Analysis of insecticide resistance and de novo transcriptome assembly of resistance associated genes in the European grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae).

The European grapevine moth Lobesia botrana (Denis & Shiffermüller 1776) is an economically important pest of the vine-growing areas worldwide. Chemical insecticides have been used for its control; however, its resistance status is largely unknown in many regions. We monitored the susceptibility of several L. botrana populations from Greece and Turkey. In addition, based on RNAseq transcriptome analysis, we identified and phylogenetically classify the cytochrome P450 genes of L. botrana, as well as analysed target site sequences and looked for the presence of known resistance mutations. Resistance against chlorantraniliprole, alpha-cypermethrin, spinetoram, etofenprox, and acetamiprid was very low (below 2.5-fold in all cases, compared to a reference strain from Greece) in all populations from Greece that were included in the study. However, resistance against indoxacarb (4-30-fold), spinosad (5-59-fold), and deltamethrin (18-30 fold) was detected in the L. botrana populations from Turkey, compared to a reference population from Turkey. De novo transcriptome assembly and manual annotation, and subsequent PCR-based analysis of insecticide target sequences (i.e. voltage-gated sodium channel - VGSC: target of pyrethroids and oxadiazines; nicotinic acetylcholine receptor subunit a6 - nAChR_α6: target of spinosad; ryanodine receptor - RyR: target of diamides; glutamate-gated chloride channel - GluCl: target of avermectins and; acetylcholinesterase - AChE: target of organophosphates) showed the absence of known resistance mutations in all specimens from both countries. Finally, the L. botrana CYPome (116 genes) was manually analysed and phylogenetically characterised, to provide resources for future studies that will aim the analysis of metabolic resistance.

The biological and physiological responses of Leptinotarsa decemlineata Say (Col. Chrysomelidae) to different potato cultivars.

BACKGROUND: The use of pest-resistant or less susceptible plant varieties is of great importance for integrated pest management programs. This study was conducted to evaluate the resistance of seven potato cultivars to a major pest, namely the Colorado potato beetle (CPB), by measuring its bio-physiological aspects. RESULTS: The longest developmental time and the lowest survival rate of CPB were detected on cultivar 'Manitou.' Although the larvae fed voraciously, the food conversion efficiency was the lowest on this cultivar leading to the lowest relative growth rate. Furthermore, larvae reared on Manitou had the lowest total protease activity and specific activity values of cathepsin B compared to the larvae reared on other cultivars. Additionally, the cluster analysis showed that the Manitou cultivar was the least suitable (partially resistant) CPB. Moreover, the results indicated that the protein content was the lowest in the Manitou cultivar; conversely, this cultivar was rich in polyphenol and flavonoid compared to Agria and Santae cultivars. CONCLUSION: The overall results in this study indicated that the Manitou cultivar might be a good candidate to be incorporated into integrated pest management programs. © 2022 Society of Chemical Industry.

Evolution of Adaptive Variation in the Mosquito Culex pipiens: Multiple Independent Origins of Insecticide Resistance Mutations.

Insecticide resistance is an informative model for studying the appearance of adaptive traits. Simultaneously, understanding how many times resistance mutations originate is essential to design effective resistance management. In the mosquito Culex pipiens, target-site resistance to the insecticide diflubenzuron (DFB) has been recently found in Italian and Turkish populations. Three point mutations confer it at the codon 1043 of the chitin synthase 1 gene (chs-1): I1043L, I1043M, and I1043F. Whether the resistant mutations originated independently from different susceptible alleles or sequentially from resistant alleles and whether resistant alleles from Italy and Turkey have originated once or multiple times remain unresolved. Here, we sequenced a fragment of the chs-1 gene carrying the resistant mutations and inferred the phylogenetic relationships among susceptible and resistant alleles. Confirming previous findings, we found the three mutations in Italy and the I1043M in Turkey. Notably, the I1043F was also found for the first time in Turkish samples, highlighting the need for extensive monitoring activities. Phylogenetic analyses are consistent with an independent origin of the I1043F, I1043M, and I1043L mutations from different susceptible alleles and with multiple independent origins of the Italian and Turkish I1043M and I1043F alleles.

