Hadi Mosallanejad

Biochemical mechanisms of methoxyfenozide resistance in the cotton leafworm Spodoptera littoralis

BACKGROUND Methoxyfenozide is a lepidopteran-specific insecticide that belongs to a new group of insecticides, the non-steroidal ecdysteroid agonists, also called moulting accelerating compounds (MACs). To investigate the risk of resistance and possible mechanisms conferring resistance to methoxyfenozide, the authors selected in the laboratory for a resistant strain of the cotton leafworm Spodoptera littoralis (Boisd.), which is a representative lepidopteran model and an important pest in cotton and vegetables worldwide, with a high risk for resistance development. RESULTS After selection with methoxyfenozide during 13 generations, toxicity data showed that the selected strain developed fivefold resistance to methoxyfenozide in comparison with the susceptible strain. Measurement of the detoxification enzymes demonstrated that the monooxygenase (MO) activity was 2.1 times higher in the selected strain, whereas there was no change for esterases and glutathione-S-transferases. When the inhibitors piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and diethyl maleate were tested as synergists, the respective synergistic ratios were 0.97, 0.96 and 1.0 for the susceptible strain, and 2.2, 0.96 and 1.1 for the resistant strain. The significant synergistic effect by PBO concurs with the increased MO activity in the selected strain. CONCLUSION Taken overall, the present study supports the importance of MO-mediated metabolism in resistance to methoxyfenozide, directing tactics to fight against resistance development for this novel group of insecticides.

Ecdysteroid signaling in ecdysteroid-resistant cell lines from the polyphagous noctuid pest Spodoptera exigua

Although dibenzoylhydrazine-type non-steroidal ecdysone agonists such as methoxyfenozide (RH-2485) have an excellent performance record, the emergence of resistance could severely compromise the efficacy of these compounds in integrated pest management programs. To investigate possible mechanisms of resistance, cell lines derived from the polyphagous noctuid pest Spodoptera exigua (Se4 cells) were selected for continuous growth in the presence of high concentrations of 20-hydroxyecdysone (20E) or methoxyfenozide. Here we describe an analysis of ecdysteroid receptor signaling in the ecdysteroid-resistant Se4 cell lines. In contrast to other ecdysteroid-resistant cell lines described in literature, our data support the existence of a normal functioning ecdysteroid receptor complex in the resistant Se4 cell lines: (1) using a recombinant BmNPV baculovirus as a transduction tool, activation of an ecdysone-responsive luciferase cassette was demonstrated; (2) the early gene HR3 is constitutively expressed in the resistant cell lines that are grown in the presence of 20E or methoxyfenozide. Quantitative RT-PCR experiments indicated that expression levels of SeEcR mRNA were comparable among sensitive and resistant cell lines. Sequencing of PCR fragments also revealed the presence of SeEcR mRNA with a wild-type ligand-binding domain in resistant cells. Finally, a possible role for the gene FTZ-F1, whose expression correlates with the absence of circulating ecdysteroids during insect development, in the resistance mechanism was investigated. However, it was observed that FTZ-F1, in contrast to what is observed during insect development, is constitutively expressed in Se4 cells and that its expression is not regulated by the addition of ecdysteroid. It is proposed that the resistance mechanism in Se4 cells resides at the coupling between the conserved hierarchical cascade of early and early-late gene expression and the differentiation program in the Se4 cell line. The use of insect cell lines for the investigation of resistance against dibenzoylhydrazine ecdysone agonists and their relevance for uncovering resistance mechanisms in insects during pest control programs is discussed.

Assessment of species specificity of moulting accelerating compounds in Lepidoptera: comparison of activity between Bombyx mori and Spodoptera littoralis by in vitro reporter and in vivo toxicity assays

BACKGROUND Dibenzoylhydrazine analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. A notable feature is their high activity against lepidopteran insects, raising the question as to whether species-specific analogues can be isolated. In this study, the specificity of ecdysone agonists was addressed through a comparative analysis in two important lepidopterans, the silkworm Bombyx mori L. and the cotton leafworm Spodoptera littoralis (Boisd.). RESULTS When collections of non-steroidal ecdysone agonists containing different mother structures (dibenzoylhydrazine, acylaminoketone, tetrahydroquinoline) were tested, in vitro reporter assays showed minor differences using cell lines derived from both species. However, when compounds with high ecdysone agonist activity were examined in toxicity assays, larvicidal activity differed considerably. Of note was the identification of three dibenzoylhydrazine analogues with > 100-fold higher activity against Bombyx than against Spodoptera larvae. CONCLUSION The present study demonstrated that species-specific ecdysone-agonist-based insecticides can be developed, but their species specificity is not based on differences in the activation of the ecdysone receptor but rather on unidentified in vivo parameters such as permeability of the cuticle, uptake/excretion by the gut or metabolic detoxification.

