Root knot nematode, Meloidogyne incognita, is an obligate sedentary endoparasite that infects a large number of crop species and causes substantial yield losses. Non-chemical based control strategies for these nematodes are gaining importance. In the present study, we have demonstrated the significance of two FMRFamide like peptide genes (flp-14 and flp-18) for infection and development of resistance to M. incognita through host-derived RNAi. The study demonstrated both in vitro and in planta validation of RNAi-induced silencing of the two genes cloned from J2 stage of M. incognita. In vitro silencing of both the genes interfered with nematode migration towards the host roots and subsequent invasion into the roots. Transgenic tobacco lines were developed with RNAi constructs of flp-14 and flp-18 and evaluated against M. incognita. The transformed plants did not show any visible phenotypic variations suggesting the absence of any off-target effects. Bioefficacy studies with deliberate challenging of M. incognita resulted in 50-80% reduction in infection and multiplication confirming the silencing effect. We have provided evidence for in vitro and in planta silencing of the genes by expression analysis using qRT-PCR. Thus the identified genes and the strategy can be used as a potential tool for the control of M. incognita. This is the first ever report that has revealed the utility of host delivered RNAi of flps to control M. incognita. The strategy can also be extended to other crops and nematodes.

BackgroundAnalysis of gene expression patterns leads to functional understanding of biological processes. Quantitative real-time PCR has become the most commonly used technique for in-depth studies of gene expression. To quantify variation in specific gene expression, accurate and reliable normalization across different samples and tissues is necessary. This can be achieved by selecting one or more suitable reference genes to compare the target mRNA transcript levels. In the present work, we illustrate the first evaluation of potential internal control or reference genes across different developmental stages of eggplant for reliable quantification of transcripts by real-time PCR.ResultsWe have evaluated the stability in expression of six candidate reference genes (18s rRNA, APRT, GAPDH, Cyclophilin, Actin, and RuBP) in a set of tissues representing six developmental stages of eggplant. The candidate genes were cloned from cDNA and analysed by real-time PCR. The expression data analyzed by three statistical methods (geNorm, NormFinder and BestKeeper) identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant. This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant.Conclusion18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant.

The complex parasitic strategy of Meloidogyne incognita appears to involve simultaneous expression of its pharyngeal gland-specific effector genes in order to colonize the host plants. Research reports related to effector crosstalk in phytonematodes for successful parasitism of the host tissue is yet underexplored. In view of this, we have used in planta effector screening approach to understand the possible interaction of pioneer genes (msp-18 and msp-20, putatively involved in late and early stage of M. incognita parasitism, respectively) with other unrelated effectors such as cell-wall modifying enzymes (CWMEs) in M. incognita. Host-induced gene silencing (HIGS) strategy was used to generate the transgenic eggplants expressing msp-18 and msp-20, independently. Putative transformants were characterized via qRT-PCR and Southern hybridization assay. SiRNAs specific to msp-18 and msp-20 were also detected in the transformants via Northern hybridization assay. Transgenic expression of the RNAi constructs of msp-18 and msp-20 genes resulted in 43.64–69.68% and 41.74–67.30% reduction in M. incognita multiplication encompassing 6 and 10 events, respectively. Additionally, transcriptional oscillation of CWMEs documented in the penetrating and developing nematodes suggested the possible interaction among CWMEs and pioneer genes. The rapid assimilation of plant-derived carbon by invading nematodes was also demonstrated using 14C isotope probing approach. Our data suggests that HIGS of msp-18 and msp-20, improves nematode resistance in eggplant by affecting the steady-state transcription level of CWME genes in invading nematodes, and safeguard the plant against nematode invasion at very early stage because nematodes may become the recipient of bioactive RNA species during the process of penetration into the plant root.