Molecular characterization and expression patterns of heat shock proteins in Spodoptera littoralis, heat shock or immune response?

The Egyptian cotton leaf worm, Spodoptera littoralis (Boisd.), is a major agricultural lepidopterous pest causing extensive damage in a variety of crops including vegetable, cotton, fodder, and fiber crops. Heat shock protein (HSP) family members play important roles in protecting insects against environmental stressors. In this study, we characterized three putative heat shock proteins (SpliHsp70, SpliHsp90, and SpliHSF) from S. littoralis and analyzed their expression levels in response to heat, cold, ultraviolet irradiation, Bacillus thuringiensis, and Spodoptera littoralis nucleopolyhedrovirus treatments. Significant upregulation of SpliHsp70 was observed in female pupae, while the highest expression levels of SpliHsp90 and SpliHSF were found in female adults. Heat shock triggered increases in SpliHsp levels compared to cold treatment. SpliHsp90 exhibited the highest expression levels during the first 30 min of UV treatment. Both bacterial and viral pathogenic agents effected the regulation of Hsps in S. littoralis. These findings suggest that SpliHsp genes might play significant roles in the response to biotic and abiotic stress, as well as in the regulation of developmental stages.

Sublethal effects of three insecticides on fitness parameters and population projection of Brevicoryne brassicae (Hemiptera: Aphididae).

The cabbage aphid, Brevicoryne brassicae (L.), is one of the major insect pests of cole crops in Iran. In most instances outbreaks are normally kept under control by application of insecticides. In this study, the sublethal effects (LC30) of three insecticides, acetamiprid, buprofezin, and thiamethoxam-lambda cyhalothrin, (TLC) were evaluated on the population growth rate of the progeny of insecticide-treated cabbage aphid adults. The age-stage, two-sex life table method was used to analyze the collected data. The results indicated that the insecticide applications affected the duration of the preadult period, their survival, reproduction, life span/longevity, and consequently, the population growth rate of the F1 generation. The indicators of the greatest sublethal effects were noted in the progeny of the TLC-treated adults. These included the lowest net reproductive rate (R0), intrinsic rate of increase (r), finite rate of increase (λ), and the longest mean generation time (T). The highest values of r, λ, R, and the lowest value of T occurred in the control group followed by, in order, the acetamiprid and buprofezin groups. These research findings will be useful in the development and implementation of future aphid management programs.

High Prevalence of Pantoea in Diaphorina citri (Hemiptera: Liviidae): Vector of Citrus Huanglongbing Disease.

As an important insect vector, Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) transmits the pathogen 'Candidatus Liberibacter asiaticus' (CLas) that is associated with citrus greening also known as Huanglongbing (HLB) disease. The bacterial endosymbionts have a potential role in shaping the host range of insect herbivores and their performance on different host plants, which might affect the endosymbiont distribution in insect populations. Here, we detected and characterized Pantoea endosymbiont in nymph and adult ACP specimens collected from Citrus reticulata Blanco and Cordia myxa L. plants. The phylogenetic tree constructed using endosymbiotic bacteria 16S ribosomal RNA sequences indicated that Pantoea sp. was closely related to Mixta calida, sharing about 98% identity and was grouped with other Mixta and Pantoea endosymbionts. Our findings showed 100% and 92.3% infection of Pantoea in adults while 61.5% and 90% infection of Pantoea in nymphs collected from C. reticulata and C. myxa plants, respectively. Understanding the interaction of endosymbiotic bacteria with ACP associated with host plants could be useful for developing an effective management strategy for both ACP and HLB disease.

Detection of Wolbachia (Rickettsiales: Anaplasmataceae) and Candidatus Liberibacter asiaticus (Rhizobiales: Rhizobiaceae) Associated With Diaphorina citri (Hemiptera: Liviidae) Collected From Citrus reticulata (Sapindales: Rutaceae) and Alternate Host, Cordia myxa (Boraginales: Boraginaceae).

