Field-evolved resistance to beta-cyfluthrin in the boll weevil: Detection and characterization.

BACKGROUND: Insecticide resistance in arthropods is an inherited trait that has become a major cause of insect pest control failure. Monitoring the level of susceptibility and characterization of the type of resistance of key pest species aims to determine the risk of resistance selection in time to take action to mitigate control failures. Seven populations of the boll weevil, Anthonomus grandis grandis, collected from cotton fields in the Semiarid and Cerrado areas of Brazil, were screened for their resistance to malathion and beta-cyfluthrin, insecticides widely recommended for control of boll weevil and other pests. RESULTS: The levels of adult mortality were variable for beta-cyfluthrin (0-82%) but invariant (100%) for malathion. Bioassays of concentration-mortality were used to determine lethal concentrations (LCs) for each insecticide. The LC-values corroborate the lack of resistance to field rates of malathion but high levels of resistance to beta-cyfluthrin from 62.7- to 439.7-fold. Weevils resistant to beta-cyfluthrin were found through genome sequencing to possess a kdr mutation through the L1014F substitution in the voltage gated-sodium channel gene. CONCLUSIONS: This study found boll weevil resistance to beta-cyfluthrin to be not mediated by carboxylesterases, but with cross-resistance to DDT and carbaryl, and kdr mutation as the major mechanism of the resistance in our samples. Caution is recommended in further use of beta-cyfluthrin against boll weevil due to potential resistance. Monitoring studies using other boll weevil populations are recommended to determine the geographic pattern and extent of pyrethroid resistance. © 2021 Society of Chemical Industry.

Increasing Anthonomus grandis susceptibility to Metarhizium anisopliae through RNAi-induced AgraRelish knockdown: a perspective to combine biocontrol and biotechnology.

BACKGROUND: The hemolymph and insect gut together have an essential role in the immune defense against microorganisms, including the production of antimicrobial peptides (AMP). AMPs are mainly induced by two specific signaling pathways, Toll and immune deficiency (IMD). Here, we characterize the expression profile of four genes from both pathways and describe the importance of AgraRelish in the immune defense of Anthonomus grandis against the entomopathogenic fungus Metarhizium anisopliae by RNA interference (RNAi). RESULTS: To characterize the pathway that is activated early during the A. grandis-M. anisopliae interaction, we assessed the expression profiles of AgraMyD88 and AgraDorsal (Toll pathway), AgraIMD and AgraRelish (IMD pathway), and several AMP genes. Interestingly, we found that IMD pathway genes are upregulated early, and Toll pathway genes are upregulated just 3 days after inoculation (DAI). Furthermore, nine AMPs were upregulated 24 h after fungus inoculation, including attacins, cecropins, coleoptericins, and defensins. AgraRelish knockdown resulted in a reduction in median lethal time (LT50 ) for M. anisopliae-treated insects of around 2 days compared to control treatments. In addition, AgraRelish remained knocked down at 3 DAI. Finally, we identified that AgraRelish knockdown increased fungal loads at 2 DAI compared to control treatments, possibly indicating a faster infection. CONCLUSIONS: Our data indicate the influence of the IMD pathway on the antifungal response in A. grandis. Combining biocontrol and RNAi could significantly improve cotton boll weevil management. Hence, AgraRelish is a potential target for the development of biotechnological tools aimed at improving the efficacy of M. anisopliae against A. grandis.

The plant WEE1 kinase is involved in checkpoint control activation in nematode-induced galls.

Galls induced by plant-parasitic nematodes involve a hyperactivation of the plant mitotic and endocycle machinery for their profit. Dedifferentiation of host root cells includes drastic cellular and molecular readjustments. In such a background, potential DNA damage in the genome of gall cells is evident. We investigated whether DNA damage checkpoint activation followed by DNA repair occurred, or was eventually circumvented, in nematode-induced galls. Galls display transcriptional activation of the DNA damage checkpoint kinase WEE1, correlated with its protein localization in the nuclei. The promoter of the stress marker gene SMR7 was evaluated under the WEE1-knockout background. Drugs inducing DNA damage and a marker for DNA repair, PARP1, were used to understand the mechanisms for coping with DNA damage in galls. Our functional study revealed that gall cells lacking WEE1 conceivably entered mitosis prematurely, disturbing the cell cycle despite the loss of genome integrity. The disrupted nuclei phenotype in giant cells hinted at the accumulation of mitotic defects. In addition, WEE1-knockout in Arabidopsis and downregulation in tomato repressed infection and reproduction of root-knot nematodes. Together with data on DNA-damaging drugs, we suggest a conserved function for WEE1 in controlling G1/S cell cycle arrest in response to a replication defect in galls.

