Oral delivery of dsHvlwr is a feasible method for managing the pest Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae)

RNA interference(RNAi)techniques have emerged as powerful tools that facilitate development of novel management strategies for insect pests,such as Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae),which is a major pest of solanaceous plants in Asia.In this study,the potential of oral delivery of in vvYro-synthesized and bacterially expressed double-stranded H.vigintioctopunctata lesswright(Iwr)gene(dsHvlwr)to manage of H.vigintioctopunctata was investigated.Our results showed that the gene Hvlwr had a 480-bp open reading frame and encoded a 160-amino acid protein.Hvlwr expression levels were greater in the fat body than other tissue types.Hvlwr silencing led to greater H.vigintioctopunctata mortality rates and appeared to be time-and partially dose-dependent,likely as a result of the number of hemocytes increasing with dsRNA concentration,but decreasing with time.Bacterially expressed dsHvlwr that was applied to leaf discs caused 88%,66%,and 36%mortality in 1st instars,3rd instars,and adults after 10,10,and 14 d,respectively;when applied to living plants,there was greater mortality in 1 st and 3rd instars,but there was no effect on adults.Furthermore,dsHvlwr led to improved plant protection against H.vigintioctopunctata.Our study shows an effective dietary RNAi response in H.vigintioctopunctata and that Hvlwr is a promising RNAi target gene for control of this pest species.     

Foliar persistence and residual activity of methoxyfenozide against beet armyworm （Lepidoptera： Noctuidae）

A greenhouse study was conducted to investigate the persistence of methoxyfenozide in pepper （Capsicum annuum L.） foliage. An aqueous suspension of methoxyfenozide was sprayed on pepper plants at concentrations of 72 and 144 mg of active ingredient （a.i.）/L. Foliage was collected at different intervals of time （0, 3, 5, 10, 20, 30, 40, and 50 days） after the treatment, and the methoxyfenozide residue was measured by high-performance liquid chromatography. The foliage was also used in bioas- says to determine the residual toxicity on and the consumption rate of the third-instar larvae of the beet armyworm, Spodoptera exigua （Htibner） （Lepidoptera： Noctuidae）. The methoxyfenozide concentrations observed after 50 days had decreased to 19 and 69μg/g per sample, corresponding to a loss of 61% and 28% from the application concentrations of 72 and 144 mg a.i./L, respectively. When fitting a first-order kinetics degradation model, the half-life （DT50） of this compound was 76 days. Both application concentrations of methoxyfenozide caused a high mortality rate （〉97%） when the larvae were fed the pepper foliage collected at all of the time intervals. Lastly, at all of the time points, the consumption rate by the larvae was reduced to between 57% and 92% for both concentrations that were bioassayed. Our results indicate that, under the present greenhouse conditions, the degradation of methoxyfenozide was slower than that reported by other authors and that its residues were highly toxic to S. exigua larvae. The implications of these results for the management programs of this pest are discussed.     

Complete protection from Henosepilachna vigintioctopunctata by expressing long double-stranded RNAs in potato plastids

RNA interference(RNAi) has emerged as a powerful technology for pest management. Previously, we have shown that plastid-mediated RNAi(PM-RNAi)can be utilized to control the Colorado potato beetle,an insect pest in the Chrysomelidae family; however,whether this technology is suitable for controlling pests in the Coccinellidae remained unknown.The coccinellid 28-spotted potato ladybird(Henosepilachna vigintioctopunctata; HV) is a serious pest of solanaceous crops. In this study, we identified thre...     

Nmnoparticle-mediated double-stranded RNA delivery system: A promising approach for sustainable pest management

RNA interference(RNAi)targeting lethal genes in insects has great potential for sustainable crop protection.Compared with traditional double-stranded(ds)RNA delivery systems,nanoparticles such as chitosan,liposomes,and cationic dendrimers offer advantages in delivering dsRNA/small interfering(si)RNA to improve RNAi efficiency,thus promoting the development and practice of RNAi-based pest management strategies.Here,we illustrate the limitations of traditional dsRNA delivery systems,reveal the mechanism of nanoparticle-mediated RNAi,summarize the recent progress and successful applications of nanoparticle-mediated RNAi in pest management,and finally address the prospects of nanoparticle-based RNA pesticides.     

Spraying compound probiotics improves growth performance and immunity and modulates gut microbiota and blood metabolites of suckling piglets

One factor that shapes the establishment of early neonatal intestinal microbiota is environmental microbial exposure, and probiotic application has been shown to promote health and growth of piglets. Thus, this study hypothesized that environmental probiotic application in early days of life would be beneficial to newborn piglets. This study aimed to investigate the effect of spraying a compound probiotic fermented liquid(CPFL) into the living environment of piglets on their early growth perform...     

