado que, pese a que los enemigos naturales reducen de manera beneficiosa los niveles de plaga, su incorporación en los sistemas planta-insecto-virus puede desencadenar complicadas interacciones con efectos no deseables (Dicke y van Loon, 2000; Jeger et al., 2011). Así, los agentes de biocontrol también pueden inducir a que los insectos vectores modifiquen su comportamiento como respuesta al ataque y, con ello, el grado de dispersión y los patrones de distribución de las virosis que transmiten (Bailey et al., 1995; Weber et al., 1996; Hodge y Powell, 2008a; Hodge et al., 2011). Además, en ocasiones el control biológico por sí solo no es suficiente para controlar determinadas plagas (Medina et al., 2008). Entre los métodos que se pueden aplicar bajo sistemas MIP están las barreras físicas que limitan la entrada de plagas al interior de los invernaderos o interfieren con su movimiento, como pueden ser las mallas anti-insecto (Álvarez et al., 2014), las mallas fotoselectivas (Raviv y Antignus, 2004; Weintraub y Berlinger, 2004; Díaz y Fereres, 2007) y las mallas impregnadas en insecticida (Licciardi et al., 2008; Martin et al., 2014). Las mallas fotoselectivas reducen o bloquean casi por completo la transmisión de radiación UV, lo que interfiere con la visión de los insectos y dificulta o impide la localización del cultivo y su establecimiento en el mismo (Raviv y Antignus, 2004; Weintraub, 2009). Se ha comprobado cómo su uso puede controlar los pulgones y las virosis en cultivo de lechuga (Díaz et al., 2006; Legarrea et al., 2012a), así como la mosca blanca, los trips y los ácaros, y los virus que estos transmiten en otros cultivos (Costa y Robb, 1999; Antignus et al., 2001; Kumar y Poehling, 2006; Doukas y Payne, 2007a; Legarrea et al., 2010). Sin embargo, no se conoce perfectamente el modo de acción de estas barreras, puesto que existe un efecto directo sobre la plaga y otro indirecto mediado por la planta, cuya fisiología cambia al desarrollarse en ambientes con falta de radiación UV, y que podría afectar al ciclo biológico de los insectos fitófagos (Vänninen et al., 2010; Johansen et al., 2011). Del mismo modo, es necesario estudiar la compatibilidad de esta estrategia con los enemigos naturales de las plagas. Hasta la fecha, los estudios han evidenciado que los agentes de biocontrol pueden realizar su actividad bajo ambientes pobres en radiación UV (Chyzik et al., 2003; Chiel et al., 2006; Doukas y Payne, 2007b; Legarrea et al., 2012c). Otro método basado en barreras físicas son las mallas impregnadas con insecticidas, que se han usado tradicionalmente en la prevención de enfermedades humanas transmitidas por mosquitos (Martin et al., 2006). Su aplicación se ha ensayado en agricultura en ciertos cultivos al aire libre (Martin et al., 2010; Díaz et al., 2004), pero su utilidad en cultivos protegidos para prevenir la entrada de insectos vectores en invernadero todavía no ha sido investigada. Los aditivos se incorporan al tejido durante el proceso de extrusión de la fibra y se liberan lentamente actuando por contacto en el momento en que el insecto aterriza sobre la malla, con lo cual el riesgo medioambiental y para la salud humana es muy limitado. Los plaguicidas que se emplean habitualmente suelen ser piretroides (deltametrina o bifentrín), aunque también se ha ensayado dicofol (Martin et al., 2010) y alfa-cipermetrina (Martin et al., 2014). Un factor que resulta de vital importancia en este tipo de mallas es el tamaño del poro para facilitar una buena ventilación del cultivo, al tiempo que se evita la entrada de insectos de pequeño tamaño como las moscas blancas (Bethke y Paine, 1991; Muñoz et al., 1999). Asimismo, se plantea la necesidad de estudiar la compatibilidad de estas mallas con los enemigos naturales. Es por ello que en esta Tesis Doctoral se plantea la necesidad de evaluar nuevas mallas impregnadas que impidan el paso de insectos de pequeño tamaño al interior de los invernaderos, pero que a su vez mantengan un buen intercambio y circulación de aire a través del poro de la malla. Así, en la presente Tesis Doctoral, se han planteado los siguientes objetivos generales a desarrollar: 1. Estudiar el impacto de la presencia de parasitoides sobre el grado de dispersión y los patrones de distribución de pulgones y las virosis que éstos transmiten. 2. Conocer el efecto directo de ambientes pobres en radiación UV sobre el comportamiento de vuelo de plagas clave de hortícolas y sus enemigos naturales. 3. Evaluar el efecto directo de la radiación UV-A sobre el crecimiento poblacional de pulgones y mosca blanca, y sobre la fisiología de sus plantas hospederas, así como el efecto indirecto de la radiación UV-A en ambas plagas mediado por el crecimiento de dichas planta hospederas. 