Insects constitute the largest and most diverse group of animals on Earth with an equally diverse virome. The main antiviral immune system of these animals is the post-transcriptional gene-silencing mechanism known as RNA(i) interference. Furthermore, this process can be artificially triggered via delivery of gene-specific double-stranded RNA molecules, leading to specific endogenous gene silencing. This is called RNAi technology and has important applications in several fields. In this paper, we review RNAi mechanisms in insects as well as the potential of RNAi technology to contribute to species-specific insecticidal strategies. Regarding this aspect, we cover the range of strategies considered and investigated so far, as well as their limitations and the most promising approaches to overcome them. Additionally, we discuss patterns of viral infection, specifically persistent and acute insect viral infections. In the latter case, we focus on infections affecting economically relevant species. Within this scope, we review the use of insect-specific viruses as bio-insecticides. Last, we discuss RNAi-based strategies to protect beneficial insects from harmful viral infections and their potential practical application. As a whole, this manuscript stresses the impact of insect viruses and RNAi technology in human life, highlighting clear lines of investigation within an exciting and promising field of research.

/pdf/rna-interference-in-insects-protecting-beneficials-and-25cybdaba5.pdf

RNA Interference in Insects: Protecting Beneficials and Controlling Pests.

The progress and future of enhancing antiviral capacity by transgenic technology in the silkworm Bombyx mori.

Rapid advances in genome engineering tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced palindromic repeats/CRISPR-associated (CRISPR/Cas) system, have enabled efficient gene knockout experiments in a wide variety of organisms. Here, we review the recent progress in targeted gene disruption techniques in the silkworm, Bombyx mori. Although efficiency of targeted mutagenesis was very low in an early experiment using ZFNs, recent studies have shown that TALENs can induce highly efficient mutagenesis of desired target genes in Bombyx. Notably, mutation frequencies induced by TALENs can reach more than 50% of G0 gametes. Thus, TALENs can now be used as a standard tool for gene targeting studies, even when mutant phenotypes are unknown. We also propose guidelines for experimental design and strategy for knockout experiments in Bombyx. Genome editing technologies will greatly increase the usefulness of Bombyx as a model for lepidopteran insects, the major agricultural pests, and lead to sophisticated breeding of Bombyx for use in sericulture and biotechnology.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/dgd.12096

Recent progress in genome engineering techniques in the silkworm, Bombyx mori

The seasonal differences in the environmental components considerably affect the genotypic expression in the form of phenotypic output of silkworm crop such as cocoon weight, shell weight, and cocoon shell ratio. The variations in the environmental conditions day to day and season to season emphasize the need of management of temperature and relative humidity for sustainable cocoon production. The present review paper discuss in details about the role of temperature and humidity on growth and development of silkworm including recent studies on heat shock protein. Study also discusses the influence of air and light on silkworm development. In addition to this study emphasis on the role of various environmental factors on embryonic development of silkworm egg, nutritional indices of silkworm larva and reproductive potential of silkworm moth. The study also highlights about the care to be required during silkworm spinning and influence of temperature and humidity on post cocoon parameters of silkworm. The study included future strategies to be taken for the management climatic condition for successful cocoon crop. The paper covers 140 references connected with the topic.

/pdf/management-of-climatic-factors-for-successful-silkworm-3c7ztx82xv.pdf

Management of Climatic Factors for Successful Silkworm (Bombyx mori L.) Crop and Higher Silk Production: A Review

RNAi is applied as a new and safe method for pest control in agriculture but efficiency and specificity of delivery of dsRNA trigger remains a critical issue. Various agents have been proposed to augment dsRNA delivery, such as engineered micro-organisms and synthetic nanoparticles, but the use of viruses has received relatively little attention. Here we present a critical view of the potential of the use of recombinant viruses for efficient and specific delivery of dsRNA. First of all, it requires the availability of plasmid-based reverse genetics systems for virus production, of which an overview is presented. For RNA viruses, their application seems to be straightforward since dsRNA is produced as an intermediate molecule during viral replication, but DNA viruses also have potential through the production of RNA hairpins after transcription. However, application of recombinant virus for dsRNA delivery may not be straightforward in many cases, since viruses can encode RNAi suppressors, and virus-induced silencing effects can be determined by the properties of the encoded RNAi suppressor. An alternative is virus-like particles that retain the efficiency and specificity determinants of natural virions but have encapsidated non-replicating RNA. Finally, the use of viruses raises important safety issues which need to be addressed before application can proceed.

https://biblio.ugent.be/publication/8540748/file/8540750.pdf

Viral Delivery of dsRNA for Control of Insect Agricultural Pests and Vectors of Human Disease: Prospects and Challenges

Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for >50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (>75% survival rate as compared to <15% in nontransgenic lines) to baculovirus infection conferred by the transgene. We also observed impaired infectivity of the occlusion bodies derived from the transgenic lines as compared to the wild-type ones. Currently, large-scale exploitation of these transgenic lines is underway to bring about economic transformation of sericulture.

