Larval Midgut Research Articles

Functional analysis of a putative Bombyx mori cypovirus miRNA BmCPV-miR-10 and its effect on virus replication.

Bombyx mori cypovirus (BmCPV) is an important pathogen of silkworm (B. mori), the economically beneficial insect. The mechanism of its interaction with host immune defence system in the process of infection is still not yet completely clear. Researches have demonstrated that virus-encoded microRNAs (miRNA) play a crucial role in regulating host-pathogen interaction, but few reports are available so far on miRNAs encoded by insect viruses, especially the RNA viruses. In this study, a putative miRNA encoded by the 10th segment of BmCPV genomic RNA, BmCPV-miR-10, was identified and functionally analysed. The expression of the putative BmCPV-miR-10 could be detected via stem-loop RT-PCR (reverse transcription-Polymerase Chain Reaction) in the midgut of silkworm larvae infected with BmCPV. BmCSDE1 (B. mori cold shock domain E1 protein) gene was predicted to be a candidate target gene for BmCPV-miR-10 with the miRNA binding site located in 3' untranslated region of its mRNA. The regulation effect of the putative BmCPV-miR-10 on BmCSDE1 was verified in HEK293 cells by lentiviral expression system, in BmN cells by transfecting BmCPV-miR-10 mimics. The qRT-PCR (quantitative real-time PCR) results showed that the putative BmCPV-miR-10 could suppress the expression of BmCSDE1. By injection of BmCPV-miR-10 mimics into the silkworm larvae infected with BmCPV, it was further proved that the putative BmCPV-miR-10 could suppress the expression of BmCSDE1 in vivo, then inhibit the expression of BmApaf-1 (B. mori apoptotic protease activating factor 1), while enhance the replication of BmCPV genomic RNAs to a certain extent. These results implied that the putative BmCPV-miR-10 could down-regulate the expression of BmCSDE1, then suppress the expression of BmApaf-1, thereby created a favourable intracellular environment for virus replication and proliferation.

Effects of fluoranthene on digestive enzymes activity and relative growth rate of larvae of lepidopteran species, Lymantria dispar L. and Euproctis chrysorrhoea L.

Fluoranthene is one of the most abundant polycyclic aromatic hydrocarbon pollutants in the environment and it may accumulate in plant leaves which are the main food source for phytophagous insect species. The aim of this study was to establish the effects of dietary fluoranthene on specific activities of digestive enzymes and expression of their isoforms in the midgut, and the relative growth rates of Lymantria dispar and Euproctis chrysorrhoea larvae. Exposure to fluoranthene led to significantly decreased trypsin activity in the midgut of larvae of both species. Leucine aminopeptidase activity decreased significantly in the midgut of L. dispar larvae exposed to the lower concentration of fluoranthene, but that enzyme activity showed the opposite trend in E. chrysorrhoea larvae. There was no pollutant induced changes in lipase activity in L. dispar, while elevated enzyme activity was recorded in the midgut of E. chrysorrhoea larvae exposed to the lower concentration of fluoranthene. Different patterns of expression of enzyme isoforms were noticed. Relative growth rates of both species significantly decreased in fluoranthene treated larvae. These responses indicate to the significance of relationships between physiological changes and fitness-related traits in L. dispar and E. chrysorrhoea larvae affected by pollutant, and contribute to understanding the mechanisms of their adjustment to stressful conditions.

Effects of inhibitors on the protease profiles and degradation of activated Cry toxins in larval midgut juices of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Midgut juice plays an important role in food digestion and detoxification in insects. In order to understand the potential of midgut juice of Cnaphalocrocis medinalis (Guenée) to degrade Bt proteins, the enzymatic activity of midgut juice and its degradation of Bt proteins (Cry2A, Cry1C, Cry1Aa, and Cry1Ac) were evaluated in this study through protease inhibitor treatments. The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), pepstatin A and leupeptin. The enzymatic activity of chymotrypsin was significantly inhibited by PMSF, and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid (EDTA), PMSF, tosyl phenylalanine chloromethyl ketone (TPCK), TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). EDTA could significantly inhibit the degradation of Cry2A by C. medinalis. EDTA, PMSF, TPCK, and TLCK could inhibit the degradation of Cry1C and Cry1Aa. EDTA, PMSF, TPCK, TLCK, and E-64 could inhibit the degradation of Cry1Ac. Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C. medinalis, which may reduce the insect's ability to degrade Bt toxins. These findings may aid the application of protease inhibitors in the management of this insect pest in the future.

