Multiple Insect Research Articles

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along a range of insecticide concentrations. This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies.

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact withthe insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects alonga range of insecticide concentrations. This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

Weight Gain of Spodoptera frugiperda Larvae (Lepidoptera: Noctuidae) on Leaf and Floral Tissues of Silphium integrifolium (Asterales: Asteraceae) Differs by Plant Genotype.

Silflower (Silphium integrifolium (Michaux)) is a native North American relative of sunflower that is undergoing domestication as a perennial oilseed crop. As silflower incurs pest damage from multiple insect species, it is necessary to screen genotypes for their effect on insect performance such that more pest tolerant/resistant accessions can be incorporated into future silflower breeding programs. We present a bioassay protocol for silflower using the generalist herbivore fall armyworm (Spodoptera frugiperda (J. E. Smith)). In this study, fall armyworm larvae were placed on leaf and flower tissue from eleven silflower genotypes, one cup plant (Silphium perfoliatum (L.) (Asterales: Asteraceae)) genotype, and an inbred sunflower line (Helianthus annuus (L.) (Asterales: Asteraceae), HA89). Caterpillar weight gained during a 4-d feeding period significantly differed on leaf and floral tissue from different silflower genotypes, between the Silphium species (silflower and cup plant), and between Silphium genotypes and annual sunflower. Two wild silflower genotypes produced lower larval weight gain on both the floral and leaf tissue than all other genotypes, suggesting these genotypes have either lower nutrition or greater resistance to fall armyworm. However, nonsignificant correlations between larval growth on floral versus leaf tissue across all plant species tested and among all silflower accessions suggest insect performances on these tissue types in silflower are independent. Along with identifying germplasm of interest for silflower breeding programs, we established an easily replicable bioassay protocol using fall armyworm on silflower floral and leaf tissues.

Parallel host‐plant‐associated differences in an extended phenotype between populations of six species of gall‐forming insects

Abstract Understanding the role of divergent host‐use in promoting population differentiation among herbivorous insects provides valuable insight into the origin of diversity in this species‐rich group. A strong signature of divergent selection in promoting population differentiation is provided by parallel phenotypic divergence across multiple insect species adapting to the same set of divergent host plants. In this study, we explored whether a key phenotype of gall‐forming insects—gall size—exhibits parallel patterns of divergence across a community of gall‐forming insect species because of adaptation to different host plant species. A previous study found parallel divergence in gall morphology across two gall‐forming insect species adapted to two sister species of oaks (Fagaceae). Here, we expand this question to four additional members of the gall‐forming insect community that specialise on the same two host plant species: Quercus geminata (Qg) and Quercus virginiana (Qv). Consistent with the previous result, all four additional gall‐forming species tended to induce larger galls on Qg than on Qv, with two of the four species exhibited statistically significant differences and one species showed marginally significant differences. Moreover, we found gall size was similar for two gall‐forming species when cross‐reared on natural versus planted hosts, ruling out environmental causes of parallel host‐associated gall size difference across multiple species. Our study documents parallel divergence in a key phenotype of gall‐forming insects associated with different host environments, shedding light on the important role of divergent host use in driving the evolution of gall morphology.

Characterization of Long Non-Coding RNAs in the Bollworm, Helicoverpa zea, and Their Possible Role in Cry1Ac-Resistance.