The sophisticated parasitic tactic of sedentary endoparasitic nematodes seems to involve the simultaneous alteration of the expression of multitude of its effector genes in order to hijack the plant metabolic and developmental pathway. In concordance with this hypothesis, we have targeted some candidate effector genes of Meloidogyne incognita to understand the possible interaction among those effectors for successful infection of the host plant. In vitro RNAi strategy was used to knock down M. incognita-specific pioneer effector genes, such as msp-18, msp-20, msp-24, msp-33 and msp-16 (known to interact with plant transcription factor), to investigate their possible effect on the expression of key cell wall-degrading enzymes (CWDE) and vice versa. Supported by the phenotypic data, intriguingly our study revealed that induced suppression of these pioneer genes cause transcriptional alteration of CWDE genes in M. incognita. This remarkable finding may provide some useful links for future research on nematode effector interaction.

The cereal cyst nematode, Heterodera avenae (Wollenweber, 1924) is one of the most important plant parasitic nematodes of cereals. It is an obligate sedentary endo parasite causing considerable crop losses in wheat, barley and oats worldwide. FMRFamide-like peptides (FLPs) play critical role as neurotransmitters or neuromodulators in the nervous system and proposed as one of the important targets for the plant parasitic nematode management. Therefore, for the first time we have cloned and characterized two neuropeptide genes (flp-12 and flp-16) from the cDNA library of feeding female of H. avenae. Sequence analysis of FLPs revealed that both the neuropeptides are closely related with the parasitic as well as free-living nematodes. The flp-12 contains putative 22 residue long signal peptide at N-terminal suggesting its association with extra-cellular functions, while flp-16 does not contain signal peptide. Besides this, we have found highly conserved motif KFEFIRF in flp-12 and RFGK motif in flp-16. These two flp genes could be interesting and potential targets for functional validation to explore their utility for designing management strategies.

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BACKGROUND Mosquito vectors exhibit variable biological characteristics that influence their response to vector control measures. Therefore, updated information on vector bionomics is important for ensuring that the currently used vector control strategies and tools are effective in view of the goal of malaria elimination in India by 2030. OBJECTIVE The objectives of the study were to assess the indoor and outdoor resting proportions of vector species, to know the host biting/feeding preference of vector species, the sites of transmission, and to get the susceptibility status of vectors agisnt insecticides used in public health programme. METHODS Mosquito collections were made from 13 districts from eight Indian states during 2017 to 2020 using different methods to deduce their densities. After morphological identification in the field, sibling species of Anopheles mosquitoes were molecularly identified using polymerase chain reaction (PCR) specific alleles. Plasmodium falciparum and Plasmodium vivax infections in the vectors were detected out using ELISA and PCR assays. Susceptibility status of the known primary malaria vectors to the insecticides commonly used in the national programme was determined using World Health Organization (WHO) protocol. RESULTS Anopheles culicifacies, one of the primary malaria vectors, was collected (man hour density 3.1-15.9) from all the states of India except the states in the northeastern region. An. fluviatilis, another primary vector, was collected from Madhya Pradesh, Maharashtra, Karnataka and Odisha states. An. culicifacies sibling species A was predominant in Haryana and Karnataka, while species C and E were predominant in Madhya Pradesh and Maharashtra. An. culicifacies was mainly endophilic in all the states except Madhya Pradesh where proportion of semigravid and gravid mosquitoes was nearly half of the unfed mosquitoes. Human blood index of An. culicifacies varied from 0.001 to 0.220 in all the study sites. The sporozoite rate of An. culicifacies was in the range of 0.06 – 4.24 except in Madhya Pradesh, where none of the vector mosquitoes was found infected with Plasmodium parasite. In the study area, An. culicifacies was resistant to DDT (<39% mortality). It has developed resistance also to malathion (49-78% mortality) in all districts except in Angul, Odisha and Palwal, Haryana and to deltamethrin in districts of Maharashtra, Gujarat, Haryana, and Karnataka states. CONCLUSIONS Our study provides crucial information on prevalence, resting behavior and sibling species composition of malaria vectors in India. It is evident from the study that development of resistance in An. culicifacies, the primary vector, to synthetic pyrethroids is increasing in the country. Result of the study also indicated towards the change in the resting behavior of An. culicifacies in Madhya Pradesh but needs larger studies to reaffirm this plausible change. Our findings will be crucial in developing vector control strategies in India, which intends to eliminate malaria by 2030.