Transcriptome and gene expression analysis of three developmental stages of the coffee berry borer, Hypothenemus hampei.

Coffee production is a global industry valued at approximately 173 billion US dollars. One of the main challenges facing coffee production is the management of the coffee berry borer (CBB), Hypothenemus hampei, which is considered the primary arthropod pest of coffee worldwide. Current control strategies are inefficient for CBB management. Although biotechnological alternatives, including RNA interference (RNAi), have been proposed in recent years to control insect pests, characterizing the genetics of the target pest is essential for the successful application of these emerging technologies. In this study, we employed RNA-seq to obtain the transcriptome of three developmental stages of the CBB (larva, female and male) to increase our understanding of the CBB life cycle in relation to molecular features. The CBB transcriptome was sequenced using Illumina Hiseq and assembled de novo. Differential gene expression analysis was performed across the developmental stages. The final assembly produced 29,434 unigenes, of which 4,664 transcripts were differentially expressed. Genes linked to crucial physiological functions, such as digestion and detoxification, were determined to be tightly regulated between the reproductive and nonreproductive stages of CBB. The data obtained in this study help to elucidate the critical roles that several genes play as regulatory elements in CBB development.

Application of Nuclear Volume Measurements to Comprehend the Cell Cycle in Root-Knot Nematode-Induced Giant Cells.

Root-knot nematodes induce galls that contain giant-feeding cells harboring multiple enlarged nuclei within the roots of host plants. It is recognized that the cell cycle plays an essential role in the set-up of a peculiar nuclear organization that seemingly steers nematode feeding site induction and development. Functional studies of a large set of cell cycle genes in transgenic lines of the model host Arabidopsis thaliana have contributed to better understand the role of the cell cycle components and their implication in the establishment of functional galls. Mitotic activity mainly occurs during the initial stages of gall development and is followed by an intense endoreduplication phase imperative to produce giant-feeding cells, essential to form vigorous galls. Transgenic lines overexpressing particular cell cycle genes can provoke severe nuclei phenotype changes mainly at later stages of feeding site development. This can result in chaotic nuclear phenotypes affecting their volume. These aberrant nuclear organizations are hampering gall development and nematode maturation. Herein we report on two nuclear volume assessment methods which provide information on the complex changes occurring in nuclei during giant cell development. Although we observed that the data obtained with AMIRA tend to be more detailed than Volumest (Image J), both approaches proved to be highly versatile, allowing to access 3D morphological changes in nuclei of complex tissues and organs. The protocol presented here is based on standard confocal optical sectioning and 3-D image analysis and can be applied to study any volume and shape of cellular organelles in various complex biological specimens. Our results suggest that an increase in giant cell nuclear volume is not solely linked to increasing ploidy levels, but might result from the accumulation of mitotic defects.

Vitellogenin knockdown strongly affects cotton boll weevil egg viability but not the number of eggs laid by females.

Vitellogenin (Vg), a yolk protein precursor, is the primary egg nutrient source involved in insect reproduction and embryo development. The Cotton Boll weevil (CBW) Anthonomus grandis Boheman, the most important cotton pest in Americas, accumulates large amounts of Vg during reproduction. However, the precise role of this protein during embryo development in this insect remains unknown. Herein, we investigated the effects of vitellogenin (AgraVg) knockdown on the egg-laying and egg viability in A. grandis females, and also characterized morphologically the unviable eggs. AgraVg transcripts were found during all developmental stages of A. grandis, with highest abundance in females. Silencing of AgraVg culminated in a significant reduction in transcript amount, around 90%. Despite this transcriptional reduction, egg-laying was not affected in dsRNA-treated females but almost 100% of the eggs lost their viability. Eggs from dsRNA-treated females showed aberrant embryos phenotype suggesting interference at different stages of embryonic development. Unlike for other insects, the AgraVg knockdown did not affect the egg-laying ability of A. grandis, but hampered A. grandis reproduction by perturbing embryo development. We concluded that the Vg protein is essential for A. grandis reproduction and a good candidate to bio-engineer the resistance against this devastating cotton pest.