Phyto-availability and speciation change of heavy metals in soils amended with lignin as micro-fertilizer

Lignin is a primary byproduct from the black liquor treatment in paper making industries, its application as micro-fertilizer in agricultural land might provide a promising alternative to sewage discharge. However, application of such a micro-fertilizer might affect the soil properties and result in soil pollution. In this study, the effects of lignin application on phyto-availability and speciation change of heavy metals in soils were investigated. Greenhouse experiments showed that lignin application improved the growth of winter wheat (Triticum aestivum L.) in all three soils investigated. The increase of the biomass for wheat shoot was 59.7%, 39.8% and 12.3% for Beijing soil, Jiangxi soil and Dongbei soil, respectively. In contrast, lignin amendment decreased the concentrations of heavy metals in wheat shoots from 2.2% to 61.0%. Sequential extraction procedure of a three-step BCR was used to investigate the fraction distribution. The extractable fractions were specified as fraction B1: water soluble,     

Greenhouse growth and acclimatization of encapsulated Hibiscus moscheutos nodal segments

Leafless nodal segments (4 ± 1 mm long) of hardy hibiscus were excised from in vitro proliferating microshoots, encapsulated in sodium alginate solidified with 50 μM CaCl₂, stored under refrigeration for 4 weeks in darkness, and then planted in the greenhouse. Planting in vermiculite and placing under intermittent mist was the best environment tested. If the encapsulated nodal segments were exposed to light for at least 2 weeks while in vitro in the laboratory prior to planting in the greenhouse, all survived, rooted, and produced shoots in the greenhouse. Rooting into the vermiculite was best if the encapsulated nodal segments were planted 1 cm deep and not covered. Anatomically, the new leaves that were produced from shoots that grew under mist in the greenhouse from encapsulated nodal segments were about the same thickness as leaves produced in vitro; had fewer intercellular spaces than the in vitro produced leaves; had palisade cells intermediate in length, and were intermediate for epicuticular wax formation between in vitro produced leaves and leaves on macrocuttings rooted in the greenhouse. The stomates on greenhouse shoots from encapsulated nodal segments closed similar to stomates on leaves on rooted macrocuttings, and were unlike in vitro produced leaves where the stomates remained open even when stressed. Storing and planting encapsulated nodal segments could allow producers to generate sufficient numbers of nodal segments, refrigerate them until needed, and facilitate greenhouse acclimatization and production of plants.     

CRISPR-Cas9 knockout screening for functional genomics

<正>Large-scale genetic screening offers a critical tool to dissect gene function and pathways underlying biological processes and human disease.Despite the success of genome-wide genetic screens in microorganisms such as yeast,the creation of genome-scale homozygous mutant libraries for metazoans with diploid genomes is time and cost consuming and low throughput.RNA interference(RNAi)has been a predominant solution for functional genomics during the past years since a double strand RNA can inactivate gene function by causing sequence-specific degradation of mRNA.However,the RNAi approach has intrinsic limitations because the suppression of target gene expression at     

Production protocol for and storage efficacy of an anthocorid predator Cardiastethus exiguus Poppius

Cardiastethus exiguus Poppius is an indigenous anthocorid predator of eggs and neonates of the notorious pest,coconut black-headed caterpillar Opisina arenosella Walker in India.At the National Bureau of Agriculturally Important Insects(Indian Council of Agricultural Research),Bangalore,India,a simple mass production protocol was developed for multiplying C.exiguus using UV-irradiated eggs of alternate laboratory host Corcyra cephalonica Stainton.Field evaluation of the predator in the states of Kerala and Karnataka indicated that this predator could bring about a significant reduction in the pest population.Subsequently,the need was felt to investigate the storage efficacy of the eggs and adults of C.exiguus so that sufficient numbers could be accumulated and transportation of the predator could be planned for field releases.Low temperature storage studies indicated that C.exiguus eggs can be safely stored for up to 5 days at 10℃ and 10 days at 15℃ and incubation period could be staggered for up to 10 and 13 days,respectively.The longevity of the C.exiguus adults was significantly reduced due to low temperature storage.However,for adult females,a storage temperature of 15℃ for 15 days could be recommended as they could live for a more than a month after removal from storage and their progeny production was comparable to that of the control adults.     

Cover Caption

The 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata (Fabricius) (Coleoptera: Coccinellidae), is a major agricultural pest that primarily causes serious damage to solanaceous plants. The destructive potential of H. vigintioctopunctata is high at the larval stages. The cover photo shows that H. vigintioctopunctata third instar larva eating on the surface of eggplant leaf; it can be easily killed after ingestion of dsHvvATPase A or dsHvvATPase E (see pages xxx-xxx). Photo provided by Li-Yuan Liu.     

Effects of Pesta-Pelletized Steinernema carpocapsae (All) on Western Corn Rootworms and Colorado Potato Beetles

Pesta-pelletized Steinernema carpocapsae (All) nematodes were used in soil treatments in the greenhouse against larvae of Western corn rootworm and prepupae of Colorado potato beetle. The pesta-pellets delivered 100,000 living nematodes/g. Infective-stage nematodes and their associated bacteria survived the pesta-pellet process, emerged from the pellets in large numbers in the soil, and reduced adult emergence of both pest insects by more than 90%.     