Docking-based generation of antibodies mimicking Cry1A/1B protein binding sites as potential insecticidal agents against diamondback moth (Plutella xylostella).

Broad use of insecticidal Cry proteins from Bacillus thuringiensis in biopesticides and transgenic crops has resulted in cases of practical field resistance, highlighting the need for novel approaches to insect control. Previously we described an anti-Cry1Ab idiotypic-antibody (B12-scFv) displaying toxicity against rice leafroller (Cnaphalocrocis medinalis) larvae, supporting the potential of antibodies for pest control. The goal of the present study was to generate insecticidal antibodies against diamondback moth (Plutella xylostella) larvae. Four genetically engineered antibodies (GEAbs) were designed in silico from B12-scFv using three-dimensional (3D) structure and docking predictions to alkaline phosphatase (ALP) as a Cry1Ac receptor in P. xylostella. Among these GEAbs, the GEAb-dVL antibody consisting of two light chains had overlapping binding sites with Cry1A and Cry1B proteins and displayed high binding affinity to P. xylostella midgut brush border membrane (BBM) proteins. Proteins in BBM identified by pull-down assays as binding to GEAb-dVL included an ABC transporter and V-ATPase subunit A protein. Despite lacking the α-helical structures in Cry1A that are responsible for pore formation, ingestion of GEAb-dVL disrupted the P. xylostella larval midgut epithelium and resulted in toxicity. Apoptotic genes were activated in gut cells upon treatment with GEAb-dVL . This study describes the first insecticidal GEAb targeting P. xylostella by mimicking Cry proteins. Data support that GEAb-dVL toxicity is associated to activation of intracellular cell death pathways, in contrast to pore-formation associated toxicity of Cry proteins. This work provides a foundation for the design of novel insecticidal antibodies for insect control. © 2021 Society of Chemical Industry.

Novel RNA Viruses from the Transcriptome of Pheromone Glands in the Pink Bollworm Moth, Pectinophora gossypiella.

Simple SummaryThe pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae), is a major pest of cotton. In this study, we analyzed the mRNA from pheromone glands of two populations in Israel. We found several virus sequences that were the same in these populations. We identified these viruses based on high-throughput sequencing data and analysis of the assembled transcripts. Through analysis of the sequences, we identified several unique viral sequences representing possible novel viral species. Two of the viral sequences were found in relatively high abundance in pheromone glands. One of the virus sequences was also found through analysis of previous transcriptome sequencing data from the midgut of pink bollworm larvae. This is the first report of these unique viral sequences found in the pink bollworm, and these viruses could be developed to help control this pest around the world, but more research is needed to determine their utility as biological control agents.In this study, we analyzed the transcriptome obtained from the pheromone gland isolated from two Israeli populations of the pink bollworm Pectinophora gossypiella to identify viral sequences. The lab population and the field samples carried the same viral sequences. We discovered four novel viruses: two positive-sense single-stranded RNA viruses, Pectinophora gossypiella virus 1 (PecgV1, a virus of Iflaviridae) and Pectinophora gossypiella virus 4 (PecgV4, unclassified), and two negative-sense single-stranded RNA viruses, Pectinophora gossypiella virus 2 (PecgV2, a virus of Phasmaviridae) and Pectinophora gossypiella virus 3 (PecgV3, a virus of Phenuiviridae). In addition, sequences derived from two negative-sense single-stranded RNA viruses that belong to Mononegavirales were found in the data. Analysis of previous transcriptome sequencing data derived from the midgut of pink bollworm larvae of a USA population only identified PecgV1, but no other viruses. High viral sequence coverages of PecgV1 and PecgV4 were observed in both field and lab populations. This is the first report of viral sequences discovered from the pink bollworm. Results from this investigation suggest that the pink bollworm harbors multiple viruses. Further investigation of the viral pathogens may help to develop novel pest management strategies for control of the pink bollworm.

