Identification of nectar sources foraged by female mosquitoes in Canada.

For many mosquito species, the females must obtain vertebrate blood to complete a gonotrophic cycle. These blood meals are frequently supplemented by feeding on sugary plant nectar, which sustains energy reserves needed for flight, mating, and overall fitness. Our understanding of mosquito nectar foraging behaviors is mostly limited to laboratory experiments and direct field observations, with little research into natural mosquito-host plant relationships done in North America. In this study, we collected nectar-fed female mosquitoes over a 2-year period in Manitoba, Canada, and amplified a fragment of the chloroplast rbcL gene to identify the plant species fed upon. We found that mosquitoes foraged from diverse plant families (e.g., grasses, trees, ornamentals, and legumes), but preferred certain species, most notably soybean and Kentucky blue grass. Moreover, there appeared to be some associations between plant feeding preferences and mosquito species, date of collection, landscape, and geographical region. Overall, this study implemented DNA barcoding to identify nectar sources forage by mosquitoes in the Canadian Prairies.

Black queen cell virus detected in Canadian mosquitoes.

Black queen cell virus (BQCV) is a ubiquitous honeybee virus and a significant pathogen to queen bee (Apis mellifera) larvae. However, many aspects of the virus remain poorly understood, including the transmission dynamics. In this study, we used next-generation sequencing to identify BQCV in Aedes vexans (n = 4,000) collected in 2019 and 2020 from Manitoba, Canada. We assembled de novo the nearly complete (>96%) genome sequence of the virus, which is the first available from North America and the first report of BQCV being harbored by mosquitoes. Phylogenetic tree reconstructions indicated that the genome had 95.5% sequence similarity to a BQCV isolate from Sweden. Sequences of a potential vector (Varroa destructor) and a microsporidian associated with BQCV (Nosema apis) were not identified in the mosquito samples, however, we did detect sequences of plant origin. We, therefore, hypothesize that the virus was indirectly acquired by mosquitoes foraging at the same nectar sources as honeybees.

Metabolic consequences of PGC-1α dysregulation in adult zebrafish muscle.

The peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) is central to the regulation of cellular and mitochondrial energy homeostasis in mammals, but its role in other vertebrates remains unclear. Indeed, previous work suggests extensive structural and functional divergence of PGC-1α in teleosts but this remains to be directly tested. Here, we describe the initial characterization of heterozygous PGC-1α mutant zebrafish lines created by CRISPR-Cas9 disruptions of an evolutionarily conserved regulatory region of the PGC-1α proximal promoter. Using qPCR, we confirmed the disruption of PGC-1α gene expression in striated muscle, leading to a simultaneous fourfold increase in mixed skeletal muscle PGC-1α mRNA levels and an opposite fourfold downregulation in cardiac muscle. In mixed skeletal muscle, most downstream effector genes were largely unaffected yet two mitochondrial lipid transporters, carnitine palmitoyltransferase-1 and -2, were strongly induced. Conversely, PGC-1α depression in cardiac muscle reduced the expression of several transcriptional regulators (estrogen-related receptor α, nuclear respiratory factor 1, and PGC-1ß) without altering metabolic gene expression. Using high-resolution respirometry, we determined that white muscle exhibited increased lipid oxidative capacity with little difference in markers of mitochondrial abundance. Finally, using whole animal intermittent respirometry, we show that mutant fish exhibit a twofold higher basal metabolism than their wild-type counterparts. Altogether, this new model confirms a central but complex role for PGC-1α in mediating energy utilization in zebrafish, and we propose its use as a valuable tool to explore the intricate regulatory pathways of energy homeostasis in a popular biomedical model.

Diet and ontogeny drastically alter the larval microbiome of the invertebrate model Galleria mellonella.

