Genome mining and functional analysis of cytochrome P450 genes involved in insecticide resistance in Leucinodes orbonalis (Lepidoptera: Crambidae).

Genome-wide analysis of cytochrome P450 monooxygenase (CYP) genes from the advanced genome project of the Leucinodes orbonalis and the expression analysis provided significant information about the metabolism-mediated insecticide resistance. A total of 72 putative CYP genes were identified from the genome and transcriptome of L. orbonalis. The genes were classified under 30 families and 46 subfamilies based on the standard nomenclature. In the present study, a novel CYP gene, CYP324F1, was identified and it has not been reported from any other living system so far. Biochemical assays showed enhanced titers (5.81-18.5-fold) of O-demethylase of CYP in five field-collected populations. We selected 34 homologous CYP gene sequences, seemed to be involved in insecticide resistance for primer design and quantitative real-time PCR studies. Among the many overexpressed genes (>10 fold), the expression levels of CYP324F1 and CYP306A1 were prominent across all the field populations as compared with the susceptible iso-female line. Oral delivery of ds-CYP324F1 and ds-CYP306A1 directed against CYP324F1 and CYP306A1 to the larvae of one of the insecticide resistance populations caused reduced expression of these two transcripts in a dose-dependent manner (53.4%-85.0%). It appears that the increased titer of O-demethylase is the result of increased transcription level of CYP genes in resistant populations. The data provide insight for identifying the novel resistance management strategies against L. orbonalis.

The miniature genome of broad mite, Polyphagotarsonemus latus (Tarsonemidae: Acari).

The broad mite, Polyphagotarsonemus latus (Tarsonemidae: Acari) is a highly polyphagous species that damage plant species spread across 57 different families. This pest has developed high levels of resistance to some commonly used acaricides. In the present investigation, we deciphered the genome information of P. latus by PacBio HiFi sequencing. P. latus is the third smallest arthropod genome sequenced so far with a size of 49.1 Mb. The entire genome was assembled into two contigs. A set of 9,286 protein-coding genes were annotated. Its compact genome size could be credited with multiple features such as very low repeat content (5.1%) due to the lack of proliferation of transposable elements, high gene density (189.1/Mb), more intronless genes (20.3%) and low microsatellite density (0.63%).

Identification and expression dynamics of CYPome across different developmental stages of Maconellicoccus hirsutus (Green).

Maconellicoccus hirsutus is a highly polyphagous insect pest, posing a substantial threat to various crop sp., especially in the tropical and sub-tropical regions of the world. While extensive physiological and biological studies have been conducted on this pest, the lack of genetic information has hindered our understanding of the molecular mechanisms underlying its growth, development, and xenobiotic metabolism. The Cytochrome P450 gene, a member of the CYP gene superfamily ubiquitous in living organisms is associated with growth, development, and the metabolism of both endogenous and exogenous substances, contributing to the insect's adaptability in diverse environments. To elucidate the specific role of the CYP450 gene family in M. hirsutus which has remained largely unexplored, a de novo transcriptome assembly of the pink mealybug was constructed. A total of 120 proteins were annotated as CYP450 genes through homology search of the predicted protein sequences across different databases. Phylogenetic studies resulted in categorizing 120 CYP450 genes into four CYP clans. A total of 22 CYP450 families and 30 subfamilies were categorized, with CYP6 forming the dominant family. The study also revealed five genes (Halloween genes) associated with the insect hormone biosynthesis pathway. Further, the expression of ten selected CYP450 genes was studied using qRT-PCR across crawler, nymph, and adult stages, and identified genes that were expressed at specific stages of the insects. Thus, the findings of this study reveal the expression dynamics and possible function of the CYP450 gene family in the growth, development, and adaptive strategies of M. hirsutus which can be further functionally validated.

Transcriptome mining and expression analysis of ABC transporter genes in a monophagous herbivore, Leucinodes orbonalis (Crambidae: Lepidoptera).

Insecticide resistance is a global concern and requires immediate attention to manage dreadful insect pests. One of the resistance mechanisms adopted by insects is through ATP-binding cassette (ABC) transporter proteins. These proteins rapidly transport and eliminate the insecticidal molecules across the lipid membranes (Phase III detoxification mechanism). In the present study, we investigated the potential role of ABC transporter genes in imparting insecticide resistance in field-collected insecticide resistant larvae of eggplant shoot and fruit borer (Leucinodes orbonalis; Crambidae: Lepidoptera). Dose-mortality bioassays against five insecticidal molecules revealed moderate to high levels of insecticide resistance (32.2. to 134.1-fold). Thirty-one genes encoding ABC transporter proteins were mined from the transcriptome resources of L. orbonalis. They were classified under eight sub-families (ABCA to ABCH). Phylogenetic analysis indicated ABCG is the most divergent, composed of nine genes as compared to many other insects. Transcriptome analysis of the insecticide resistant and susceptible strains of L. orbonalis revealed differential expression of 13 ABC transporter genes. The altered expression of these genes was further validated using qRT-PCR. The knockdown studies indicated the involvement of ABCD1 and ABCG2 genes in chlorantraniliprole resistance in the insecticide-resistant strain of L. orbonalis. This study unveils the additional mechanisms employed by L. orbonalis in resisting insecticide toxicity through accelerated excretion mode. These ABCD and ABCG family genes could be candidate targets in developing genome-assisted pest management strategies in the future.