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important insect pest of the citrus crop worldwide. It vectors the pathogen 'Candidatus Liberibacter asiaticus' (CLas) that causes a serious disease known as citrus greening. Here, we tested the infection frequency of Wolbachia and CLas from 100 D. citri individuals collected from two host plants belonging to families Rutaceae (Citrus reticulata Blanco) and Boraginaceae (Cordia myxa L.) using molecular methods. The following trend of endosymbionts infection in adult D. citri was found; 85.4% (35/41) by Wolbachia, and 19.5% (8/41) by CLas collected from C. reticulata plants and 65.4% (17/26) by Wolbachia, and 15.4% (4/26) by CLas in case of C. myxa plant. However, 61.5% (8/13) nymphs collected from C. reticulata and 20.0% (4/20) collected from C. myxa plants were infected by Wolbachia, while no nymph was infected by CLas collected from either host plants. Findings from this work represent the first report of CLas presence in D. citri feeding on C. myxa plants. By studying the presence of CLas with other endosymbiotic bacteria, future basic and applied research to develop control strategies can be prioritized.

Detection of diflubenzuron and pyrethroid resistance mutations in Culex pipiens from Mugla, Turkey.

Chemical insecticides are the main tool for vector control worldwide with pyrethroids and the larvicide diflubenzuron (DFB) used extensively against the primary West Nile virus vector Culex pipiens. In this study we monitored the presence, frequency and geographical distribution of the Chitin synthase 1 gene mutations I1043L, I1043M associated with striking diflubenzuron resistance and the knock down resistance mutation L1014F associated with pyrehtroid resistance, in Cx. pipiens populations from Turkey. DFB resistance mutations I1043L, I1043M were found at high frequencies ranging from 15.7% to 37.5% and 25% to 52.7% in all study sites (first report for the presence of DFB resistant mutations, anywhere outside Italy). The L1014F pyrethroid resistance mutation was also present at relatively high frequencies, ranging from 40% to 50%. The high frequency of both DFB and pyrethroid resistance mutations are possibly associated with the intense use of these insecticides in agricultural and mosquito control applications in the region. Given the limited number of available insecticides for vector control these findings are of major importance for public health in Turkey and neighboring countries.

Identification of a putative antifreeze protein gene that is highly expressed during preparation for winter in the sunn pest, Eurygaster maura.

A cDNA library generated from the fat body of field-collected, diapausing adults of the sunn pest, Eurygaster maura revealed the presence of a transcript that encodes a protein that shares the distinct physiochemical and structural features of an insect antifreeze protein. The transcript, which is most abundant in the midgut, accumulates in adults as they leave the fields in late summer and migrate to surrounding mountainous areas to overwinter. Transcript abundance again declines when adults return to the fields the following spring. This winter pattern of abundance suggests that this protein may be critical for winter survival in the cold regions where the bug enters its obligatory diapause.

Identification and coordinated expression of perilipin genes in the biological cycle of sunn pest, Eurygaster maura (Hemiptera: Scutelleridae): implications for lipolysis and lipogenesis.

The sunn pest, Eurygaster spp., is one of the most destructive pests of grains in Asia, Europe and Africa. The nymphs and adults feed voraciously in the field by late-spring, followed by migration of adults into mountains for diapause, which includes estivation by late summer and hibernation during winter. Adults migrate back to the field by the end of diapause in mid-spring, where they mate and lay eggs. To understand how sunn pest survives and maintains basic metabolic functions without feeding for 7 months during diapause, this study focused on lipid metabolism as the major source of energy production, and the primary organ of lipid metabolism, the fat body. Studies on lipid metabolism revealed two major factors referred to perilipin protein family, Lipid Storage Droplet Protein 1 (LSD1) and Lipid Storage Droplet Protein 2 (LSD2), which are involved in hydrolysis and accumulation of lipids, respectively. In this study, two LSD (EmLSD1-2) orthologues in the hemimetabolous Eurygaster maura were identified. EmLSD1 and EmLSD2 genes were expressed in multiple tissues, but primarily in fat body. Both genes were continuously expressed throughout the insect's life cycle but peaked in the 4th nymphal stage. Their expression patterns were in accordance with the biological roles of LSDs. EmLSD1 expression peaked in non-feeding stages supporting its lipolytic role, while the highest level of EmLSD2 expression was in feeding stages supporting its lipogenetic role. Expression patterns of both genes differed in females and males. Overall, expression patterns of EmLSDs provide clues to understanding the interesting life cycle of sunn pest.