Larvae of the greater wax moth (Galleria mellonella) are an emerging animal model to study the innate immune response and biodegradation of plastic polymers. Both of these complex biological processes are likely impacted by the plasticity of host-microbe interactions, which remains understudied in lepidopterans. Consequently, we carried out 16S rRNA sequencing to explore the effect diet (natural, artificial) has on the bacterial assemblages of G. mellonella in different tissues (gut, fat bodies, silk glands) throughout development (eggs, six instar stages, adults). The microbiome was rich in diversity, with Proteobacteria and Firmicutes being the most represented phyla. Contrary to other lepidopterans, G. mellonella appears to possess a resident microbiome dominated by Ralstonia. As larvae progress through development, the bacterial assemblages become increasingly shaped by the caterpillar's diet. In particular, a number of bacteria genera widely associated with the G. mellonella microbiome (e.g., Enterococcus and Enterbacter) were significantly enriched on an artificial diet. Overall, these results indicate that the G. mellonella microbiome is not as simplistic and homogenous as previously described. Rather, its bacterial communities are drastically affected by both diet and ontogeny, which should be taken into consideration in future studies planning to use G. mellonella as model species.

Role of the intestinal microbiome in low-density polyethylene degradation by caterpillar larvae of the greater wax moth, Galleria mellonella.

Recently, a few insects, including the caterpillar larva of the greater wax moth Galleria mellonella, have been identified as avid 'plastivores'. These caterpillars are able to ingest and metabolize polyethylene at unprecedented rates. While it appears that G. mellonella plays an important role in the biodegradation process, the contribution of its intestinal microbiome remains poorly understood and contested. In a series of experiments, we present strong evidence of an intricate relationship between an intact microbiome, low-density polyethylene (LDPE) biodegradation and the production of glycol as a metabolic by-product. First, we biochemically confirmed that G. mellonella larvae consume and metabolize LDPE, as individual caterpillars fed on polyethylene excreted glycol, but those excretions were reduced by antibiotic treatment. Further, while the gut bacterial communities remained relatively stable regardless of diet, we showed that during the early phases of feeding on LDPE (24-72 h), caterpillars exhibited increased microbial abundance relative to those starved or fed on their natural honeycomb diet. Finally, by isolating and growing gut bacteria with polyethylene as their exclusive carbon source for over 1 year, we identified microorganisms in the genus Acinetobacter that appeared to be involved in this biodegradation process. Taken collectively, our study indicates that during short-term exposure, the intestinal microbiome of G. mellonella is intricately associated with polyethylene biodegradation in vivo.

A Very Hungry Caterpillar: Polyethylene Metabolism and Lipid Homeostasis in Larvae of the Greater Wax Moth (Galleria mellonella).

Larvae of the greater wax moth (Galleria mellonella) possess the remarkable ability to consume and rapidly degrade low-density polyethylene. Previous studies have investigated the involvement of the animal's microbiome, but little is known about the host's actual role and if it benefits from biodegradation of this synthetic polymer. We used a combination of RNA sequencing and biochemical approaches to assess caterpillars fed honeycomb, fed polyethylene (PE), or starved for up to 72 h. Sequencing of gut transcripts revealed PE-fed larvae retain an expression profile consistent with normal intestinal function but also show distinct molecular signatures indicative of enhanced fatty acid metabolism (FAM). Further, quantification of total lipid content validated the impact of a PE diet on FAM; in contrast to lipid-depleted starved animals, PE-fed caterpillars maintain lipid reserves similar to honeycomb-fed larvae. Additionally, we found the activity of putative enzymes involved in lipid oxidation (e.g., alcohol dehydrogenase) are considerably higher in PE-fed larvae, indicating that on a functional level, these caterpillars are inducing pathways to effectively metabolize PE. Overall, we put forward a hypothesized model where the similarity in chemical structure between PE and its natural honeycomb diet has endowed larvae of G. mellonella with the extraordinary capability to derive energy from PE as an exclusive food source through pre-existing metabolic pathways.

Exposure Assessment in Millimeter-Wave Reverberation Chamber Using Murine Phantoms.

