Starvation-Induced Changes to the Midgut Proteome and Neuropeptides in Manduca sexta.

Starvation is a complex physiological state that induces changes in protein expression to ensure survival. The insect midgut is sensitive to changes in dietary content as it is at the forefront of communicating information about incoming nutrients to the body via hormones. Therefore, a DIA proteomics approach was used to examine starvation physiology and, specifically, the role of midgut neuropeptide hormones in a representative lepidopteran, Manduca sexta. Proteomes were generated from midguts of M. sexta fourth-instar caterpillars, starved for 24 h and 48 h, and compared to fed controls. A total of 3047 proteins were identified, and 854 of these were significantly different in abundance. KEGG analysis revealed that metabolism pathways were less abundant in starved caterpillars, but oxidative phosphorylation proteins were more abundant. In addition, six neuropeptides or related signaling cascade proteins were detected. Particularly, neuropeptide F1 (NPF1) was significantly higher in abundance in starved larvae. A change in juvenile hormone-degrading enzymes was also detected during starvation. Overall, our results provide an exploration of the midgut response to starvation in M. sexta and validate DIA proteomics as a useful tool for quantifying insect midgut neuropeptide hormones.

The highly improved genome of Ixodes scapularis with X and Y pseudochromosomes.

Ixodes scapularis, the black-legged tick, is the principal vector of the Lyme disease spirochete, Borrelia burgdorferi, and is responsible for most of the ∼470,000 estimated Lyme disease cases annually in the USA. Ixodes scapularis can transmit six additional pathogens of human health significance. Because of its medical importance, I. scapularis was the first tick genome to be sequenced and annotated. However, the first assembly, I. scapularis Wikel (IscaW), was highly fragmented because of the technical challenges posed by the long, repetitive genome sequences characteristic of arthropod genomes and the lack of long-read sequencing techniques. Although I. scapularis has emerged as a model for tick research because of the availability of new tools such as embryo injection and CRISPR-Cas9-mediated gene editing yet the lack of chromosome-scale scaffolds has slowed progress in tick biology and the development of tools for their control. Here we combine diverse technologies to produce the I. scapularis Gulia-Nuss (IscGN) genome assembly and gene set. We used DNA from eggs and male and female adult ticks and took advantage of Hi-C, PacBio HiFi sequencing, and Illumina short-read sequencing technologies to produce a chromosome-level assembly. In this work, we present the predicted pseudochromosomes consisting of 13 autosomes and the sex pseudochromosomes: X and Y, and a markedly improved genome annotation compared with the existing assemblies and annotations.

Shifts in sage-grouse arthropod food sources across grazing and environmental gradients in upland meadow communities.

Groundwater dependent systems are extremely important habitats for a wide variety of taxa in the Great Basin of North America. The impacts of grazing on these habitats cause shifts in resources and subsequent change in species composition. The Greater sage-grouse, a keystone species of Great Basin ecosystems, rear offspring in these areas during spring and summer months using forbs and arthropods. To examine the impact of grazing on arthropod abundance in these ecosystems, seven meadows, each made up of three unique vegetative communities, were grazed at three intensities across two years (2019-2020) and monitored for environmental variables and abundance of arthropods during peak sage-grouse utilization periods. Additionally, the relationship of field measurements and near-surface digital cameras (phenocams) was examined to better understand how remote sensing technologies can be used to monitor these insect abundance shifts on larger scales. Arthropod taxa abundance responded differently to grazing management and environmental variables. Coleoptera abundance during peak sage-grouse usage periods increased roughly 50% in some meadows with increased grazing intensity. For year-to-year environmental variability in precipitation, Lepidoptera abundance was 114% higher in the drier year, while Coleoptera was 39% lower. Near-surface cameras had varied success with predicting peak insect abundance levels. Lepidoptera and Coleoptera capture rates had strong correlations with phenological indices derived from phenocams, while Formicidae had much weaker relationships.

Trypsin, the Major Proteolytic Enzyme for Blood Digestion in the Mosquito Midgut.

When a female mosquito takes a blood meal, proteolytic activity surges in the midgut. Trypsin-like serine proteases are the major endoproteolytic enzyme induced by feeding in mosquitoes. The mosquito midgut lacks trypsin activity before the blood meal, but in most anautogenous mosquitoes, trypsin activity increases continuously up to 30 h after feeding and subsequently returns to baseline levels by 60 h. Trypsin activity in mosquitoes is restricted entirely to the posterior midgut lumen, where blood is stored and digested. Trypsin enzyme activity can be quantitatively measured using the artificial Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride substrate, a method described in our associated protocol.