/pdf/engineering-silkworms-for-resistance-to-baculovirus-through-3n7k35n6ot.pdf

Engineering Silkworms for Resistance to Baculovirus Through Multigene RNA Interference

Mulberry (Morus spp.), being an economically important tree, is cultivated in China, India, Thailand, Brazil, Uzbekistan and other Countries across the globe, for its leaves to feed monophagous mulberry silkworm (Bombyx mori). The sustainability of silk industry is directly correlated with the production and continuous supply of high-quality mulberry leaves. In India, it is cultivated on large scale in tropical, sub-tropical and temperate regions under irrigated conditions for silkworm rearing. Drought, low temperature, high salinity and alkalinity, being experienced in widespread areas, are the major abiotic stresses, causing reduction in its potential foliage yield and quality. Further, climate change effects may worsen the productivity of mulberry in near future, not only in India but also across the globe. Although traditional breeding methods contributed immensely towards the development of abiotic stress-tolerant mulberry varieties, still there is lot of scope for implementation of modern genomic and molecular biology tools for accelerating mulberry genetic improvement programmes. This review discusses omics approaches, molecular breeding, plant tissue culture and genetic engineering techniques exploited for mulberry genetic improvement for abiotic stress tolerance. However, high-throughput biotechnological tools such as RNA interference, virus-induced gene silencing, epigenomics and genome editing tools need to be utilized in mulberry to accelerate the progress of functional genomics. The application of genomic tools such as genetic engineering, marker-assisted selection and genomic selection in breeding programmes can hasten the development of climate resilient and productive mulberry varieties leading to the vertical and horizontal expansion for quality silk production.

Improvement of abiotic stress adaptive traits in mulberry (Morus spp.): an update on biotechnological interventions.

The production of quality mulberry leaf and subsequent production of quality silk is hampered due to the incidence of various insect pests. The present study analyses the population dynamics of Diaphania pulverulentalis (leaf roller), a serious pest of mulberry in a sericulture seed farm. The results indicated that maximum population buildup of the pest was recorded during rainy season. High humidity coinciding with low temperature because of southwest and northeast monsoon was conducive for breeding and multiplication of the pest. Correlation studies revealed that there was a significant negative correlation between increase in temperature and pest infestation. All other weather factors recorded from the study location have a positive correlation with incidence of the pest. The regression model developed also supported the relationship between the pest population buildup and weather factors.

/pdf/association-of-climatic-factors-on-population-dynamics-of-5gk7fjpxad.pdf

Association of Climatic Factors on Population Dynamics of Leaf Roller, Diaphania pulverulentalis Hampson (Lepidoptera: Pyralidae) in Mulberry Plantations of Sericulture Seed Farm

Silkworm is a poikilothermic insect, whose growth and development is significantly affected by high temperatures. The current study aimed to identify bivoltine breeds tolerant of the high temperature conditions that occur in the tropics. Percentage pupa- tion at high temperatures and heat shock responses of silkworms were used as measures of thermotolerance. Thermotolerance of 20 silkworm breeds was assessed by rearing them at 36°C. Based on percentage pupation, three breeds, namely Nistari (multivoltine), SK4C (bivoltine) and CSR2 (bivoltine) were designated tolerant, moderately tolerant and susceptible, respectively. To understand the heat shock responses and the molecular mechanisms underlying thermotolerance, the tissue specific expression profiles of the nine heat shock protein (Hsp) genes were determined in the three breeds after a heat shock of 1 h at 36°C and a 2 h recovery period by perform- ing real-time qPCR. The level of expression of Hsp genes was significantly increased in heat shocked tissues and gradually decreased during the recovery period. The greatest increase in the expression of Hsp genes was recorded in fat body followed by mid gut and silk gland. Of the three breeds, Nistari showed the highest expression of Hsp genes and SK4C a moderate expression relative to CSR2. The qPCR results showed that the transcript levels of sHsp20.4 and 20.1, and Hsp70 were increased by 10.3, 9.7 and 2.3 times, respectively, in Nistari compared to CSR2. Similarly the expression of sHsp20.4 and 20.1, and Hsp70 were increased by 3.5, 2.3 and 1.5 times, respectively in SK4C compared to CSR2. The expression levels of Hsps during heat shock corresponded to the percentage pupation recorded for the three breeds at a high temperature. It is suggested that the Hsps and their levels of expression may play an important role in increasing the survival of silkworm larvae at high temperatures. This study identified SK4C as a bivoltine breed, which is highly tolerant of high temperature measured in terms of percentage pupation (of the bivoltine breeds) and higher levels of expression of Hsp genes compared to CSR2. The importance of SK4C as a thermotolerant bivoltine parent for breeding new bivoltine hybrids tolerant of high temperatures is discussed.

/pdf/analysis-of-transcripts-of-heat-shock-protein-genes-in-5corjwb2fu.pdf

V. Sivaprasad

Papers

Engineering Silkworms for Resistance to Baculovirus Through Multigene RNA Interference

Improvement of abiotic stress adaptive traits in mulberry (Morus spp.): an update on biotechnological interventions.

Association of Climatic Factors on Population Dynamics of Leaf Roller, Diaphania pulverulentalis Hampson (Lepidoptera: Pyralidae) in Mulberry Plantations of Sericulture Seed Farm

Analysis of transcripts of heat shock protein genes in silkworm, Bombyx mori (Lepidoptera: Bombycidae).

Colloidal Textile Dye-Based Dipstick Immunoassay for the Detection of Nuclear Polyhedrosis Virus (BmNPV) of Silkworm, Bombyx mori L.