This study deals with the design and calibration of the first mode-stirred reverberation chamber (RC) in the 60-GHz-band adapted for in vivo bioelectromagnetic studies. In addition to the interface for electromagnetic and thermal dosimetry, the interfaces for lighting and ventilation were integrated into the RC walls while preserving acceptable shielding. The RC with mechanical and electronic steering capabilities is characterized in the 55-65 GHz range. To this end, murine skin-equivalent phantoms of realistic shape were designed and fabricated. Their complex permittivity is within ±12% of the target value of murine skin (6.19-j5.81 at 60 GHz). The quality factor of the RC loaded with an animal cage, bedding litter, and five murine phantoms was found to be 1.2 × 104 . The losses inside the RC were analyzed, and it was demonstrated that the main sources of the power dissipation were the phantoms and mice cage. The input power required to reach the average incident power density of 1 and 5 mW/cm2 was found to be 0.23 and 1.14 W, respectively. Surface heating of the mice models was measured in the infrared (IR) range using a specifically designed interface, transparent at IR and opaque at millimeter waves (mmW). Experimental results were compared with an analytical solution of the heat transfer equation and to full-wave computations. Analytical and numerical results were in very good agreement with measurements (the relative deviation after 90 min of exposure was within 4.2%). Finally, a parametric study was performed to assess the impact of the thermophysical parameters on the resulting heating. Bioelectromagnetics. 2020;41:121-135. © 2020 Bioelectromagnetics Society.

Metabolic regulation by the PGC-1α and PGC-1ß coactivators in larval zebrafish (Danio rerio).

The peroxisome proliferator activated receptor γ coactivator-1 (PGC-1) family is composed of three coactivators whose role in regulating mammalian bioenergetics regulation is clear, but is much less certain in other vertebrates. Current evidence suggests that in fish, PGC-1α and PGC-1ß may exhibit much less redundancy in the control of fatty acid oxidation and mitochondrial biogenesis compared to mammals. To assess these roles directly, we knocked down PGC-1α and PGC-1ß expression with morpholinos in zebrafish embryos, and we investigated the resulting molecular and physiological phenotypes. First, we found no effects of either morpholinos on larval hatching, heart rates and oxygen consumption over the first few days of development. Next, at 3 days post fertilization (dpf), we confirmed by real time PCR a specific knock down of both coactivators, that resulted in a significant reduction in the transcript levels of citrate synthase (CS), 3-hydroxyacyl-CoA dehydrogenase (HOAD), and medium-chain acyl-coenzyme A dehydrogenase (MCAD) in both morphant groups. However, there was no effect on transcription factors' gene expression except for a marked reduction in estrogen related receptor α (ERRα) transcripts in PGC-1α morphants. Finally, we assessed whole embryonic enzyme activity for CS, cytochrome oxidase (COX), HOAD and carnitine palmitoyltransferase I (CPT-1) at 4 dpf. The only significant effect of the knockdown was a reduced CS activity in PGC-1α morphants and a counterintuitive increase of cytochrome oxidase activity in PGC-1ß morphants. Overall, our results indicate that in larval zebrafish, PGC-1α and PGC-1ß both play a role in regulating expression of important mitochondrial genes potentially through ERRα.

Evolution of urea transporters in vertebrates: adaptation to urea's multiple roles and metabolic sources.

In the two decades since the first cloning of the mammalian kidney urea transporter (UT-A), UT genes have been identified in a plethora of organisms, ranging from single-celled bacteria to metazoans. In this review, focusing mainly on vertebrates, we first reiterate the multiple catabolic and anabolic pathways that produce urea, then we reconstruct the phylogenetic history of UTs, and finally we examine the tissue distribution of UTs in selected vertebrate species. Our analysis reveals that from an ancestral UT, three homologues evolved in piscine lineages (UT-A, UT-C and UT-D), followed by a subsequent reduction to a single UT-A in lobe-finned fish and amphibians. A later internal tandem duplication of UT-A occurred in the amniote lineage (UT-A1), followed by a second tandem duplication in mammals to give rise to UT-B. While the expected UT expression is evident in excretory and osmoregulatory tissues in ureotelic taxa, UTs are also expressed ubiquitously in non-ureotelic taxa, and in tissues without a complete ornithine-urea cycle (OUC). We posit that non-OUC production of urea from arginine by arginase, an important pathway to generate ornithine for synthesis of molecules such as polyamines for highly proliferative tissues (e.g. testis, embryos), and neurotransmitters such as glutamate for neural tissues, is an important evolutionary driving force for the expression of UTs in these taxa and tissues.