Analyzing Blood Digestion: Estimation of Active Trypsin Levels in the Mosquito Midgut.

The Nα-benzoyl-dl-arginine 4-nitroanilide hydrochloride (BApNA) assay is widely used to quantify trypsin in mosquito midguts and is highly sensitive. BApNA is a chromogenic substrate for proteolytic enzymes such as trypsin and amidase. Hydrolysis of BApNA at the bond between the arginine and the p-nitroaniline moieties releases the chromophore p-nitroaniline, which is detected by colorimetric analysis. The intensity of the color is directly proportional to the amount of trypsin in the solution. Here, we present a trypsin measurement assay specifically using the BApNA substrate.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis.

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here. This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

Cas9-mediated gene editing in the black-legged tick, Ixodes scapularis, by embryo injection and ReMOT Control.

Despite their capacity to acquire and pass on an array of debilitating pathogens, research on ticks has lagged behind other arthropod vectors, such as mosquitoes, largely because of challenges in applying available genetic and molecular tools. CRISPR-Cas9 is transforming non-model organism research; however, successful gene editing has not yet been reported in ticks. Technical challenges for injecting tick embryos to attempt gene editing have further slowed research progress. Currently, no embryo injection protocol exists for any chelicerate species, including ticks. Herein, we report a successful embryo injection protocol for the black-legged tick, Ixodes scapularis, and the use of this protocol for genome editing with CRISPR-Cas9. We also demonstrate that the ReMOT Control technique could be successfully used to generate genome mutations outside Insecta. Our results provide innovative tools to the tick research community that are essential for advancing our understanding of the molecular mechanisms governing pathogen transmission by tick vectors and the underlying biology of host-vector-pathogen interactions.

Characterization of Anopheles stephensi Odorant Receptor 8, an Abundant Component of the Mouthpart Chemosensory Transcriptome.

Several mosquito species within the genus Anopheles are vectors for human malaria, and the spread of this disease is driven by the propensity of certain species to feed preferentially on humans. The study of olfaction in mosquitoes is important to understand dynamics of host-seeking and host-selection; however, the majority of these studies focus on Anopheles gambiae or An. coluzzii, both vectors of malaria in Sub-Saharan Africa. Other malaria vectors may recognize different chemical cues from potential hosts; therefore, in this study, we investigated An. stephensi, the south Asian malaria mosquito. We specifically focused on the mouthparts (primarily the maxillary palp and labella) that have been much less investigated compared to the antennae but are also important for host-seeking. To provide a broad view of chemoreceptor expression, RNAseq was used to examine the transcriptomes from the mouthparts of host-seeking females, blood-fed females, and males. Notably, AsOr8 had a high transcript abundance in all transcriptomes and was, therefore, cloned and expressed in the Drosophila empty neuron system. This permitted characterization with a panel of odorants that were selected, in part, for their presence in the human odor profile. The responsiveness of AsOr8 to odorants was highly similar to An. gambiae Or8 (AgOr8), except for sulcatone, which was detected by AsOr8 but not AgOr8. Subtle differences in the receptor sensitivity to specific odorants may provide clues to species- or strain-specific approaches to host-seeking and host selection. Further exploration of the profile of An. stephensi chemosensory proteins may yield a better understanding of how different malaria vectors navigate host-finding and host-choice.

Genetic Manipulation of Ticks: A Paradigm Shift in Tick and Tick-Borne Diseases Research.

Ticks are obligate hematophagous arthropods that are distributed worldwide and are one of the most important vectors of pathogens affecting humans and animals. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind other arthropod vectors, such as mosquitoes. This is largely because of challenges in applying functional genomics and genetic tools to the idiosyncrasies unique to tick biology, particularly techniques for stable genetic transformations. CRISPR-Cas9 is transforming non-model organism research; however, successful germline editing has yet to be accomplished in ticks. Here, we review the ancillary methods needed for transgenic tick development and the use of CRISPR/Cas9, the most promising gene-editing approach, for tick genetic transformation.

The sugar substitute erythritol shortens the lifespan of Aedes aegypti potentially by N-linked protein glycosylation.