Simple SummaryMultiple caterpillar pest species have become resistant to transgenic crops. These crops were originally engineered to make a bacteria protein that would kill the caterpillar when the insect eats the plant. This project focused on comparing gene expression patterns in a bollworm caterpillar resistant strain compared to a susceptible bollworm strain. Expression differences were found in long non-coding RNAs, sequences that do not make proteins but can regulate making proteins. There were increased and decreased levels of different long non-coding RNAs in the resistant strain. Proximity relationships of these non-coding RNAs to protein coding-genes that have functions known to cause resistance were also found. Proximity is one way long non-coding RNA regulates the making of proteins and could be a mechanism of how these insects became resistant. The potential of using these discoveries in managing insect pest resistance levels in the field is discussed.Multiple insect pest species have developed field resistance to Bt-transgenic crops. There has been a significant amount of research on protein-coding genes that contribute to resistance, such as the up-regulation of protease activity or altered receptors. However, our understanding of the role of non-protein-coding mechanisms in Bt-resistance is minimal, as is also the case for resistance to chemical pesticides. To address this problem relative to Bt, RNA-seq was used to examine statistically significant, differential gene expression between a Cry1Ac-resistant (~100-fold resistant) and Cry1Ac-susceptible strain of Helicoverpa zea, a prevalent caterpillar pest in the USA. Significant differential expression of putative long non-coding RNAs (lncRNAs) was found in the Cry1Ac-resistant strain (58 up- and 24 down-regulated gene transcripts with an additional 10 found only in resistant and four only in susceptible caterpillars). These lncRNAs were examined as potential pseudogenes and for their genomic proximity to coding genes, both of which can be indicative of regulatory relationships between a lncRNA and coding gene expression. A possible pseudogenic lncRNA was found with similarities to a cadherin. In addition, putative lncRNAs were found significantly proximal to a serine protease, ABC transporter, and CYP coding genes, potentially involved in the mechanism of Bt and/or chemical insecticide resistance. Characterization of non-coding genetic mechanisms in Helicoverpa zea will improve the understanding of the genomic evolution of insect resistance, improve the identification of specific regulators of coding genes in general (some of which could be important in resistance), and is the first step for potentially targeting these regulators for pest control and resistance management (using molecular approaches, such as RNAi and others).

Woodland, cropland and hedgerows promote pollinator abundance in intensive grassland landscapes, with saturating benefits of flower cover

Abstract Pollinating insects provide economic value by improving crop yield. They are also functionally and culturally important across ecosystems outside of cropland. To understand landscape‐level drivers of pollinator declines, and guide policy and intervention to reverse declines, studies must cover (a) multiple insect and plant taxa and (b) a range of agricultural and semi‐natural land uses. Furthermore, in an era of woodland restoration initiatives and rewilding ideologies, the contribution of woodland and woody linear features (WLFs; e.g. hedgerows) to pollinator abundance demands further investigation. We demonstrate fine‐scale analysis of high‐quality, co‐located measurements from a national environmental survey. We relate pollinator transect counts to ground‐truth habitat and WLF maps across 300 1 km squares in Wales, UK. We look at effects of habitat type, flower cover, WLF density and habitat diversity on summer abundance (July and August) of eight insect groups, representing three insect orders (Lepidoptera, Hymenoptera and Diptera). Compared with improved grassland (the dominant habitat in Wales), pollinator abundance is consistently higher in cropland and woodland—especially broadleaved woodland. For mining bees and two hoverfly groups, abundance is predicted to be at least 1.5× higher in woodland ecosystems than elsewhere. Furthermore, we estimate contributions of WLFs to abundance in agriculturally improved habitats to be up to 14% for honeybees and up to 21% for hoverflies. The abundance of all insect groups increases with flower cover, which is a key mechanism through which woodland, cropland and grassland support pollinators. Importantly, we observe diminishing returns of increasing flower cover for abundance of non‐Apis pollinator groups, expecting roughly twice the increase in abundance per % flower cover from 0% to 5%, as compared with 10% to 15%. However, the shape of the relationship was inverted for honeybees, which showed steeper increases in abundance at higher flower cover. Synthesis and applications. We provide a holistic view of the drivers of pollinator abundance in Wales, in which flower cover, woodland, hedgerows and cropland are critical. We propose a key role for woodland creation, hedge‐laying and farmland heterogeneity within future land management incentive schemes. Finally, we suggest targeting of interventions to maximise benefits for non‐Apis pollinators. Specifically, increasing floral provision in areas where existing flower cover is low—for example, in flower‐poor improved grasslands—could effectively increase pollinator abundance and diversity while prioritising wild over managed species.

Three-Dimensional Tracking of Multiple Small Insects by a Single Camera.

Many systems to monitor insect behavior have been developed recently. Yet most of these can only detect two-dimensional behavior for convenient analysis and exclude other activities, such as jumping or flying. Therefore, the development of a three-dimensional (3D) monitoring system is necessary to investigate the 3D behavior of insects. In such a system, multiple-camera setups are often used to accomplish this purpose. Here, a system with a single camera for tracking small insects in a 3D space is proposed, eliminating the synchronization problems that typically occur when multiple cameras are instead used. With this setup, two other images are obtained via mirrors fixed at other viewing angles. Using the proposed algorithms, the tracking accuracy of five individual drain flies, Clogmia albipunctata (Williston) (Diptera: Psychodidae), flitting about in a spherical arena (78 mm in diameter) is as high as 98.7%, whereas the accuracy of 10 individuals is 96.3%. With this proposed method, the 3D trajectory monitoring experiments of insects can be performed more efficiently.