Patterns of fuel use during locomotion in mammals revisited: the importance of aerobic scope.

Fuel selection patterns during exercise are thought to be conserved among sea-level native mammals when intensity is expressed relative to maximum aerobic capacity (V̇(O2,max)). However, this claim is based on data from only a few species larger than rats, and has never been tested statistically. Thus, we investigated fuel use in a small mammal (Mus musculus, CD-1 strain), and combined these data with published data on rats, dogs, goats and humans to evaluate the robustness of the mammalian fuel selection model. We found that mice rely less on carbohydrates to power moderate intensity exercise at the same % V̇(O2,max) than larger mammals. We suggest that this difference is due to a decline in aerobic scope (O2 available for exercise above resting metabolism) as body size decreases. We propose a redefined fuel use model that reflects changes in fractional aerobic scope with body size. Our results indicate that exercise defined as percent aerobic scope is a better predictor of fuel use across a wide range of quadruped species from mice to dogs and running humans.

Ontogeny of ornithine-urea cycle gene expression in zebrafish (Danio rerio).

Although the majority of adult teleosts excrete most of their nitrogenous wastes as ammonia, several fish species are capable of producing urea early in development. In zebrafish, it is unclear whether this results from a functional ornithine-urea cycle (O-UC) and, if so, how it might be regulated. This study examined the spatiotemporal patterns of gene expression of four major O-UC enzymes: carbamoyl phosphate synthase III (CPSIII), ornithine transcarboxylase, arginosuccinate synthetase, and arginosuccinate lyase, using real-time PCR and whole mount in situ hybridization. In addition, we hypothesized that CPSIII gene expression was epigenetically regulated through methylation of its promoter, a widespread mode of differential gene regulation between tissues and life stages in vertebrates. Furthermore, to assess CPSIII functionality, we used morpholinos to silence CPSIII in zebrafish embryos and assessed their nitrogenous waste handling during development, and in response to ammonia injections. Our results suggest that mRNAs of O-UC enzymes are expressed early in zebrafish development and colocalize to the embryonic endoderm. In addition, the methylation status of CPSIII promoter is not consistent with the patterns of expression observed in developing larvae or adult tissues, suggesting other means of transcriptional regulation of this enzyme. Finally, CPSIII morphants exhibited a transient reduction in CPSIII enzyme activity 24 h postfertilization, which was paralleled by reduced urea production during development and in response to an ammonia challenge. Overall, we conclude that the O-UC is functional in zebrafish embryos, providing further evidence that the capacity to produce urea via the O-UC is widespread in developing teleosts.

Nitrogen metabolism of the intestine during digestion in a teleost fish, the plainfin midshipman (Porichthys notatus).