Adult male and female mosquitoes consume sugar as floral and extrafloral nectar. Earlier work demonstrated that mosquito populations and their vector potential are dependent upon the availability of sugar sources. Thus, a novel method of vector control may involve targeting sugar-feeding mosquitoes. Multiple human-safe sugar substitutes are already approved by the U.S. Food and Drug Administration and are readily available. However, plant-based sugar substitutes such as stevia (erythritol) have been shown to affect lifespan in other flies. Therefore, the current study was carried out to test the potential of commercially available sugar substitutes to adversely affect the survival, fecundity, and metabolism of adult Aedes aegypti mosquitoes. Of the four sugar substitutes tested, erythritol (Stevia), sucralose (Splenda), aspartame (Equal), and saccharin (Sweet'N Low), only erythritol negatively affected mosquito longevity and fecundity. The effect on fecundity was probably due in part to a corresponding decrease in glycogen and lipid levels over time in mosquitoes fed on erythritol. Comparative mosquito head transcriptomes indicated upregulation of a gene in the mannose biosynthesis pathway in females fed on erythritol, suggesting that N-linked glycosylation might be responsible for the negative impact of erythritol feeding in mosquitoes. Mosquitoes preferred sucrose when a choice was given but were not averse to erythritol. Our results suggest the possibility of using erythritol alone or in combination with sucrose as a component of attractive toxic sugar baits for a human-safe approach for mosquito control.

Blood Digestion by Trypsin-Like Serine Proteases in the Replete Lyme Disease Vector Tick, Ixodes scapularis.

Ixodes scapularis is the major vector of Lyme disease in the Eastern United States. Each active life stage (larva, nymph, and adult) takes a blood meal either for developing and molting to the next stage (larvae and nymphs) or for oviposition (adult females). This protein-rich blood meal is the only food taken by Ixodes ticks and therefore efficient blood digestion is critical for survival. Studies in partially engorged ticks have shown that the initial stages of digestion are carried out by cathepsin proteases within acidic digestive cells. In this study, we investigated the potential role of serine proteases in blood digestion in replete ticks. RNA interference was used for functional analysis and a trypsin-benzoyl-D, L-arginine 4-nitoanilide assay was used to measure active trypsin levels. Hemoglobinolytic activity was determined in vitro, with or without a serine protease inhibitor. Our data suggest that trypsin levels increase significantly after repletion. Knockdown of serine proteases negatively impacted blood feeding, survival, fecundity, levels of active trypsin in the midgut, and resulted in lower hemoglobin degradation. Incubation of midgut extract with a trypsin inhibitor resulted in 65% lower hemoglobin degradation. We provide evidence of the serine proteases as digestive enzymes in fully engorged, replete females. Understanding the digestive profile of trypsin during blood meal digestion in I. scapularis improves our understanding of the basic biology of ticks and may lead to new methods for tick control.

Unlocking the genetic basis of monarch butterflies' use of medicinal plants.

If there was any doubt of the primary role that plant secondary metabolites play in host-parasite co-evolution, the "From the Cover" paper by Tan et al. (2019) featured in this issue of Molecular Ecology will lay these doubts to rest. The group's previous work on monarch butterflies (Danaus plexippus) infected with the protozoan pathogen Ophryocystis elektroscirrha (OE) demonstrated higher survival and lower spore load on high cardenolide-producing milkweed (Asclepias curassavica) (Figure 1a) compared with low cardenolide-producing milkweed (A. incarnata) (de Roode, Pedersen, Hunter, & Altizer, 2008) (Figure 1b). The mechanism of this protective effect is not directly clear, but a leading hypothesis is that the cardenolides confer protection through toxicity to the parasite. However, the role of the caterpillar immune system in managing this parasite is largely unknown. Novel insights into the influence of toxic plant metabolites on caterpillar immunity are explored in Tan et al. (2019). Using transcriptomics to probe this model system, the authors found that herbivore immune genes were down-regulated and detoxification genes were up-regulated when larvae were reared on the milkweed species with high cardenolide concentrations (A. curassavica). Surprisingly, immune genes were not significantly up- or down-regulated in response to protozoan infection alone. This tantalizing result suggests that sequestered plant metabolites, not immunity, is reining in protozoan infections in these larvae, and promoting survival. As the authors point out, the strategy to invest in sequestration may come at a cost, which is to the detriment of the immune response (Smilanich, Dyer, Chambers, & Bowers, 2009). However, the cost becomes worth the investment when chemical sequestration takes on an antipathogen role. The novelty of the Tan et al. (2019) paper is that they show the investment in sequestration leading to a possible divestment in immunity.