Assessment of combining biosynthesized silver nanoparticles using Bacillus thuringiensis and gamma irradiation for controlling Pectinophora gossypiella (saunders) (lepidoptera: Gelechiidae)

Purpose Combining gamma irradiation and nanotechnology has become one of the most promising new approaches for area-wide (AW) pest management in recent years. The laboratory trials were conducted to determine the combining effects of BT-AgNPs and gamma irradiation for controlling P. gossypiella. Radio-sensitivity of male pupae at different doses of gamma radiation and the effectiveness of biosynthesized silver nanoparticles using Bacillus thuringiensis on larval instar were assayed. Additionally, the ultrastructure changes on the alimentary canal of 4th instar larvae were studied to evaluate the impact of the combined approach at a cellular level. Materials and methods Laboratory- rearing technique was used for rearing Pectinophora gossypiella. The irradiation process was achieved at Co60 - Gamma Chamber (4000 A). Alanine dosimeters were used for measuring the average absorbed dose and dose mapping. Preparation of Silver nanoparticles (AgNPs) using Bacillus thuringiensis (Bt) and their characterization has been investigated. The treated 4th instar larvae by gamma irradiation or ∕and BT-AgNPs were dissected under the stereo microscope. The alimentary canal was obtained anatomically and Transmission Electron Microscope) was used in examining the stained sections. Results Based on the nonhatching eggs produced by irradiated males’ pupae, the values of effective doses were calculated. The effective doses ranged from 16 to 291 Gy for the ED25 - ED75. The sterility index reached 74.1% when irradiated with males by 291 Gy crossed with nonirradiated females and the adult emergence decreased to be 35.3%. The insecticidal potential of Bt-AgNPs on the 2nd and 4th larval instars was dose-dependent and its LC50 toxicity value was 0. 3 and 0. 4 mg/ml, respectively. The lethal concentration LC50 of the 2nd instar larvae increased the larval and pupal mortality to 55% and 44.4%, respectively, and reduced the adult emergence to be 55.6%. The combining effects of Bt-AgNPs with 291 Gy induced 100% pupae mortality and there was no adult emergence in F1 generation. Such effects also severed the ultrastructure deformity of the midgut of the 4th instar larvae after the two-day post-treatment. Conclusions The combining effects are recommended as an effective IPM program to control P. gossypiella by releasing sterile males (derived from pupae irradiated with 291 Gy) crossing with the normal females in the field, and reducing the fertility of the population to 31.2%. Subsequently, the resulted larvae treated with LC50 of Bt-AgNPs prevented the adult emergence and stopped the life cycle of P. gossypiella.

Identification and Characterization of Genes for the Allo A Lectins in Japanese Rhinoceros Beetle (Trypoxylus dichotomus [Allomyrina dichotoma]).

The Japanese rhinoceros beetle (Trypoxylus dichotomus [Allomyrina dichotoma]) produces the lectins allo A-I and allo A-II, which have strong N-acetyllactosamine (Galβ1-4GlcNAc)-binding activity. It has been suggested that the two lectins are formed from three subunits (α, β, and γ), with allo A-I comprising α and γ subunits and allo A-II comprising β and γ subunits. Here, we determined the cDNA sequences of these subunits using both conventional polymerase chain reaction (PCR)-cloning-sequencing and transcriptome-sequencing analyses. For the α and β subunits, one gene (locus) for each was predicted, whereas for the γ subunit, two types of cDNA sequences were obtained, which we named γ1 and γ2. These two types probably have distinct loci. Average nucleotide sequence identities among the subunits ranged from 87.6% (between α and γ1) to 92.6% (between γ1 and γ2), suggesting that they form a gene family. Although no homology was found between the sequences of allo A and other known lectin proteins in a protein database search, some unknown proteins containing the DUF3421 domain were identified. Those DUF3421 domain-encoding proteins are upregulated in the insect larval midgut. Thus, we infer that allo A genes also play an important role in larvae and that their lectin activity may have been obtained collaterally.