4. Caracterización de diversas mallas impregnadas en deltametrina y bifentrín con diferentes propiedades y selección de las óptimas para el control de pulgones, mosca blanca y sus virosis asociadas en condiciones de campo. Estudio de su compatibilidad con parasitoides. ABSTRACT Insect vectors of plant viruses are the main agents causing major economic losses in vegetable crops grown under protected environments. This Thesis focuses on the implementation of new alternatives to chemical control of insect vectors under Integrated Pest Management programs. In Spain, biological control is the main pest control strategy used in a large part of greenhouses where horticultural crops are grown. The first study aimed to increase our knowledge on how the presence of natural enemies such as Aphidius colemani Viereck may alter the dispersal of the aphid vector Aphis gossypii Glover (Chapter 4). In addition, it was investigated if the presence of this parasitoid affected the spread of aphid-transmitted viruses Cucumber mosaic virus (CMV, Cucumovirus) and Cucurbit aphid-borne yellows virus (CABYV, Polerovirus) infecting cucumber (Cucumis sativus L). SADIE methodology was used to study the distribution patterns of both the virus and its vector, and their degree of association. Results suggested that parasitoids promoted aphid dispersal in the short term, which enhanced CMV spread, though consequences of parasitism suggested potential benefits for disease control in the long term. Furthermore, A. colemani significantly limited the spread and incidence of the persistent virus CABYV in the long term. The flight activity of pests Myzus persicae (Sulzer), Bemisia tabaci (Gennadius) and Tuta absoluta (Meyrick), and natural enemies A. colemani and Sphaerophoria rueppellii (Weidemann) under UV-deficient environments was studied under field conditions (Chapter 5). One-chamber tunnels were covered with cladding materials with different UV transmittance properties. Inside each tunnel, insects were released from tubes placed in a platform suspended from the ceiling. Specific targets were located at different distances from the platform. The ability of aphids and whiteflies to reach their targets was diminished under UV-absorbing barriers, suggesting a reduction of vector activity under this type of nets. Fewer aphids reached distant traps under UV-absorbing nets, and significantly more aphids could fly to the end of the tunnels covered with non-UV blocking materials. Unlike aphids, differences in B. tabaci captures were mainly found in the closest targets. The oviposition of lepidopteran T. absoluta was also negatively affected by a UV-absorbing cover. The photoselective barriers were compatible with parasitism and oviposition of biocontrol agents. Apart from the direct response of insects to UV radiation, plant-mediated effects influencing insect performance were investigated (Chapter 6). The impact of UV-A radiation on the performance of aphid M. persicae and whitefly B. tabaci, and growth and leaf physiology of host plants pepper and eggplant was studied under glasshouse conditions. Plants were grown inside cages covered by transparent and UV-A-opaque plastic films. Plant growth and insect fitness were monitored. Leaves were harvested for chemical analysis. Pepper plants responded directly to UV-A by producing shorter stems whilst UV-A did not affect the leaf area of either species. UV-A-treated peppers had higher content of secondary metabolites, soluble carbohydrates, free amino acids and proteins. Such changes in tissue chemistry indirectly promoted aphid performance. For eggplants, chlorophyll and carotenoid levels decreased with supplemental UVA but phenolics were not affected. Exposure to supplemental UV-A had a detrimental effect on whitefly development, fecundity and fertility presumably not mediated by plant cues, as compounds implied in pest nutrition were unaltered. Lastly, the efficacy of a wide range of Long Lasting Insecticide Treated Nets (LLITNs) was studied under laboratory and field conditions. This strategy aimed to prevent aphids and whiteflies to enter the greenhouse by determining the optimum mesh size (Chapter 7). This new approach is based on slow release deltamethrin- and bifenthrin-treated nets with large hole sizes that allow improved ventilation of greenhouses. All LLITNs produced high mortality of M. persicae and A. gossypii although their efficacy decreased over time with sun exposure. It was necessary a net with hole size of 0.29 mm2 to exclude B. tabaci under laboratory conditions. The feasibility of two selected nets was studied in the field under a high insect infestation pressure in the presence of CMV- and CABYV-infected cucumber plants. Besides, the compatibility of parasitoid A. colemani with bifenthrin-treated nets was studied in parallel field experiments. Both nets effectively blocked the invasion of aphids and reduced the incidence of both viruses, however they failed to exclude whiteflies. We found that our LLITNs were compatible with parasitoid A. colemani. As shown, the role of natural enemies has to be taken into account regarding the dispersal of insect vectors and subsequent spread of plant viruses. The additional benefits of novel physicochemical barriers, such as photoselective and insecticide-impregnated nets, need to be considered in Integrated Pest Management programs of vegetable crops grown under protected environments.