Dynamics of Insulin Signaling in the Black-Legged Tick, Ixodes scapularis.

Insulin-like peptides (ILPs) have been identified in several invertebrates, particularly insects, and work on these ILPs has revealed many roles including regulation of energy homeostasis, growth, development, and lifespan to name a few. However, information on arthropod ILPs outside of insects is sparse. Studies of Ixodid tick ILPs are particularly scarce, despite their importance as vectors of infectious agents, most notably Lyme disease. The recent publication of the genome of the black-legged tick, Ixodes scapularis, has advanced opportunities to study this organism from a molecular standpoint, a resource sorely needed for an organism with challenging life history requirements for study in the laboratory, such as a long life cycle and obligate, prolonged, blood-feeding at each life stage. Through bioinformatics searches of the tick genome and other available I. scapularis databases, we identified four putative ILP sequences. Full-length sequences of these ILP transcripts were confirmed, and quantitative RT-PCR was used to examine expression levels of these ILPs in different life stages, feeding states, and adult tissues. This work serves as an initial characterization of ILP expression in ticks and provides the foundation for further exploration of the roles of ILPs in these important arthropod vectors.

Insulin-Like Peptide Signaling in Mosquitoes: The Road Behind and the Road Ahead.

Insulin signaling is a conserved pathway in all metazoans. This pathway contributed toward primordial metazoans responding to a greater diversity of environmental signals by modulating nutritional storage, reproduction, and longevity. Most of our knowledge of insulin signaling in insects comes from the vinegar fly, Drosophila melanogaster, where it has been extensively studied and shown to control several physiological processes. Mosquitoes are the most important vectors of human disease in the world and their control constitutes a significant area of research. Recent studies have shown the importance of insulin signaling in multiple physiological processes such as reproduction, innate immunity, lifespan, and vectorial capacity in mosquitoes. Although insulin-like peptides have been identified and functionally characterized from many mosquito species, a comprehensive review of this pathway in mosquitoes is needed. To fill this gap, our review provides up-to-date knowledge of this subfield.

Nutritional Quality during Development Alters Insulin-Like Peptides' Expression and Physiology of the Adult Yellow Fever Mosquito, Aedes aegypti.

Mosquitoes have distinct developmental and adult life history, and the vectorial capacity of females has been shown to be affected by the larval nutritional environment. However, little is known about the effect of developmental nutrition on insulin-signaling and nutrient storage. In this study, we used Aedes aegypti, the yellow fever mosquito, to determine whether larval nutrition affects insulin gene expression. We also determined the traits regulated by insulin signaling, such as stored-nutrient levels and fecundity. We raised mosquito larvae on two different diets, containing either high protein or high carbohydrates. Development on a high-carbohydrate diet resulted in several life-history phenotypes indicative of suboptimal conditions, including increased developmental time and decreased fecundity. Additionally, our data showed that insulin transcript levels are affected by a high-carbohydrate diet during development. Females, not males, reared on high-carbohydrate diets had much higher transcript levels of insulin-like peptide 3 (ILP3), a mosquito equivalent of human insulin, and these females more readily converted sugar meals into lipids. We also found that AaILP4, not AaILP3, transcript levels were much higher in the males after a sugar meal, suggesting sex-specific differences in the insulin-signaling pathway. Our findings suggest a conserved mechanism of carbohydrate-mediated hyperinsulinemia in animals.

Insulin receptor knockdown blocks filarial parasite development and alters egg production in the southern house mosquito, Culex quinquefasciatus.