Histological, histochemical and energy disorders caused by R-limonene on Aedes aegypti L. larvae (Diptera: Culicidae)

The study evaluated the effects of R-limonene, in sublethal concentration, on the histology, histochemistry, biochemistry, and carbohydrates and proteins levels in the third instar Aedes aegypti larvae. The R-limonene (LC50 of 27 ppm) and control groups were analyzed 12 and 24 h after the beginning of treatments. The midgut of the control larvae was composed of cylindrical and elongated cells with a spherical and central nucleus and regenerative cells with a pyramidal shape. After 12 h of treatment, columnar cells, protuberances, and cytoplasmic vacuolization were found. However, 24 h after treatment, complete disorganization of the epithelium was observed. There was a positive reaction in all treatments for the presence of glycogen. However, the midgut of larvae treated with R-limonene showed higher levels. For the total protein, positive marking occurred in all groups evaluated, with higher levels in treatments and the lowest in control 12 h. The levels of total protein and glycogen increased in the treated larvae compared to the 12 h control. Besides, a reduction in total sugar levels was observed in the treated larvae compared to controls 12 and 24 h, being more evident in the last one. Therefore, these results demonstrate that R-Limonene caused pathological changes in the epithelium of the A. aegypti midgut at histophysiological and biochemical levels.

Growth inhibition of Spodoptera frugiperda larvae by camptothecin correlates with alteration of the structures and gene expression profiles of the midgut

BackgroundSpodoptera frugiperda is a serious pest that causes devastating losses to many major crops, including corn, rice, sugarcane, and peanut. Camptothecin (CPT) is a bioactive secondary metabolite of the woody plant Camptotheca acuminata, which has shown high toxicity to various pests. However, the effect of CPT against S. frugiperda remains unknown.ResultsIn this study, bioassays have been conducted on the growth inhibition of CPT on S. frugiperda larvae. Histological and cytological changes were examined in the midgut of larvae fed on an artificial diet supplemented with 1.0 and 5.0 µg/g CPT. The potential molecular mechanism was explored by comparative transcriptomic analyses among midgut samples obtained from larvae under different treatments. A total of 915 and 3560 differentially expressed genes (DEGs) were identified from samples treated with 1.0 and 5.0 µg/g CPT, respectively. Among the identified genes were those encoding detoxification-related proteins and components of peritrophic membrane such as mucins and cuticle proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that part of DEGs were involved in DNA replication, digestion, immunity, endocrine system, and metabolism.ConclusionsOur results provide useful information on the molecular basis for the impact of CPT on S. frugiperda and for future studies on potential practical application.

Effects of Plectranthus barbatus leaf extract on survival, digestive proteases, midgut morphophysiology and gut microbiota homeostasis of Aedes aegypti larvae

Aedes aegypti can transmit dengue, chikungunya, zika and yellow fever. Its control represents the main strategy for reducing the spreading of these diseases. In the present work, Plectranthus barbatus leaf saline extract (SE), decoction and infusion were studied for their effects on the survival of A. aegypti third instar larvae (L3), as well as for the presence of secondary metabolites, lectins, and trypsin inhibitors. The effects of SE (non-heated or heated) on digestive proteases, permeability of peritrophic membrane, morphophysiology, microbiota growth and melanogenesis of L3 are reported. SE killed L3 (LC50 of 0.48%, w/v), while decoction and infusion were not larvicides. HPLC analysis identified caffeic acid and flavonoids in SE, decoction, and infusion. SE also contained a ribose/galactose-binding lectin, trypsin inhibitor activities and polypeptides (34, 63 and 66 kDa) resistant to hydrolysis by L3 gut proteases. The incubation of SE with larval proteases did not alter lectin and trypsin inhibitor activities. Treatment of L3 with SE (at LC50) increased the permeability of peritrophic membrane and prevented the larvae from passing into the fourth instar. The morphology of larval midgut was not affected by SE, but the content of neutral polysaccharides in epithelial cells was reduced. SE stimulated in vitro the proliferation of L3 midgut microbiota and increased in vivo the production of melanin by larvae. The heating (100°C, 5 h) of SE increased lectin, trypsin inhibitor and larvicidal activities. In conclusion, SE larvicidal activity may be linked to the presence of lectin and trypsin inhibitor and involves the inhibition of digestive proteases, damage to larval midgut morphophysiology and induction of dysbiosis.