Control of insect vectors and plant viruses in protected crops by novel pyrethroid-treated nets

Long-lasting insecticide-treated nets (LLITNs) constitute a novel alternative that combines physical and chemical tactics to prevent insect access and the spread of insect-transmitted plant viruses in protected enclosures. This approach is based on a slow-release insecticide-treated net with large hole sizes that allow improved ventilation of greenhouses. The efficacy of a wide range of LLITNs was tested under laboratory conditions against Myzus persicae, Aphis gossypii and Bemisia tabaci. Two nets were selected for field tests under a high insect infestation pressure in the presence of plants infected with Cucumber mosaic virus and Cucurbit aphid-borne yellows virus. The efficacy of Aphidius colemani, a parasitoid commonly used for biological control of aphids, was studied in parallel field experiments.

When defense backfires: Detrimental effect of a plant’s protective trichomes on an insect beneficial to the plant

The plant Mentzelia pumila (family Loasaceae) has leaves and stems densely covered with tiny hooked trichomes. The structures entrap and kill insects and therefore are most probably protective. But they are also maladaptive in that they incapacitate a coccinellid beetle (Hippodamia convergens) that preys upon an aphid enemy (Macrosiphum mentzeliae) of the plant. The adaptive benefit provided by the trichomes is evidently offset by a cost.

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal “clip” domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.

Antibody-mediated inhibition of the growth of larvae from an insect causing cutaneous myiasis in a mammalian host

Many insects feed on blood or tissue from mammalian hosts. One potential strategy for the control of these insects is to vaccinate the host with antigens derived from the insect. The larvae of the fly Lucilia cuprina feed on ovine tissue and tissue fluids causing a cutaneous myiasis associated with considerable host morbidity and mortality. A candidate vaccine antigen, peritrophin 95, was purified from the peritrophic membrane, which lines the gut of these larvae. Serum from sheep vaccinated with peritrophin 95 inhibited growth of first-instar L. cuprina larvae that fed on this serum. Growth inhibition was probably caused by antibody-mediated blockage of the normally semipermeable peritrophic membrane and the subsequent development of an impervious layer of undefined composition on the gut lumen side of the peritrophic membrane that restricted access of nutrients to the larvae. The amino acid sequence of peritrophin 95 was determined by cloning the DNA complementary to its mRNA. The deduced amino acid sequence codes for a secreted protein containing a distinct Cys-rich domain of 317 amino acids followed by a mucin-like domain of 139 amino acids. The Cys-rich domain may be involved in binding chitin. This report describes a novel immunological strategy for the potential control of L. cuprina larvae that may have general application to the control of other insect pests.