Effect of Polyphenolic Compounds of Rosa Canina (L.) Against The Acetylcholinesterase Activity of Rhopalosiphum Padi (L.) (Homoptera: Aphididae)

Rhopalosiphum padi (L.) is among the most aggressive cereal pests worldwide. The pest causes economically heavy crop loss. Chemical insecticides were used for the control of multiple insects. However, the harmful consequences of these chemical products are well known. Seeking more ecological alternatives, respect to the environment is, therefore, a necessity. The phytochemicals are known for their various biological activities of pharmaceutical and agri-food interest. Several plant species have been found to have effective bioinsecticide properties against a variety of insects. In this study, the plant Rosa canina (L.) has made the objective of an in-silico research using molecular docking by screening the inhibitory potential of its polyphenolic compounds against the enzyme acetylcholinesterase of R. padi (L.). The enzyme 3D structure was first modeled, then its stereochemical quality was validated. The result of molecular docking allowed the selection of seven phytocompounds (Ellagic acid, Dihydroquercetin, Bilobalide A, Luteolin 5-methyl ether, Rosmarinic acid, Kaempferol, and Quercetin) with binding energy lower than that of the commercial insecticide Malathion. These components showed intense links with the catalytic site key residues of the enzyme, indicating their high inhibitor potential. The environmental and health safety of these components and their bio-availability were also validated by the verification of several pharmacokinetic and ADMET criteria, suggesting the interest of the plant R. canina (L.) as promising bioinsecticide material against the pest R. padi (L.).

Ecological requirements drive the variable responses of wheat pests and natural enemies to the landscape context

Abstract Semi‐natural habitats (SNH) are considered essential for pest‐suppressive landscapes, but their influence on crop pests and natural enemies can be highly variable. Instead of SNH per se, the availability of resources, such as pollen and nectar, may be more relevant for supporting pest control. Here, we assessed the spatiotemporal variation of multiple insect pests (cereal leaf beetles and aphids) and natural enemies (predators and aphid parasitoids) in wheat fields and their responses to landscape context and flower availability. We combined detailed information on pollen use by natural enemies with the specific distribution of pollen‐providing plants across a gradient of landscape composition and configuration. The abundance of wheat pests was tightly linked to wheat development stage. Syrphids colonised the fields early in the season, while the abundance of other enemies increased later in the season. The responses of pests to landscape structure were variable and, while some pests had low abundances in landscapes with high edge density and SNH cover, Sitobion avenae abundance was positively associated with SNH cover. Lacewings, syrphids and cereal leaf beetles were abundant in landscapes with diverse and abundant flower resources, whereas the abundance of parasitoids and Nabis sp. was driven by aphid abundance. We detected no significant indirect effects of landscape on pests via natural enemies. Synthesis and applications. Our findings highlight the need for conservation biological control to go beyond ‘one size fits all’ and consider the specific ecology of the involved organisms, even for a single crop type. Landscapes with high edge density and flowering woody plants may support natural enemies, in particular syrphids, which colonised the fields early in the season. Incentives for pest‐suppressive landscapes should focus on tailored strategies that disfavour dominant cereal pests and simultaneously enhance natural enemies according to their ecological requirements.

Plasticity in induced resistance to sequential attack by multiple herbivores in Brassica nigra

In nature, plants interact with multiple insect herbivores that may arrive simultaneously or sequentially. There is extensive knowledge on how plants defend themselves against single or dual attack. However, we lack information on how plants defend against the attack of multiple herbivores that arrive sequentially. In this study, we investigated whether Brassica nigra L. plants are able to defend themselves against caterpillars of the late-arriving herbivore Plutella xylostella L., when plants had been previously exposed to sequential attack by four other herbivores (P. xylostella, Athalia rosae, Myzus persicae and Brevicoryne brassicae). We manipulated the order of arrival and the history of attack by four herbivores to investigate which patterns in sequential herbivory determine resistance against the fifth attacker. We recorded that history of sequential herbivore attack differentially affected the capability of B. nigra plants to defend themselves against caterpillars of P. xylostella. Caterpillars gained less weight on plants attacked by a sequence of four episodes of attack by P. xylostella compared to performance on plants that were not previously damaged by herbivores. The number of times the plant was attacked by herbivores of the same feeding guild, the identity of the first attacker, the identity and the guild of the last attacker as well as the order of attackers within the sequence of multiple herbivores influenced the growth of the subsequent herbivory. In conclusion, this study shows that history of sequential attack is an important factor determining plant resistance to herbivores.