Digestion affects nitrogen metabolism in fish, as both exogenous and endogenous proteins and amino acids are catabolized, liberating ammonia in the process. Here we present a model of local detoxification of ammonia by the intestinal tissue of the plainfin midshipman (Porichthys notatus) during digestion, resulting in an increase in urea excretion of gastrointestinal origin. Corroborating evidence indicated whole-animal ammonia and urea excretion increased following feeding, and ammonia levels within the lumen of the midshipman intestine increased to high levels (1.8±0.4 µmol N g(-1)). We propose that this ammonia entered the enterocytes and was detoxified to urea via the ornithine-urea cycle (O-UC) enzymes, as evidenced by a 1.5- to 2.9-fold post-prandial increase in glutamine synthetase activity (0.14±0.05 and 0.28±0.02 µmol min(-1) g(-1) versus 0.41±0.03 µmol min(-1) g(-1)) and an 8.7-fold increase in carbamoyl phosphate synthetase III activity (0.3±1.2 versus 2.6±0.4 nmol min(-1) g(-1)). Furthermore, digestion increased urea production by isolated gastrointestinal tissue 1.7-fold, supporting our hypothesis that intestinal tissue synthesizes urea in response to feeding. We further propose that the intestinal urea may have been excreted into the intestinal lumen via an apical urea transporter as visualized using immunohistochemistry. A portion of the urea was then excreted to the environment along with the feces, resulting in the observed increase in urea excretion, while another portion may have been used by intestinal ureolytic bacteria. Overall, we propose that P. notatus produces urea within the enterocytes via a functional O-UC, which is then excreted into the intestinal lumen. Our model of intestinal nitrogen metabolism does not appear to be universal as we were unab le to activate the O-UC in the intestine of fed rainbow trout. However, literature values suggest that multiple fish species could follow this model.

Seasonal turnover and insights into the overwintering biology of wireworms (Coleoptera: Elateridae) in the Canadian Prairies.

BACKGROUND: The long-lived terricolous larvae of click beetles, colloquially called wireworms, pose a significant threat to agriculture worldwide. Several economically important pest species have been documented in the Canadian Prairies, including Hypnoidus bicolor, Limonius californicus and Hypnoidus abbreviatus. However, most monitoring activities are performed in the early spring and there is evidence from other geographical regions of seasonal shifts in wireworm species composition and prevalence. Further, little is known about the overwintering physiology or behaviors of wireworms, which undoubtedly contribute to their population dynamics. RESULTS: We surveyed wireworm populations from four Manitoban fields six times throughout the 2020 and 2021 growing seasons. Both Hypnoidus species were active throughout the spring and summer; however, L. californicus did not become active until later in the spring. Chill-coma recovery assays indicated Hypnoidus species recovered quicker than L. californicus from cold acclimation. Vertical migration assays simulating progressively lower ambient temperatures experienced by overwintering larvae identified H. bicolor throughout the soil profile, with L. californicus preferentially found at cooler, shallower depths. We speculate that these differences in species distribution within the soil column are due to the higher levels of putative cryoprotectants (for example, trehalose, sorbitol, glucose, glycerol) in L. californicus, as identified by targeted liquid chromatography tandem mass spectrometry. CONCLUSION: Our findings of a stark seasonal turnover in wireworm species prevalence and composition in the Canadian Prairies should be incorporated into future integrated pest management and surveillance activities. This study also advances our understanding of wireworm overwintering biology, which should be factored into current management approaches. © 2022 His Majesty the King in Right of Canada. Pest Management Science © 2022 Society of Chemical Industry. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

Fat on plastic: Metabolic consequences of an LDPE diet in the fat body of the greater wax moth larvae (Galleria mellonella).

The caterpillar larvae of the greater wax moth (Galleria mellonella) are avid plastivores, as when provided a diet of low-density polyethylene (LDPE) they actively feed on it. Recent work has highlighted the importance of their microbiome in the putative biodegradation of this plastic polymer, though the impact of plastic metabolism on the insect host is less clear. In the present study, we undertook an integrative approach spanning all levels of biological organization to explore the effects of a plastic diet on the metabolic physiology of this animal model of plastic biodegradation. We demonstrate that an LDPE diet is not sufficient to maintain optimal larval growth and survival. In addition, we confirm that plastic fed waxworms retain their fat body lipid stores in a manner proportional to their individual polyethylene consumption suggesting a direct effect of LDPE biodegradation. At the functional level, the oxidative capacity of the fat body of LDPE-fed larvae is maintained reflecting unaltered metabolic function of the tissue. Finally, metabolomic analyses confirmed fat body lipid stores maintenance in LDPE-fed worms, but uncovered various other nutritional deficiencies. Overall, this work unveils novel insights in the complex interplay between LDPE biodegradation and the metabolic physiology of this model plastivore.