Next generation sequencing based transcriptome analysis of septic-injury responsive genes in the beetle Tribolium castaneum.

Beetles (Coleoptera) are the most diverse animal group on earth and interact with numerous symbiotic or pathogenic microbes in their environments. The red flour beetle Tribolium castaneum is a genetically tractable model beetle species and its whole genome sequence has recently been determined. To advance our understanding of the molecular basis of beetle immunity here we analyzed the whole transcriptome of T. castaneum by high-throughput next generation sequencing technology. Here, we demonstrate that the Illumina/Solexa sequencing approach of cDNA samples from T. castaneum including over 9.7 million reads with 72 base pairs (bp) length (approximately 700 million bp sequence information with about 30× transcriptome coverage) confirms the expression of most predicted genes and enabled subsequent qualitative and quantitative transcriptome analysis. This approach recapitulates our recent quantitative real-time PCR studies of immune-challenged and naïve T. castaneum beetles, validating our approach. Furthermore, this sequencing analysis resulted in the identification of 73 differentially expressed genes upon immune-challenge with statistical significance by comparing expression data to calculated values derived by fitting to generalized linear models. We identified up regulation of diverse immune-related genes (e.g. Toll receptor, serine proteinases, DOPA decarboxylase and thaumatin) and of numerous genes encoding proteins with yet unknown functions. Of note, septic-injury resulted also in the elevated expression of genes encoding heat-shock proteins or cytochrome P450s supporting the view that there is crosstalk between immune and stress responses in T. castaneum. The present study provides a first comprehensive overview of septic-injury responsive genes in T. castaneum beetles. Identified genes advance our understanding of T. castaneum specific gene expression alteration upon immune-challenge in particular and may help to understand beetle immunity in general.

Molecular characterization of Ephestia kuehniella (Lepidoptera: Pyralidae) transferrin and its response to parasitoid Venturia canescens (Hymenoptera: Ichneumonidae Gravenhorst).

In the present study, we characterized a full-length cDNA encoding a putative iron-binding protein transferrin from the lepidopteran Mediterranean flour moth (EkTrf, 2397 bp). The putative EkTrf is 683 amino acids with a molecular mass of approximately 76 kDa. The deduced amino acid sequence showed significant homology with other insect transferrins from Chilo suppressalis (76%), Galleria mellonella (75%), Plutella xylostella (72%), Manduca sexta (74%), Bombyx mori (73%), Spodoptera litura and (72%), Choristoneura fumiferana (71%). Northern blot analysis indicated that Ephestia transferrin mRNA was expressed in the last larval instars of both males and females and in the pupal developmental stages. EkTrf is expressed predominantly in the fat body and ovary tissues. Analysis of parasitized larva by the endoparasitoid Venturia canescens suggests that transferrin expression is induced following parasitoid challenge. Expression of EkTrf levels also increased upon bacterial infection at 6 h post treatment and remained high until 24 h. Similarly to other insect transferrins, EkTrf may play a role in immunity through its iron-binding capacity.

Infections with immunogenic trypanosomes reduce tsetse reproductive fitness: potential impact of different parasite strains on vector population structure.

The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6-8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the fly's lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite-host coevolution, and disease prevalence are discussed.

Molecular aspects of transferrin expression in the tsetse fly (Glossina morsitans morsitans).