A modular environmental and economic assessment applied to the production of Hermetia illucens larvae as a protein source for food and feed

PurposeThe inclusion of insect protein into the food system has been proposed as a promising solution to ensure future food security and mitigate negative environmental impacts related to food production. However, the market volume for edible insects in Europe is still small; consequently, producers need a decision-support system to ensure the sustainable upscaling of the sector. The study analyzed environmental and economic impacts of insect production to identify the most eco-efficient production scenarios.MethodsA novel modular eco-efficiency assessment approach was developed to analyze the production of dried Hermetia illucens larvae. An exemplary, industrial-scale insect production system was disaggregated into a total of 29 module variants that can be combined into 4608 distinct production scenarios, which are characterized by different feeds, energy efficiencies, and processing technologies. Environmental life cycle and cost assessments were carried out for each module variant, and eco-efficiency assessment was used to jointly assess these two sustainability dimensions. Additionally, the influence of the insect feed on the production system performance and impact was investigated by employing feed-specific scaling factors. These were used to aggregate module results into production scenario results.Results and discussionThe most eco-efficient production scenarios include energy-efficient rearing facilities that rely on blanching and microwave drying for processing. The insect feed is the largest contributor to the environmental impacts and costs, but from an eco-efficiency standpoint, the choice of feed might not be crucial. Waste-type feeds (e.g., manure, fruit, and vegetable waste) have low environmental impacts and costs, but the production scenarios based on these feeds are less efficient. The low impacts of the feed are offset by higher impacts during the rearing and processing stages. Conversely, scenarios based on higher quality feeds (e.g., by-products like wheat middlings or distiller’s grains) require less resources, but the initial feed impacts and costs are higher. Moreover, of the feed types studied, only highly processed ones, such as compound chicken feed, should be avoided for insect rearing.ConclusionsThe developed modular assessment approach is efficient in assessing multiple potential insect production scenarios. It can be adapted to incorporate additional variations of the production system via additional modules. Limitations include the potential for redundant module combinations and the up-front time investment needed. Finally, the results are sensitive to methodological choices: thus, these should be carefully considered and communicated during the design of the modular assessment system.

What structures diurnal visitation rates to flowering trees in an Afrotropical lowland rainforest understory?

Abstract As environmental change and degradation accelerate, perturbing insect populations, we need to better understand the resource use dynamics of diverse wild pollinators. Most tropical trees are adapted for biotic pollination, yet we still know little about plant–pollinator interactions in African rainforests. We addressed this gap from a community perspective, identifying what floral traits – colour, scent, reward accessibility and visibility – structure visitation patterns among insect functional groups to tree species flowering in the understory of Korup National Park, Cameroon. To understand how visitor groups share resources, we used joint modelling that explicitly considers zero‐inflation in visitation rates and correlation among visitor groups. Most tree species had exposed floral rewards, and all were visited by multiple insect groups among which ants, bees, beetles, and flies were most abundant. Visitation rates varied more among tree species than among individual trees. Floral scent differences were important for structuring visits, particularly for flies, bees, and ants. Ant and bee visitation rates decreased while fly visits increased marginally throughout the season, correlated with the dry to wet season transition. Comparison with other lowland tropical understories suggests flies may be uniquely diverse and important to this system, and differences in seasonality and forest structure may be drivers of community differences. Floral scent is likely a key functional trait structuring flower–insect interactions in tropical forest environments and should be emphasised in future studies. Lastly, a joint modelling approach can elucidate community structure, particularly in communities with ecologically generalised interactions.