Effect of Pheromone-Mediated Mating Disruption on Pest Population Density of Maruca vitrata (Fabricius) (Crambidae: Lepidoptera).

The legume pod borer (Maruca vitrata) is one of the most serious legume pests due to its wide host range and high damage potential. Pheromone components on M. vitrata have been previously identified, allowing research on more environmentally friendly IPM tools for its control. M. vitrata produces a three-component pheromone blend containing (E, E)-10,12-hexadecadienal (major), (E, E)-10,12-hexadecadienol (minor), (E)-10-hexadecenal (minor). This study focused on the efficacy of synthetic pheromone lures and their blend components for mating disruption in M. vitrata. Under laboratory conditions, the mating behavior of M. vitrata pairs was observed from 18:00 to 02:00 h in an interval of 20 min to assess the efficacy of different pheromone lures. The scotophase behavior results show that the complete pheromone blend (E, E)-10,12-hexadecadienal + (E, E)-10,12-hexadecadienol + (E)-10-hexadecenal with a blend ratio of 1:1:1 effectively disrupted mating. The impact on mating disruption was evident from the lower fecundity and egg hatch/eclosion. The same lures were evaluated in a small-scale caged field study. The results show that the pheromone blend of (E, E)-10,12-hexadecadienal + (E, E)-10,12-hexadecadienol + (E)-10-hexadecenal in a1:1:1 ratio significantly disrupted the normal mating, leading to lower flower and pod damage and higher mung bean yield.

Differential toxicity of Bacillus thuringiensis strains and their crystal toxins against high-altitude Himalayan populations of diamondback moth, Plutella xylostella L.

BACKGROUND: Diamondback moth, Plutella xylostella (L.), is a major insect pest of crucifers in the biodiversity-rich north-western Indian Himalayan hills. The present investigation was aimed at determining the susceptibility pattern of P. xylostella populations collected from different locations of this region to autochthonous and standard Bacillus thuringiensis strains. RESULTS: Among the reference as well as indigenous B. thuringiensis strains tested, sub spp. kurstaki HD-1, kurstaki HD-73, galleriae HD-8, local galleriae/colmeri strain BtOa1 and some of their Cry1 class toxins were found to be highly toxic. Surprisingly, the sub sp. tolworthi HD-125, local tolworthi strain BtHa1 and Cry9 class toxins were found to be non-toxic. Midgut homogenate from fourth-instar larvae was found to activate 130 kDa protoxin from the local tolworthi strain BtHa1 into 68 kDa toxin, but failed to exert any larval mortality, probably owing to lack of receptor binding. CONCLUSION: The present study provides valuable baseline susceptibility data for the deployment of B. thuringiensis-based control methods, as well as for future monitoring of development of resistance in P. xylostella to B. thuringiensis in this ecologically sensitive region.

Establishing the role of detoxifying enzymes in field-evolved resistance to various insecticides in the brown planthopper (Nilaparvata lugens) in South India.

The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the major pests of rice throughout Asia. Extensive use of insecticides for suppressing N. lugens has resulted in the development of insecticide resistance leading to frequent control failures in the field. The aim of the present study was to evaluate resistance in the field populations of N. lugens from major rice growing states of South India to various insecticides. We also determined the activity of detoxifying enzymes (esterases [ESTs], glutathione S-transferases [GSTs], and mixed-function oxidases [MFOs]). Moderate levels of resistance were detected in the field populations to acephate, thiamethoxam and buprofezin (resistance factors 1.05-20.92 fold, 4.52-14.99 fold, and 1.00-18.09 fold, respectively) as compared with susceptible strain while there were low levels of resistance to imidacloprid (resistance factor 1.23-6.70 fold) and complete sensitivity to etofenoprox (resistance factor 1.05-1.66 fold). EST activities in the field populations were 1.06 to 3.09 times higher than the susceptible strain while for GST and MFO the ratios varied from 1.29 to 3.41 and 1.03 to 1.76, respectively. The EST activity was found to be correlated to acephate resistance (r = 0.999, P ≥ 0.001). The high selection pressure of organophosphate, neonicotinoid, and insect growth regulator (IGR) in the field is likely to be contributing for resistance in BPH to multiple insecticides, leading to control failures. The results obtained will be beneficial to IPM recommendations for the use of effective insecticides against BPH.