Modular evolution of PGC-1alpha in vertebrates.

In mammals, the peroxisome proliferator activated receptor (PPAR)gamma coactivator-1alpha (PGC-1alpha) is a central regulator of mitochondrial gene expression, acting in concert with nuclear respiratory factor-1 (NRF-1) and the PPARs. Its role as a "master regulator" of oxidative capacity is clear in mammals, but its role in other vertebrates is ambiguous. In lower vertebrates, although PGC-1alpha seems to play a role in coordinating the PPARalpha axis as in mammals, it does not appear to be involved in NRF-1 regulation of mitochondrial content. To evaluate the evolutionary patterns of this coactivator in fish and mammals, we investigated the evolutionary trajectories of PGC-1alpha homologs in representative vertebrate lineages. A phylogeny of the PGC-1 paralogs suggested that the family diversified through repeated genome duplication events early in vertebrate evolution. Bayesian and maximum likelihood phylogenetic reconstructions of PGC-1alpha in representative vertebrate species revealed divergent evolutionary dynamics across the different functional domains of the protein. Specifically, PGC-1alpha exhibited strong conservation of the activation/PPAR interaction domain across vertebrates, whereas the NRF-1 and MEF2c interaction domains experienced accelerated rates of evolution in actinopterygian (fish lineages) compared to sarcopterygians (tetrapod lineages). Furthermore, analysis of the amino acid sequence of these variable domains revealed successive serine- and glutamine-rich insertions within the teleost lineages, with important ramifications for PGC-1alpha function in these lineages. Collectively, these results suggest modular evolution of the PGC-1alpha protein in vertebrates that could allow for lineage-specific divergences in the coactivating capabilities of this regulator.

Effects of MP Polyethylene Microparticles on Microbiome and Inflammatory Response of Larval Zebrafish.

Plastic polymers have quickly become one of the most abundant materials on Earth due to their low production cost and high versatility. Unfortunately, some of the discarded plastic can make its way into the environment and become fragmented into smaller microscopic particles, termed secondary microplastics (MP). In addition, primary MP, purposely manufactured microscopic plastic particles, can also make their way into our environment via various routes. Owing to their size and resilience, these MP can then be easily ingested by living organisms. The effect of MP particles on living organisms is suspected to have negative implications, especially during early development. In this study, we examined the effects of polyethylene MP ingestion for four and ten days of exposure starting at 5 days post-fertilization (dpf). In particular, we examined the effects of polyethylene MP exposure on resting metabolic rate, on gene expression of several inflammatory and oxidative stress linked genes, and on microbiome composition between treatments. Overall, we found no evidence of broad metabolic disturbances or inflammatory markers in MP-exposed fish for either period of time. However, there was a significant increase in the oxidative stress mediator L-FABP that occurred at 15 dpf. Furthermore, the microbiome was disrupted by MP exposure, with evidence of an increased abundance of Bacteroidetes in MP fish, a combination frequently found in intestinal pathologies. Thus, it appears that acute polyethylene MP exposure can increase oxidative stress and dysbiosis, which may render the animal more susceptible to diseases.

Climbing Waterfalls: How Metabolism and Behavior Impact Locomotor Performance of Tropical Climbing Gobies on Reunion Island.

The life cycle of gobies of the Sicydiinae subfamily depends on climbing waterfalls. Two sympatric sicydiines species from Reunion Island, Sicyopterus lagocephalus (SIL) and Cotylopus acutipinnis (COA), employ different climbing modes. SIL uses a steady "inching" mode interrupted by short rest periods, whereas COA exhibits short "power-burst" undulatory movements punctuated by longer rest periods. Consequently, we explored the relationship between climbing performance and metabolic activity in these two species. We demonstrated that the two climbing modes are supported by different ecophysiological profiles that promote the interspecific variability of locomotor performance. More specifically, SIL performed better than COA during a climbing experiment because of its inching climbing mode, supported by a generally greater metabolic capacity and a higher potential for oxidative metabolism. Interestingly, we did not detect any difference in metabolic fuel storage and lactate production during climbing in either species, suggesting that these species can maintain fuel reserves and limit lactate accumulation through extensive rest times. Overall, this study provides new insights into the ecophysiology of these two emblematic species and suggests that the better climbing capacity of SIL is supported by its muscular metabolic capacity.