Impact of a biorational pesticide on the pecan aphid complex and its natural enemies

The pecan weevil, Curculio caryae (Horn) (Coleoptera: Curculionidae), and the pecan aphid (Hemiptera: Aphididae) complex: black pecan aphid, Melanocallis caryaefoliae (Davis), blackmargined aphid, Monellia caryella (Fitch), and yellow pecan aphid, Monelliopsis pecanis Bissell are major pests of pecan production. Management is primarily based on chemical insecticides, and alternative control methods need to be explored. Prior field tests showed that the microbial insecticide Grandevo® (based on Chromobacterium subtsugae) can suppress C. caryae at similar rates to insecticides, and a laboratory test showed that Grandevo is toxic to M. caryaefoliae. However, it was unknown if Grandevo applications targeted towards C. caryae also impact the pecan aphid complex and aphid natural enemies. Hence, we initiated a field study to determine the impact of two rates of Grandevo (2.24 and 3.36 kg/ha) on aphid, Coccinellidae (Coleoptera) and Chrysopidae (Neuroptera) populations. Both Grandevo rates, in both years of the study, suppressed M. caryaefoliae in comparison to the negative control. Only the standard chemical insecticide regimen suppressed M. caryella and M. pecanis combined populations during both years of the experiment. In 2016, both Grandevo rates had more abundant natural enemy populations compared with the chemical insecticide regimen, and the higher Grandevo rate (3.36 kg/ha) had more natural enemies than the non-treated control. In 2017, all treatments had comparable populations of natural enemies. These findings suggest that a microbial insecticide can manage multiple insect pests while preserving the natural enemy community.

The First Report on the Transovarial Transmission of Microsporidian Nosema bombycis in Lepidopteran Crop Pests Spodoptera litura and Helicoverpa armigera.

Microsporidia are ubiquitous fungi-related parasites infecting nearly all vertebrates and invertebrates. Microsporidian Nosema bombycis is a natural pathogen of multiple insects, including the silkworm and many agricultural and forest pests. N. bombycis can transovarially transmit in silkworm and cause huge economic losses to the sericulture. However, it remains unclear whether N. bombycis vertically transmits in the crop pests Spodoptera litura and Helicoverpa armigera. Here, we investigated the infection of N. bombycis in S. litura and H. armigera to illuminate its infectivity and transovarial transmission. In result, tissue examination with light microscopy revealed that the fat body, midgut, malpighian tubules, hemolymph, testis, and ovary were all infected in both pest pupae. Immunohistochemical analysis (IHA) of the ovariole showed that a large number of parasites in maturation and proliferation presented in follicle cell, nurse cell, and oocyte, suggesting that N. bombycis can infect and multiply in these cells and probably transovarially transmit to the next generations in both pests. Microscopic examination on the egg infection rate demonstrated that 50% and 38% of the S. litura and H. armigera eggs were congenitally infected, respectively. IHA of both eggs manifested numerous spores and proliferative pathogens in the oocyte, confirming that N. bombycis can invade into the female germ cell from the parent body. After hatching of the infected eggs, we detected the infection in offspring larvae and found large quantities of proliferative pathogens, confirming that N. bombycis can transovarially transmit in S. litura and H. armigera, and probably persists in both pest populations via congenital infection. In summary, our work, for the first time, proved that N. bombycis is able to vertically transmit in S. litura and H. armigera via infecting the oocyte in the parent, suggesting that N. bombycis could be a biological insecticide for controlling the population of crop pests.

Characterizing the Xenoma of Vairimorpha necatrix Provides Insights Into the Most Efficient Mode of Microsporidian Proliferation.