Physical and Chemical Barriers in the Larval Midgut Confer Developmental Resistance to Virus Infection in Drosophila.

Insects can become lethally infected by the oral intake of a number of insect-specific viruses. Virus infection commonly occurs in larvae, given their active feeding behaviour; however, older larvae often become resistant to oral viral infections. To investigate mechanisms that contribute to resistance throughout the larval development, we orally challenged Drosophila larvae at different stages of their development with Drosophila C virus (DCV, Dicistroviridae). Here, we showed that DCV-induced mortality is highest when infection initiates early in larval development and decreases the later in development the infection occurs. We then evaluated the peritrophic matrix as an antiviral barrier within the gut using a Crystallin-deficient fly line (Crys−/−), whose PM is weakened and becomes more permeable to DCV-sized particles as the larva ages. This phenotype correlated with increasing mortality the later in development oral challenge occurred. Lastly, we tested in vitro the infectivity of DCV after incubation at pH conditions that may occur in the midgut. DCV virions were stable in a pH range between 3.0 and 10.5, but their infectivity decreased at least 100-fold below (1.0) and above (12.0) this range. We did not observe such acidic conditions in recently hatched larvae. We hypothesise that, in Drosophila larvae, the PM is essential for containing ingested virions separated from the gut epithelium, while highly acidic conditions inactivate the majority of the virions as they transit.

Larvae of longhorned beetles (Coleoptera; Cerambycidae) have evolved a diverse and phylogenetically conserved array of plant cell wall degrading enzymes

AbstractLonghorned beetles (Cerambycidae) are the most diverse group of predominantly wood‐feeding (xylophagous) insects on Earth. Larvae of most species feed within tissues of plants made up of large amounts of plant cell wall (PCW), which is notoriously difficult to digest. To efficiently access nutrients from their food source, cerambycid larvae have to deconstruct PCW polysaccharides – such as cellulose, hemicelluloses and pectin – requiring them to possess a diversity of plant cell wall degrading enzymes (PCWDEs) in their digestive tract. Genomic data for Cerambycidae are mostly limited to notorious forest pests and are lacking for most of the taxonomic groups. Consequently, our understanding of the distribution and evolution of cerambycid PCWDEs is quite limited. We addressed the numbers, kinds and evolution of cerambycid PCWDEs by surveying larval midgut transcriptomes from 23 species representing six of the eight recognized subfamilies of Cerambycidae and each with very diverse host types (i.e., gymnosperms, angiosperms, xylem, phloem, fresh or dead plant tissues). Using these data, we identified 340 new putative PCWDEs belonging to ten carbohydrate active enzyme families, including two gene families (GH7 and GH53) not previously reported from insects. The remarkably wide range of PCWDEs expressed by Cerambycidae should allow them to break down most PCW polysaccharides. Moreover, the observed distribution of PCWDEs encoded in cerambycid genomes agreed more with phylogenetic relationship of the species studied than with the taxonomic origin or quality of the host plant tissues.