Estimating the relative roles of top-down and bottom-up forces on insect herbivore populations: A classic study revisited

Although most ecologists agree that both top-down and bottom-up forces (predation and resource limitation, respectively) act in concert to influence populations of herbivores, it has proven difficult to estimate the relative contributions of such forces in terrestrial systems. Using a combination of time–series analysis of population counts recorded over 16 years and experimental data, we present the first estimates of the relative roles of top-down and bottom-up forces on the population dynamics of two terrestrial insect herbivores on the English oak (Quercus robur). Data suggest that temporal variation in winter moth, Operophtera brumata, density is dominated by time-lagged effects of pupal predators. By comparison, spatial variation in O. brumata density is dominated by host–plant quality. Overall, top-down forces explain 34.2% of population variance, bottom-up forces explain 17.2% of population variance, and 48.6% remains unexplained. In contrast, populations of the green oak tortrix, Tortrix viridana, appear dominated by bottom-up forces. Resource limitation, expressed as intraspecific competition among larvae for oak leaves, explains 29.4% of population variance. Host quality effects explain an additional 5.7% of population variance. We detected no major top-down effects on T. viridana populations. An unknown factor causing a linear decline in T. viridana populations over the 16-year study period accounts for most of the remaining unexplained variance. We discuss the observed differences between the insect species and the utility of time–series analysis as a tool in assessing the relative importance of top-down and bottom-up forces on herbivore populations.

Rhodopsin kinase: Expression in baculovirus-infected insect cells, and characterization of post-translational modifications*

Structure–function studies of rhodopsin kinase (RK; EC 2.7.1.125) require a variety of mutants. Therefore, there is need for a suitable system for the expression of RK mutant genes. Here we report on a study of expression of the RK gene in baculovirus-infected Sf21 cells and characterization of the enzyme produced as purified to near homogeneity. Particular attention has been paid to the post-translational modifications, autophosphorylation and isoprenylation, found in the native bovine RK. The protein produced has been purified using, successively, heparin-Sepharose, Mono Q, and Mono S FPLC (fast protein liquid chromatography) and was obtained in amounts of about 2 mg from 1 liter of cell culture. The enzyme from the last step of purification was obtained in two main fractions that differ in the level of phosphorylation. The protein peak eluted first carries two phosphate groups per protein, whereas the second protein peak is monophosphorylated. Further, while both peaks are isoprenylated, the isoprenyl groups consist of mixtures of C5, C10, C15, and C20 isoprenyl moieties. From these results, we conclude that the above expression system is suitable for some but not all aspects of structure–function studies.

Midgut-specific immune molecules are produced by the blood-sucking insect Stomoxys calcitrans

We have cloned and sequenced two defensins, Smd1 and Smd2, from anterior midgut tissue of the blood-sucking fly Stomoxys calcitrans. The DNA and N-terminal protein sequences suggest both are produced as prepropeptides. Smd1 differs from the classic defensin pattern in having an unusual six-amino acid-long N-terminal sequence. Both Smd1 and Smd2 have lower pI points and charge than insect defensins derived from fat body/hemocytes. Northern analysis shows both of these defensin molecules are tissue specific; both are produced by the anterior midgut tissue and, unlike the other insect defensins reported to date, neither appears to be expressed in fat body or hemocytes. Northern analysis also shows that mRNAs for both defensins are constitutively produced in the anterior midgut tissues and that these transcripts are up-regulated in response to sterile as well as a lipopolysaccharide-containing blood meal. However, anti-Gram-negative biological activity in the midgut is substantially enhanced by lipopolysaccharide. These findings suggest that the insect midgut has its own tissue-specific immune mechanisms and that this invertebrate epithelium is, like several vertebrate epithelia, protected by specific antibacterial peptides.

A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis

A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the midgut and caeca. The levels of CYP4C7 mRNA in the CA, measured by ribonuclease protection assays, were linked to the activity cycle of the glands. In adult females, CYP4C7 expression increased immediately after the peak of JH synthesis, reaching a maximum on day 7, just before oviposition. mRNA levels then declined after oviposition and during pregnancy. The CYP4C7 protein was produced in Escherichia coli as a C-terminal His-tagged recombinant protein. In a reconstituted system with insect NADPH cytochrome P450 reductase, cytochrome b5, and NADPH, the purified CYP4C7 metabolized (2E,6E)-farnesol to a more polar product that was identified by GC-MS and by NMR as (10E)-12-hydroxyfarnesol. CYP4C7 converted JH III to 12-trans-hydroxy JH III and metabolized other JH-like sesquiterpenoids as well. This ω-hydroxylation of sesquiterpenoids appears to be a metabolic pathway in the corpora allata that may play a role in the suppression of JH biosynthesis at the end of the gonotrophic cycle.

Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells

Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase δ or ɛ. Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase δ-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.