Microsporidia are a group of obligated intracellular parasites that can infect nearly all vertebrates and invertebrates, including humans and economic animals. Microsporidian Vairimorpha necatrix is a natural pathogen of multiple insects and can massively proliferate by making tumor-like xenoma in host tissue. However, little is known about the subcellular structures of this xenoma and the proliferation features of the pathogens inside. Here, we characterized the V. necatrix xenoma produced in muscle cells of silkworm midgut. In result, the whitish xenoma was initially observed on the 12th day post infection on the outer surface of the midgut and later became larger and numerous. The observation by scanning electronic microscopy showed that the xenoma is mostly elliptical and spindle with dense pathogen-containing protrusions and spores on the surface, which were likely shedding off the xenoma through exocytosis and could be an infection source of other tissues. Demonstrated with transmission electron microscopy and fluorescent staining, the xenoma was enveloped by a monolayer membrane, and full of vesicle structures, mitochondria, and endoplasmic reticulum around parasites in development, suggesting that high level of energy and nutrients were produced to support the massive proliferation of the parasites. Multiple hypertrophic nuclei were found in one single xenoma, indicating that the cyst was probably formed by fusion of multiple muscle cells. Observed by fluorescence in situ hybridization, pathogens in the xenoma were in merongony, sporogony, and octosporogony, and mature stages. And mature spores were pushed to the center while vegetative pathogens were in the surface layer of the xenoma. The V. necatrix meront usually contained two to three nuclei, and sporont contained two nuclei and was wrapped by a thick membrane with high electron density. The V. necatrix sporogony produces two types of spores, the ordinary dikaryotic spore and unicellular octospores, the latter of which were smaller in size and packed in a sporophorous vesicle. In summary, V. necatrix xenoma is a specialized cyst likely formed by fusion of multiple muscle cells and provides high concentration of energy and nutrients with increased number of mitochondria and endoplasmic reticulum for the massive proliferation of pathogens inside.

Development and Validation of a Three-Dimensional Printed Multifunctional Trap for Surveillance of Mosquitoes.

An essential component of vector-borne disease monitoring programs is mosquito surveillance. Surveillance efforts employ various collection traps depending on mosquito species and targeted life-history stage, i.e., eggs, larvae, host-seeking, resting, or gravid adults. Surveillance activities often use commercial traps, sometimes modified to accept specific mosquito species attractants. The advent of widely available and affordable 3D printing technology allows the construction of novel trap designs and components. The study goal was to develop and assess a cost-effective, multipurpose, 6-volt mosquito trap integrating features of both host-seeking and gravid mosquito traps to collect undamaged live specimens: a multifunctional mosquito trap (MMT). We tested the MMT in comparison to commercial traps, targeting gravid Aedes albopictus, host-seeking Ae. albopictus, and total number of host-seeking mosquitos regardless of species. Field evaluations found the MMT performed as well as or better than comparable commercial traps. This project demonstrates an easy to construct, inexpensive, and versatile mosquito trap, potentially useful for surveying multiple mosquito species and other hematophagous insects by varying attractants into the MMT.

Molecular and pharmacological characterization of biogenic amine receptors from the diamondback moth, Plutella xylostella.

Insect biogenic amines play important roles in mediating behavioral and physiological processes. They exert their effects by binding to biogenic amine receptors (BARs), which are specific receptor proteins in the G-protein-coupled receptor superfamily. BAR genes have been cloned and characterized from multiple model insects, including Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Apis mellifera and Tribolium castaneum. However, relatively little work has addressed the molecular properties, expression profiles, and pharmacological characterization of BARs from other insects, including important pests. In this study, we cloned 17 genes encoding putative biogenic amine receptor proteins from Plutella xylostella, a global pest of Brassica crops. These PxBAR genes were five octopamine receptors (PxOA1, PxOA2B1, PxOA2B2, PxOA2B3, and PxOA3), three tyramine receptors (PxTAR1A, PxTAR1B, and PxTAR2), four dopamine receptors (PxDOP1, PxDOP2, PxDOP3, and PxDopEcR), and five serotonin receptors (Px5-HT1A , Px5-HT1B , Px5-HT2A , Px5-HT2B , and Px5-HT7 ). All PxBARs showed considerable sequence identity with orthologous BARs, and phylogenetic analysis clustered the receptors within their respective groups while preserving organismal evolutionary relationships. We investigated their molecular properties and expression profiles, and pharmacologically characterized the dopamine receptor, PxDOP2. Our study provides important information and resources on biogenic amine receptors from P. xylostella, which suggests potential target sites for controlling this pest species. © 2021 Society of Chemical Industry.

Proteomic Identification of Bombyx mori Organelles Using the Engineered Ascorbate Peroxidase APEX and Development of Silkworm Organelle Proteome Database (SilkOrganPDB).