Cp1/cathepsin L is required for autolysosomal clearance in Drosophila

ABSTRACT Macroautophagy/autophagy is a highly conserved lysosomal degradative pathway important for maintaining cellular homeostasis. Much of our current knowledge of autophagy is focused on the initiation steps in this process. Recently, an understanding of later steps, particularly lysosomal fusion leading to autolysosome formation and the subsequent role of lysosomal enzymes in degradation and recycling, is becoming evident. Autophagy can function in both cell survival and cell death, however, the mechanisms that distinguish adaptive/survival autophagy from autophagy-dependent cell death remain to be established. Here, using proteomic analysis of Drosophila larval midguts during degradation, we identify a group of proteins with peptidase activity, suggesting a role in autophagy-dependent cell death. We show that Cp1/cathepsin L-deficient larval midgut cells accumulate aberrant autophagic vesicles due to a block in autophagic flux, yet later stages of midgut degradation are not compromised. The accumulation of large aberrant autolysosomes in the absence of Cp1 appears to be the consequence of decreased degradative capacity as they contain undigested cytoplasmic material, rather than a defect in autophagosome-lysosome fusion. Finally, we find that other cathepsins may also contribute to proper autolysosomal degradation in Drosophila larval midgut cells. Our findings provide evidence that cathepsins play an essential role in the autolysosome to maintain basal autophagy flux by balancing autophagosome production and turnover. Abbreviations: 26-29-p: 26-29kD-proteinase; ADCD: autophagy-dependent cell death; Atg8a: Autophagy-related protein 8a; Cp1/cathepsin L: Cysteine proteinase-1; CtsB: Cathepsin B; cathD: cathepsin D; CtsF: Cathepsin F; GFP: green fluorescent protein; LAMP1: lysosomal-associated membrane protein 1; Mitf: microphthalmia associated transcription factor; PCA: principal component analysis; RNAi: RNA interference; RPF: relative to puparium formation

Zein based-nanoparticles loaded botanical pesticides in pest control: An enzyme stimuli-responsive approach aiming sustainable agriculture

Nanoencapsulation of biopesticides is an important strategy to increase the efficiency of these compounds, reducing losses and adverse effects on non-target organisms. This study describes the preparation and characterisation of zein nanoparticles containing the botanical compounds limonene and carvacrol, responsive to proteolytic enzymes present in the insects guts. The spherical nanoparticles, prepared by the anti-solvent precipitation method, presented in the nanoparticle tracking analysis (NTA) a concentration of 4.7 × 1012 ± 1.3 × 1011 particles.mL−1 and an average size of 125 ± 2 nm. The formulations showed stability over time, in addition to not being phytotoxic to Phaseolus vulgaris plants. In vivo tests demonstrated that formulations of zein nanoparticles containing botanical compounds showed higher mortality to Spodoptera frugiperda larvae. In addition, the FTIC probe (fluorescein isothiocyanate) showed wide distribution in the larvae midgut, as well as being identified in the feces. The trypsin enzyme, as well as the enzymatic extract from insects midgut, was effective in the degradation of nanoparticles containing the mixture of botanical compounds, significantly reducing the concentration of nanoparticles and the changes in size distribution. The zein degradation was confirmed by the disappearance of the protein band in the electrophoresis gel, by the formation of the lower molecular weight fragments and also by the greater release of FTIC after enzymes incubation. In this context, the synthesis of responsive nanoparticles has great potential for application in pest management, increasing the selectivity and specificity of the system and contributing to a more sustainable agriculture.

Ovicidal, Larvicidal and Biochemical Effects of Thyme, Thymus vulgaris, ON the Cotton Leafworm, Spodoptera littoralis (Boisd.)

Ovicidal,larvicidal and biochemical effects of Thyme oil,Thymus vulgaris,and the carbamate insecticide,Methomyl,on the cotton leafworm, Spodoptera littoralis (boisd.) (Lepidoptera: Noctuidae)were studied.The calculated percentages of reduction in egg hatching were 32.5, and 57.1% for thyme oil tested at concentrations of 2 and 4% and 21.3, and 28.2% for Methomyl tested at concentrations of 2 and 4ppm, respectively.The calculated percentages of reduction were 100% for thyme oil and Methomyl tested at concentrations of 12% and 20ppm, respectively.Larval feeding assay showed that the calculated LC50 values for thyme oil were 0.701, 0.324 and 0.108% after 24, 48 and 72hrs, respectively.The calculated LC50 values for methomyl were 1.60, 0.918 and 0.51ppm after 24, 48 and 72hrs,respectively.Topical assay showed that the calculated LC50 values were 3.601, 3.601, and 2.089% after 24, 48, and 72hrs, respectively.Among methomyl, the calculated LC50 values were 2.12, 1.28 and 0.75ppm after 24, 48 and 72hrs, respectively. The in vitro inhibitory activity of Thyme oil and Methomyl on acetylcholinesterase (AChE) were also tested in larval head and midgut of S. littoralis in this study.Concentration of 6% of Thyme oil caused 68.6 and 68.7% inhibition of AChE in heads and midgut of the 4th larval instar of S. littoralis, respectively. While concentration of 20 ppm of methomyl caused 81.8 and 80.4% inhibition of AChE in heads and midgut of the 4th larval instar of S. littoralis, respectively. I50 values for larval midgut AChE was about 1.3 and 1.52 folds more resistant to inhibition by thyme oil and methomyl,respectively,than those of larval head AChE.