Preferência alimentar e desempenho de Brontocoris tabidus Signoret (Hemiptera, Pentatomidae) em plantas hospedeiras / Feeding preference and performance of Brontocoris tabidus (Signoret) (Hemiptera, Pentatomidae) among host plants

O comportamento de escolha entre plantas pelo percevejo predador Brontocoris tabidus (Signoret) (Hemiptera, Pentatomidae) e a relação com o ganho de peso e seu desempenho foram investigados com plantas de algodoeiro, Gossypium hirsutum L. (Malvaceae), caruru, Amaranthus lividus L. (Amaranthaceae) e feijão, Phaseolus vulgaris L. (Fabaceae). Adultos do predador foram permitidos livre escolha entre plantas e, posteriormente, mantidos sobre as mesmas plantas escolhidas inicialmente para a alimentação. Também, o desenvolvimento e a reprodução foram obtidos confinando os predadores com e sem presa mais plantas. Os resultados mostraram que B. tabidus não demonstrou preferência para alimentação entre as plantas estudadas. O tempo de alimentação (106 a 115 minutos) e ganho de peso (28 a 30 por cento do peso vivo) foram similares em relação à alimentação em plantas. Entretanto, a longevidade das fêmeas foi menor quando mantidas em feijão, comparadas com algodoeiro e caruru. A duração da fase ninfal do predador variou entre as plantas, bem como a produção de descendentes demonstrando menor desempenho sobre plantas de feijão. As ninfas alimentadas, apenas, de plantas não atingiram a fase adulta e viveram até 6,5 dias, sendo maior a sobrevivência em algodoeiro e caruru. Os resultados mostram que apesar de B. tabidus não apresentar seleção da planta hospedeira para alimentação, estas podem contribuir diferentemente na sua história de vida com destaque para o algodão e o caruru entre as plantas estudadas.

The choice between cotton, Gossypium hirsutum L. (Malvaceae), green bean, Phaseolus vulgaris L. (Fabaceae) and pigweed, Amaranthus lividus L. (Amaranthaceae) and its relationship as first feeding, weight gain and biological performance of the predator Brontocoris tabidus (Signoret) (Hemiptera, Pentatomidae) adults were studied. Adults of the predator were allowed to choose between plants and, immediately paired and reared on the same selected plants plus prey. Furthermore, nymphal development and adult stage were obtained on the same plants plus prey. The results show that B. tabidus do not exhibit host plant preference among tested plants. Likewise, feeding period (106 to 115 minutes) and weight gain (28 to 30 percent of body weight) were statistically equal among plants. However, predators at adult stage only or nymphal and adult stages maintained on young green bean plants plus prey exhibited lower performance compared to the other plants. Nymphs feeding exclusively on plants lived, on average, up to 6.5 days but did not reach adult stage and lived longer on cotton plants and pig weed. The results indicated that adults of the predator B. tabidus do not exhibit selection on host plant to feed although different results in its life history was attained among the studied plants with better results for cotton and pigweed.

Effects of chloride channel blockers on hypotonicity-induced contractions of the rat trachea.

1. We have investigated the inhibitory effects of blockers of volume-activated (Cl(vol)) and calcium-activated (Cl(Ca)) chloride channels on hypotonic solution (HS)-induced contractions of rat trachea, comparing their effects with those of the voltage-dependent calcium channel (VDCC) blocker nifedpine. 2. HS elicited large, stable contractions that were partially dependent on the cellular chloride gradient; a reduction to 41.45+/-7.71% of the control response was obtained when extracellular chloride was removed. In addition, HS-induced responses were reduced to 26.8+/-5.6% of the control by 1 microm nifedipine, and abolished under calcium-free conditions, indicating a substantial requirement for extracellular calcium entry, principally via VDCCs. 3. The established Cl(vol) blockers tamoxifen (