A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust

Application of RNA interference (RNAi) for insect pest management is limited by variable efficiency of RNAi in different insect species. In Locusta migratoria, RNAi is highly efficient through injection of dsRNA, but oral delivery of dsRNA is much less effective. Efforts to understand this phenomenon have shown that dsRNA is more rapidly degraded in midgut fluid than in hemolymph due to nuclease enzyme activity. In the present study, we identified and characterized two full-length cDNAs of double-stranded RNA degrading enzymes (dsRNase) from midgut of L. migratoria, which were named LmdsRNase2 and LmdsRNase3. Gene expression analysis revealed that LmdsRNase2 and LmdsRNase3 were predominantly expressed in the midgut, relatively lower expression in gastric caeca, and trace expression in other tested tissues. Incubation of dsRNA in midgut fluid from LmdsRNase3-suppressed larvae or control larvae injected with dsGFP resulted in high levels of degradation; however, dsRNA incubated in midgut fluid from LmdsRNase2-suppressed larvae was more stable, indicating LmdsRNase2 is responsible for dsRNA degradation in the midgut. To verify the biological function of LmdsRNase2 in vivo, nymphs were injected with dsGFP, dsLmdsRNase2 or dsLmdsRNase3 and chitinase 10 (LmCht10) or chitin synthase 1 (LmCHS1) dsRNA were orally delivered. Mortality associated with reporter gene knockdown was observed only in locusts injected with dsLmdsRNase2 (48% and 22%, for dsLmCht10 and dsLmCHS1, respectively), implicating LmdsRNase2 in reducing RNAi efficiency. Furthermore, recombinantly expressed LmdsRNase2 fusion proteins degraded dsRNA rapidly, whereas LmdsRNase3 did not. These results suggest that rapid degradation of dsRNA by dsRNase2 in the midgut is an important factor causing low RNAi efficiency when dsRNA is orally delivered in the locust.     

Combining genetic engineering and traditional breeding to provide elevated resistance in potatoes to Colorado potato beetle

The sustainable deployment of resistant crop varieties is a critical issue for the implementation of biotechnology in crop pest management. Feeding, biomass accumulation, and mortality were evaluated for susceptible, insecticide‐resistant, and Bacillus thuringiensis (Bt) Cry 3A‐selected Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera, Chrysomelidae) larvae fed on: cultivated potato, a Solanum chacoense line expressing leptine glycoalkaloids, a transformed line expressing Bt toxin, or the leptine line transformed to express Bt toxin. Larvae selected for resistance to Bt‐Cry3A performed better on Bt foliage, but not as well on the leptine foliage, compared to susceptible or insecticide‐resistant larvae. Neither leptine nor Bt toxin completely inhibited the feeding and growth of 3rd and 4th instars of all three strains of Colorado potato beetle. However, for all three strains of Colorado potato beetle on leptine + Bt foliage, feeding was almost zero, growth was zero or negative, and mortality was near 100%.     

Age-specific bioassays and fecundity of Phthorimaea operculella (Lepidoptera: Gelechiidae) reared on cry1Ac -transgenic potato plants

Variation in the susceptibility of lepidopterous pest larvae of different ages to transgenic crops and the potential for survivors to reproduce could have important consequences for the development of resistance in such pests. Experiments were undertaken in the laboratory to determine if larvae of the potato tuber moth, Phthorimaea operculella, of different ages (0 (< 1 day old), 3, 5, 7 days) varied in their susceptibility to cry1Ac9–transgenic potato (Solanum tuberosum) foliage grown in the glasshouse or field. The survival and fecundity of larvae reared on transgenic tubers was also determined in the laboratory. There were no apparent differences in susceptibility of larvae of different ages to transgenic foliage. Larvae fed glasshouse or field‐grown non‐transgenic foliage had significantly larger relative growth indices and more larvae pupated, than those fed transgenic foliage, regardless of larval age. Eggs from a laboratory colony were placed on transgenic or non‐transgenic tubers to measure survival and fecundity. Between 6% and 15% of eggs placed on transgenic tubers developed into pupae for three of the four transgenic potato lines tested. On one transgenic line, only six adults emerged from 1300 eggs. In contrast, between 71% and 97% of the eggs placed on non‐transgenic tubers developed into pupae. Male and female pupae from transgenic lines weighed less than those from non‐transgenic lines. The fecundity of females from two of four transgenic lines was lower than from the non‐transgenic parent cultivar. Although larvae of different ages did not exhibit any overall age‐dependent pattern of increasing or decreasing susceptibility to transgenic foliage of glasshouse or field‐grown plants, the ability of larvae to survive and reproduce on transgenic tubers suggests this pest has the ability to evolve resistance to the transgenic plants used in the present study.