Ecdysone signaling and transcript signature in Drosophila cells resistant against methoxyfenozide.

Methoxyfenozide (RH-2485) is a non-steroidal ecdysteroid agonist with a dibenzoylhydrazine structure, representing a group used as novel biorational insecticides in the control of insect pests. Here we report on the selection of Drosophila melanogaster S2 cells for resistance to inhibition of cell proliferation by methoxyfenozide by ∼ 1000-fold over 4 months. Cells were exposed to gradually increasing concentrations of methoxyfenozide and selected out based on the ecdysteroid-sensitive response for cell proliferation. In the resistant cells, the ecdysteroid receptor (EcR/USP) complex was no longer active in the presence of methoxyfenozide. But when resistant cells were relaxed from pressure in methoxyfenozide-free medium, induction of the reporter construct was observed. In parallel, EcR/USP functionality was also restored when resistant cells were rescued by a Drosophila EcR plasmid. However, it was striking that in the resistant cells the ecdysteroid-sensitive response for cell proliferation was not restored upon methoxyfenozide withdrawal, indicating permanent changes in the physiology of the cells during selection. To investigate changes in gene expression caused by inactivation of the EcR/USP complex in resistant cells, Drosophila oligo 14kv1 microarrays were used and probed with cDNAs from resistant cells in the presence and absence of ecdysone agonist on one hand and from unselected sensitive cells on the other hand. A selection of 324 differentially expressed genes was assigned covering diverse functions as transport, enzyme activity, cytoskeleton organization, cell cycle machinery, transcription/translation and ecdysteroid signaling. Besides the identification of (primary and secondary) target genes of the EcR/USP signaling pathway, this analysis also allows to gain insights into the mechanism of resistance and on the crosstalk between ecdysteroid signaling and cell proliferation-linked processes.

Plasticity in the gut microbial community and uptake of Enterobacteriaceae (Gammaproteobacteria) in Bombus terrestris bumblebees' nests when reared indoors and moved to an outdoor environment

Bombus nests consisting of one queen, brood, and worker adults, are produced indoors for biological pollination in agriculture. In this study, we investigated the gut microbial community in workers of Bombus terrestris when the environment is stable (indoors) or variable (outdoors). When nests were reared indoors under standardized conditions, we identified a small gut microbial community consisting of Neisseriaceae, Orbaceae, Lactobacillaceae, and Bifidobacteriaceae, and the age of bumblebee nests and workers did not affect the alpha and beta diversity, confirming a stable microbiota. Secondly, when indoor-reared nests were moved to outdoors, we observed a major shift in the microbial community, especially in the newborn workers fully developed in the outdoor conditions, with a significant colonization of Enterobacteriaceae. Our new findings are discussed in relation to host-associated core and non-core bacteria in bumblebees including possible implications for host functioning.

Gamma irradiation of pollen and eradication of Israeli acute paralysis virus

Honeybees and bumblebees are the most important pollinators of agricultural crops. For this purpose honeybees and bumblebees are reared and transported. A pathogen-free status of bees in general, is crucial. Indeed anthropogenic transports of hosts carrying parasites could alter the natural host/pathogen association, inducing an extra pathogenic stress. Therefore the creation of a pathogen-free rearing environment is needed. For bumblebees this is possible, as these species are reared in a closed environment. Although, a link remains between reared bumblebees and the outside bee community, as honeybee-collected pollen is essential food for bumblebee mass rearing. Here we evaluated if gamma irradiation can minimize the risk of this potential route of exposure and can inactivate viral particles present in honeybee-collected pollen. We show that 16.9kGy gamma irradiation induced a 100-1000 fold reduction on the ability of IAPV to cause mortality after injections. This result opens avenues toward rearing pathogen-free bumblebees and towards eliminating the risks of pathogen spillover to native wild bee species.

Effects of pollution on nematode assemblage structure and diversity on beaches of the northern Persian Gulf

This study is the first to present data on local (=beach) and gamma diversity of beach nematode assemblages from the Persian Gulf. We investigated four beaches near the city of Bandar Abbas, Iran. On each beach, we sampled three stations at 50-m intervals, with increasing distance from a local pollution source, mostly domestic sewage. A total of 39 genera from 17 families was recorded. This gamma diversity is low and suggests that the entire area experiences substantial stress. Five genera together comprised 75% of nematode abundance. There were significant differences in abundance as well as genus diversity between locations, but these did not unequivocally correlate with known drivers of benthic assemblage structure like sediment granulometry and hydrodynamics. The location exposed to the strongest local pollution input had the lowest nematode diversity and a very low abundance at the station nearest the pollution source. Distance from local pollution sources also significantly impacted genus diversity, but this pattern was only pronounced in two of the four beaches. Our data demonstrate that local sources of anthropogenic disturbance are a major driver of assemblage diversity and structure in this area, despite an overarching effect of natural (salinity, temperature) and anthropogenic stressors.