Iron is an essential element for metabolic processes intrinsic to life, and yet the properties that make iron a necessity also make it potentially deleterious. To avoid harm, iron homeostasis is achieved via proteins involved in transport and storage of iron, one of which is transferrin. We describe the temporal and spatial aspects of transferrin (GmmTsf) expression and its transcriptional regulation in tsetse where both the male and female are strictly hematophagous. Using Northern, Western and immunohistochemical analysis, we show that GmmTsf is abundant in the hemolymph and is expressed in the adult developmental stages of male and female insects. It is preferentially expressed in the female milk gland tubules and its expression appears to be cyclical and possibly regulated in synchrony with the oogenic and/or larvigenic cycle. Although no mRNA is detected, GmmTsf protein is present in the immature stages of development, apparently being transported into the intrauterine larva from the mother via the milk gland ducts. Transferrin is also detected in the vitellogenic ovary and the adult male testes, further supporting its classification as a vitellogenic protein. Similar to reports in other insects, transferrin mRNA levels increase upon bacterial challenge in tsetse suggesting that transferrin may play an additional role in immunity. Although transferrin expression is induced following bacterial challenge, it is significantly reduced in tsetse carrying midgut trypanosome infections. Analysis of tsetse that have cured the parasite challenge shows normal levels of GmmTsf. This observation suggests that the parasite in competing for the availability of limited dietary iron may manipulate host gene expression.

Molecular characterization of iron binding proteins from Glossina morsitans morsitans (Diptera: Glossinidae).

The regulation of iron is critical for maintaining homeostasis in the tsetse fly (Diptera: Glossinidae), in which both adult sexes are strict blood feeders. We have characterized the cDNAs for two putative iron-binding proteins (IBPs) involved in transport and storage; transferrin (GmmTsf1) and ferritin from Glossina morsitans morsitans. GmmTsf1 transcripts are detected in the female fat body and in adult reproductive tissues, and only in the adult developmental stage in a bloodmeal independent manner. In contrast, the ferritin heavy chain (GmmFer1HCH) and light chain (GmmFer2LCH) transcripts are expressed ubiquitously, suggesting a more general role for these proteins in iron transport and storage. Protein domain predictions for each IBP suggest both the conservation and loss of several motifs present in their vertebrate homologues. In concert with many other described insect transferrins (Tfs), putative secreted GmmTsf1 maintains 3 of the 5 residues necessary for iron-binding in the N-terminal lobe, but exhibits a loss of this iron-binding ability in the C-terminal lobe as well as a loss of large sequence blocks. Both putative GmmFer1HCH and GmmFer2LCH proteins have signal peptides, similar to other insect ferritins. GmmFer2LCH has lost the 5'UTR iron-responsive element (IRE) and, thus, translation is no longer regulated by cellular iron levels. On the other hand, GmmFer1HCH maintains both the conserved ferroxidase center and the 5'UTR IRE; however, transcript variants suggest a more extensive regulatory mechanism for this subunit.

Molecular aspects of viviparous reproductive biology of the tsetse fly (Glossina morsitans morsitans): regulation of yolk and milk gland protein synthesis.

Tsetse fly (Diptera: Glossinidae) viviparous reproductive physiology remains to be explored at the molecular level. Adult females carry their young in utero for the duration of embryonic and larval development, all the while supplying their offspring with nutrients in the form of a "milk" substance secreted from a modified accessory gland. Flies give birth to fully developed third instar larvae that pupariate shortly after birth. Here, we describe the spatial and temporal expression dynamics of two reproduction-associated genes and their products synthesized during the first and second gonotrophic cycles. The proteins studied include a putative yolk protein, Glossina morsitans morsitans yolk protein 1 (GmmYP1) and the major protein found in tsetse "milk" secretions (Glossina morsitans morsitans milk gland protein, GmmMGP). Developmental stage and tissue-specific expression of GmmYP1 show its presence exclusively in the reproductive tract of the fly during oogenesis, suggesting that GmmYP1 acts as a vitellogenic protein. Transcripts for GmmMGP are present only in the milk gland tissue and increase in coordination with the process of larvigenesis. Similarly, GmmMGP can be detected at the onset of larvigenesis in the milk gland, and is present during the full duration of pregnancy. Expression of GmmMGP is restricted to the adult stage and is not detected in the immature developmental stages. These phenomena indicate that the protein is transferred from mother to larvae as nourishment during its development. These results demonstrate that both GmmYP1 and GmmMGP are involved in tsetse reproductive biology, the former associated with the process of oogenesis and the latter with larvigenesis.