Molecular evidence for the inhibition of cytochrome p450s and cholinesterases in ticks by the repellent DEET.

For more than 50 years DEET (N,N-Diethyl-m-toluamide) has been considered the gold standard of repellents. It is applied to the skin or clothing to deter mosquitoes and other blood-sucking invertebrate pests from approaching and/or settling, and ultimately it provides temporary protection from bites. Despite rampant global use, surprisingly little is understood about DEET's mode of action and the molecular targets of the active ingredient. Furthermore, the theories into its mechanism for repellency are largely based off fruit fly and mosquito research. Since ticks possess a unique sensory structure, the Haller's organ, the specific genes and pathways associated with DEET avoidance may differ from insects. In these studies, we collected American dog ticks (Dermacentor variabilis) from four natural populations within Manitoba, Canada. We first carried out behavior assays, which showed DEET effectively repelled the ticks. RNA sequencing revealed that DEET caused a rapid and substantial reduction in the abundance of transcripts encoding cytochrome P450 and acetylcholinesterase genes, which gradually recovered over the 24 h time course. Finally, enzymatic kinetics provided functional support for DEET's role as an effective inhibitor of P450 s. While many facets of its mode of action remain to be worked out, our study provides valuable insights into the molecular underpinnings of DEET's repellence in ticks.

The Mexican bean beetle (Epilachna varivestis) regurgitome and insights into beetle-borne virus specificity.

For nearly 400 million years, insects and plants have been embattled in an evolutionary arms race. Insects have developed diverse feeding strategies and behaviors in an effort to circumvent and overcome an extensive collection of plant defense tactics. Sap-sucking insects often inject saliva into hosts plants, which contains a suite of effector proteins and even microbial communities that can alter the plant's defenses. Lacking salivary glands, leaf-feeding beetles represent an interesting group of phytophagous insects. Feeding beetles regurgitate onto leaf surfaces and it is thought that these oral secretions influence insect-plant interactions and even play a role in virus-vector specificity. Since the molecular and biological makeup of the regurgitant is virtually unknown, we carried out RNA sequencing and 16S rDNA analysis on a major soybean pest, Epilachna varivestis, to generate the first ever beetle "regurgitome" and characterize its microbiome. Interestingly, the regurgitant is comprised of a rich molecular assortment of genes encoding putative extracellular proteins involved in digestion, molting, immune defense, and detoxification. By carrying out plant inoculation assays, we reinforced the fundamental role of the regurgitant in beetle-borne virus specificity. Ultimately, these studies begin to characterize the importance of regurgitant in virus transmission and beetle-plant interactions.

Sensing and responding to energetic stress: Evolution of the AMPK network.

AMP-activated protein kinase is an enzyme that mediates communication between cellular energy status and diverse effector proteins, particularly those that play roles in determining the metabolic phenotype. By phosphorylating metabolic enzymes, transcriptional regulators and proteins involved in cellular structure, it can modify energy metabolism in both the short term and long term. Its basic features are highly conserved, with homologues in all eukaryotes. Gene and/or genome duplications endowed early vertebrates with paralogs of AMPK subunits, though the nature of their subfunctionalization remains uncertain, even in mammals. While most research focuses on the role of the enzyme in human health, a great deal can be learned from comparative studies targeting non-traditional model animals. Fish, in particular, are interesting models because of the diversity in the metabolic properties and complex relationships between metabolism and environmental challenges. In this review, we examine what is known about AMPK structure and function though the lens of comparative physiology, looking for opportunities to better understand how this vital energy sensor has evolved in animals.