Morphology of immature stages of Aphelinus maculatus Yasnosh (Hymenoptera: Aphelinidae) related to host aphid characteristics and larval taxonomic significance

The aphid parasitoid, Aphelinus maculatus Yasnosh (Hymenoptera: Aphelinidae), first described as a new record in China in 2016, is one of the most important natural enemies of the Chinese wolf-berry aphids. In order to relate its larval development stages to morphological changes of both the parasitoid and its host aphid during the parasitoid development, and to explore the larval taxonomic significance, a laboratory dissection study was conducted under the light microscope. There are three larval instars during immature development of A. maculatus. Instars differ in body shape, mandibles and numbers of respiratory spiracles. The first instar larva of A. maculatus is elongate in shape, the second instar larva is thick spindle-shaped to spindle-shaped, and the third instar larva is spherical-shaped with a small tail. Mandibles of the first and second instar larvae of A. maculatus are almost invisible, and their pharynxes are well developed. Mandibles of the third instar larva are well developed and are made of a pair of dark yellow sharp pointed spines each projecting from a transparent broad triangular base. The third instar larva possesses seven pairs of respiratory spiracles with one pair on the mesothorax and other six pairs successively on the second to seventh abdominal segments. As the larval development proceeds, a brown patch (the midgut of parasitoid larva) in the center of host aphid abdomen becomes larger in size and darker in color. The host aphid turns entirely black as a mummy and dies at the prepupal stage of the parasitoid, pine-nut shaped meconium is excreted at this time. The life cycle of A. maculatus from eggs to adults lasts 11.5–14 days at the temperature of 26 ± 1 °C. Morphological features of mandibles, respiratory spiracles and meconium are useful in the larval taxonomy of aphelinids.

Antimicrobial Resistance Genes in Bacteria Isolated From Japanese Honey, and Their Potential for Conferring Macrolide and Lincosamide Resistance in the American Foulbrood Pathogen Paenibacillus larvae.

American foulbrood (AFB) is the most serious bacterial disease of honey bee brood. Spores of the causative agent Paenibacillus larvae are ingested by bee larvae via brood foods and germinated cells proliferate in the larval midgut. In Japan, a macrolide antibiotic, tylosin, is used as the approved prophylactic for AFB. Although tylosin-resistant P. larvae has yet to be found in Japan, it may emerge in the future through the acquisition of macrolide resistance genes from other bacteria, and bacteria latent in brood foods, such as honey, may serve as a source of resistance genes. In this study, to investigate macrolide resistance genes in honey, we attempted to isolate tylosin-resistant bacteria from 53 Japanese honey samples and obtained 209 isolates from 48 samples in the presence of 1 μg/ml of tylosin. All isolates were Gram-positive spore-forming bacteria mainly belonging to genera Bacillus and Paenibacillus, and 94.3% exhibited lower susceptibility to tylosin than Japanese P. larvae isolates. Genome analysis of 50 representative isolates revealed the presence of putative macrolide resistance genes in the isolates, and some of them were located on mobile genetic elements (MGEs). Among the genes on MGEs, ermC on the putative mobilizable plasmid pJ18TS1mac of Oceanobacillus strain J18TS1 conferred tylosin and lincomycin resistance to P. larvae after introducing the cloned gene using the expression vector. Moreover, pJ18TS1mac was retained in the P. larvae population for a long period even under non-selective conditions. This suggests that bacteria in honey is a source of genes for conferring tylosin resistance to P. larvae; therefore, monitoring of bacteria in honey may be helpful to predict the emergence of tylosin-resistant P. larvae and prevent the selection of resistant strains.