Silkworm Bombyx mori is an economically important insect and a lepidopteran model. Organelle proteome is vital to understanding gene functions; however, it remains to be identified in silkworm. Here, using the engineered ascorbate peroxidase APEX, we constructed transgenic B. mori embryo cells (BmE) expressing APEX-NLS, COX4-APEX, APEX-Rev, and APEX-KDEL in nucleus, mitochondrial matrix (MM), cytosol, and endoplasmic reticulum (ER), and isolated the biotin-labeled proteins using streptavidin-affinity purification, respectively. The isolated proteins were determined using LC-MS/MS and annotated by searching B. mori genomes downloaded from GenBank, SilkBase, SilkDB 2.0, and SilkDB 3.0, resulting in 842, 495, 311, and 445 organelle proteins identified, respectively. We mapped the 296 MM proteins annotated in the GenBank data to mitochondrial protein databases of the fly, human, and mouse, and found that 140 (47%) proteins are homologous to 80 fly proteins, and 65 (22%) proteins match to 31 and 29 human and mouse proteins, respectively. Protein orthology was predicted in multiple insects using OrthoMCL, producing 460 families containing 839 proteins we identified. Out of 460 families, 363 were highly conserved and found in all insects, leaving only three proteins without orthology in other insects, indicating that the identified proteins are highly conserved and probably play important roles in insects. A gene ontology enrichment analysis by clusterProfiler revealed that the nucleus proteins significantly enriched in cellular component terms of nucleus and nucleolus, the MM proteins markedly enriched in molecular function terms of nucleotide binding, and the cytosol proteins mainly enriched in biological process terms of small molecule metabolism. To facilitate the usage and analysis of our data, we developed an open-access database, Silkworm Organelle Proteome Database (SilkOrganPDB), which provides multiple modules for searching, browsing, downloading, and analyzing these proteins, including BLAST, HMMER, Organelle Proteins, Protein Locations, Sequences, Gene Ontology, Homologs, and Phylogeny. In summary, our work revealed the protein composition of silkworm BmE organelles and provided a database resource helpful for understanding the functions and evolution of these proteins.

The lectin Orysata induces phosphatase-mediated and carbohydrate-independent aggregation of insect cells

Lectins, or carbohydrate-binding proteins, can cause agglutination of particular cells. This process is mediated by the interaction of the carbohydrate-binding domain with sugar structures on the cell surface, and this binding can be inhibited by pre-incubation of the lectin with its specific sugars. However, when incubated with insect cells, Orysata, a mannose-binding lectin from rice, caused aggregation of the cells, independent from carbohydrate binding activity. This phenomenon was observed for multiple insect cell lines, confirming the robustness of this phenotype. While the carbohydrate-dependent agglutination of red blood cells happens within minutes, the carbohydrate-independent aggregation of insect cells requires longer incubation times. Further analysis with the galactose-binding lectins SSA and Jacalin, validated the robustness of this lectin-induced, carbohydrate-independent aggregation in different insect cell lines. Since proteomic analysis revealed no changes in the proteome after treatment with the lectins, this cell aggregation is likely caused by the (in) activation or re-organization of the existing surface proteins. The use of inhibitors of phosphorylation and dephosphorylation, staurosporine (STS) and a phosphatase inhibitor (PPI) cocktail, pointed to dephosphorylation as a key mechanism in the lectin-induced, carbohydrate-independent aggregation of insect cells. Similar to contact inhibition, cell proliferation in cell aggregates was decreased. Analysis of the marker for cell proliferation, cyclin E, confirmed that aggregated cells enter a quiescent state. The current data offer a new perspective on the mechanism by which lectins execute their activities, specifically through lectin-induced phosphatase-mediated cell aggregation and proliferation inhibition, independent from their carbohydrate-binding activity.

The essential role of Dnmt1 in gametogenesis in the large milkweed bug Oncopeltus fasciatus.

Given the importance of DNA methylation in protection of the genome against transposable elements and transcriptional regulation in other taxonomic groups, the diversity in both levels and patterns of DNA methylation in the insects raises questions about its function and evolution. We show that the maintenance DNA methyltransferase, DNMT1, affects meiosis and is essential to fertility in milkweed bugs, Oncopeltus fasciatus, while DNA methylation is not required in somatic cells. Our results support the hypothesis that Dnmt1 is required for the transition of germ cells to gametes in O. fasciatus and that this function is conserved in male and female gametogenesis. They further suggest that DNMT1 has a function independent of DNA methylation in germ cells. Our results raise thequestion as to how a gene that is so critical to fitness across multiple insect species is able to diverge widely across the insect tree of life.