Lymphatic filariasis, commonly known as elephantiasis, is a painful and profoundly disfiguring disease. Wuchreria bancrofti (Wb) is responsible for >90% of infections and the remainder are caused by Brugia spp. Mosquitoes of the genera Culex (in urban and semi-urban areas), Anopheles (in rural areas of Africa and elsewhere), and Aedes (in Pacific islands) are the major vectors of W. bancrofti. A preventive chemotherapy called mass drug administration (MDA), including albendazole with ivermectin or diethylcarbamazine citrate (DEC) is used in endemic areas. Vector control strategies such as residual insecticide spraying and long-lasting insecticidal nets are supplemental to the core strategy of MDA to enhance elimination efforts. However, increasing insecticide resistance in mosquitoes and drug resistance in parasite limit the effectiveness of existing interventions, and new measures are needed for mosquito population control and disruption of mosquito-parasite interactions to reduce transmission. Mosquito insulin signaling regulates nutrient metabolism and has been implicated in reduced prevalence and intensity of malaria parasite, Plasmodium falciparum, infection in mosquitoes. Currently no data are available to assess how insulin signaling in mosquitoes affects the development of multi-cellular parasites, such as filarial nematodes. Here, we show that insulin receptor knockdown in blood fed C. quinquefasciatus, the major vector of Wb in India, completely blocks the development of filarial nematode parasite to the infective L3 stage, and results in decreased ecdysteroid production and trypsin activity leading to fewer mosquito eggs. These data indicate that a functional mosquito insulin receptor (IR) is necessary for filarial parasite development and mosquito reproduction. Therefore, insulin signaling may represent a new target for the development of vector control or parasite blocking strategies.

Isolation of an insulin-like peptide from the Asian malaria mosquito, Anopheles stephensi, that acts as a steroidogenic gonadotropin across diverse mosquito taxa.

Many insulin-like peptides (ILPs) have been identified in insects, yet only a few were isolated in their native form for structural and functional studies. Antiserum produced to ILP3 in Aedes aegypti was used in a radioimmunoassay to monitor the purification of an ILP from heads of adult An. stephensi and recognized the ILP in other immunoassays. The structure of the purified peptide matched that predicted for the ILP3 in this species. The native form stimulated ecdysteroid production by ovaries isolated from non-blood fed females. Synthetic forms of An. stephensi ILP3 and ILP4 similarly activated this process in a dose responsive manner. This function was first established for ILP3 and ILP4 homologs in Aedes aegypti, thus suggesting their structural and functional conservation in mosquitoes. We tested the extent of conservation by treating ovaries of An. gambiae, Ae. aegypti, and Culex quinquefasciatus with the An. stephensi ILPs, and both the native and synthetic ILP3 were stimulatory, as was the ILP4. Taken together, these results offer the first evidence for ILP functional conservation across the Anophelinae and Culicinae subfamilies.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice.

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Comparative pharmacological characterization of D1-like dopamine receptors from Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus suggests pleiotropic signaling in mosquito vector lineages.

BACKGROUND: Small molecule antagonists of mosquito dopamine receptors (DARs) are under investigation as a new class of vector-selective insecticides. Antagonists that inhibit the D1-like DARs AaDOP2 and CqDOP2 from the mosquitoes Aedes aegypti L. and Culex quinquefasciatus Say, respectively, also cause larval mortality in bioassays. Here, we report on the orthologous DAR, AgDOP2, from the malaria mosquito Anopheles gambiae Giles that was cloned and pharmacologically characterized in HEK293 cells. Larval bioassays were then conducted to examine the potential of DAR antagonist insecticides against Anopheles vectors. FINDINGS: Previous in vitro cAMP accumulation assays demonstrated Gαs coupling for AaDOP2 and CqDOP2 and dose-dependent inhibition by DAR antagonists. We observed a negligible response of AgDOP2 in the cAMP assay, which prompted an investigation of alternative coupling for mosquito DARs. In an in vitro IP-One Gαq second messenger assay of calcium signaling, dopamine stimulation increased IP1 accumulation in AaDOP2-, CqDOP2- and AgDOP2-expressing cells, and DAR antagonists inhibited IP1 signaling in a dose-dependent manner. In larval bioassays, DAR antagonists caused considerable mortality of An. gambiae larvae within 24 h post-exposure. CONCLUSIONS: In vitro data reveal pleiotropic coupling of AaDOP2 and CqDOP2 to Gαq and Gαs. In contrast, AgDOP2 appeared to selectively couple to Gαq signaling. In vitro antagonist studies revealed general conservation in pharmacology between mosquito DARs. In vivo data suggest potential for DAR antagonist insecticides against An. gambiae. Sequence conservation among the DOP2 receptors from 15 Anopheles species indicates utility of antagonists to control residual malaria transmission. AgDOP2 Gαq-dependent signaling could be exploited for An. gambiae control via pathway specific antagonists.