Peritrophic matrix-degrading proteins are dispensable virulence factors in a virulent Melissococcus plutonius strain

European foulbrood (EFB) caused by Melissococcus plutonius is a major bacterial disease of honey bees. Strains of the causative agent exhibit genetic heterogeneity, and the degree of virulence varies among strains. In bee larvae orally infected with the highly virulent strains, ingested bacterial cells colonize the larval midgut and proliferate within the sac of the peritrophic matrix (PM), a barrier lining the midgut epithelium. However, the barrier is degraded during the course of infection, and M. plutonius cells eventually directly interact with the midgut epithelium. As M. plutonius possesses genes encoding putative PM-degrading proteins (enhancin, a chitin-binding domain-containing protein and endo-α-N-acetylgalactosaminidase), we constructed PM-degrading protein gene-knockout mutants from a highly virulent M. plutonius strain and investigated their role in the pathogenesis of EFB. In larvae infected with the triple-knockout mutant, which has no PM-degrading protein genes, M. plutonius that proliferated in the larval midguts was confined to the sac of the PM. However, the midgut epithelial cells degenerated over time, and the mutant killed approximately 70–80% of bee brood, suggesting that although the PM-degrading proteins are involved in the penetration of the PM by M. plutonius, they are not indispensable virulence factors in the highly virulent M. plutonius strain.

Estrogen-Related Receptor Influences the Hemolymph Glucose Content by Regulating Midgut Trehalase Gene Expression in the Last Instar Larvae of Bombyx mori.

The expression of trehalase in the midgut of insects plays an important role in glucose supply to the hemolymph. Energy metabolism is usually regulated by the estrogen-related receptor (ERR). A decrease in ATP levels is caused by the ERR hindering glycolysis. However, the relationship between trehalose accumulation and ERR expression is still unclear. Here, we found that silkworm ERR (BmERR) is concentrated and BmERR expression is strongly correlated with trehalase in the midgut during the last instar silkworm larval stage. We cloned the promoter of the trehalase from Bombyx mori (BmTreh) and found that the ERR bound directly to the core response elements of the promoter. Cell level interference and the overexpression of ERR can reduce or enhance BmTreh transcription and promoter activity. Overexpressed transgenic BmERR can significantly increase the expression of BmTreh in the midgut of the last instar silkworm larvae, thereby hydrolyzing trehalose into glucose and releasing it into the hemolymph. Additionally, increased hemolymph glucose content reduces silkworm pupa weight but does not affect silk protein production from the silk gland. Our results suggest a novel function for BmERR through its involvement in BmTreh regulation and expand the understanding of ERR functions in insect trehalose metabolism.

Biological and Histological Alterations in The Larvae of Culex pipiens L. (Diptera: Culicidae) Induced by Imidcaloproid and Tannic Acid

The mosquito family Culicidae is the most common disease vector in the Diptera order. Almost all of them are bloodsuckers that cause a lot of major diseases, such as malaria, yellow fever and elephantiasis. The development of resistance in mosquitos to pyrethroid, organophosphate, and carbamate created the need for alternative insecticides. Therefore, 3rd instar Culex pipiens larvae were treated with the median lethal concentrations (LC50) of imidacloprid (IMI) and tannic acid. Biological tests were performed by following up the treated insects from the 3rd larval instar till the adult stages. The biological test showed that tannic acid was more effective than imidacloprid by increasing the larval period and decreasing both pupation percentage and pupal duration. The histopathological effect of the tested compound on the larvae's midgut epithelium was studied using light and transmission electron microscopy. A histological study showed that the treated larvae had cytopathological alterations of the midgut epithelium, muscular layers, epithelial cells and internal organelles. Both IMI and tannic acid showed nearly the same effect on the larvae histology. Based on these results, tested compounds could be used in the Integrated Pest Management (IPM) programs for greater effectiveness.