While most insect herbivores are selective feeders, a small proportion of them feed on a wide range of plants. This polyphagous habit requires overcoming a remarkable array of defenses, which often necessitates an adaptation period. Efforts for understanding the mechanisms involved mostly focus on the insect's phenotypic plasticity. Here, we hypothesized that the adaptation process might partially rely on transient associations with bacteria. To test this, we followed in a field-like experiment, the adaptation process of Bemisia tabaci, a generalist sap feeder, to pepper (a less-suitable host), after switching from watermelon (a suitable host). Amplicon sequencing of 16S rRNA transcripts from hundreds of dissected guts revealed the presence of active ``core'' and ``transient'' bacterial communities, dominated by the phyla Proteobacteria, Actinobacteria, and Firmicutes, and increasing differences between populations grown on watermelon and pepper. Insects grown on pepper for over two generations presented a significant increase in specific genera, mainly Mycobacterium, with a predicted enrichment in degradative pathways of xenobiotics and secondary metabolites. This result correlated with a significant increase in the insect's survival on pepper. Taken together, our findings suggest that gut-associated bacteria can provide an additional flexible metabolic ``tool-box'' to generalist sap feeders for facilitating a quick host switching process.

Like most insects, those that feed on both prey and plant materials harbor symbiotic bacteria in their body. Yet the involvement of bacteria in the feeding habits of these omnivorous consumers has yet to be investigated. In the present study, we took the first step toward testing the hypothesis that bacterial symbionts are involved in the feeding habits of the omnivorous bugMacrolophus pygmaeus. We (I) characterized the microbiome (the assembly of bacteria and fungi) ofM. pygmaeus, and (II) determined the identity and location of the most dominant bacteria species within the host body. We found thatM. pygmaeusmicrobiome is dominated by twoRickettsiaspecies,R. belliandR. limoniae. These bacteria are found in high numbers in the digestive system of the bug, each exhibiting a unique distribution pattern, and for the most part, do not share the same cells in the gut. These results strongly suggest that the host bug may gain some nutritional benefits by hosting the two dominant symbiotic bacteria in its gut. Bacterial symbionts in arthropods are common, vary in their effects, and can dramatically influence the outcome of biological control efforts.Macrolophus pygmaeus(Heteroptera: Miridae), a key component of biological control programs, is mainly predaceous but may also display phytophagy.M. pygmaeushosts symbioticWolbachia, which induce cytoplasmic incompatibility, and twoRickettsiaspecies,R. belliiandR. limoniae, which are found in all individuals tested. To test possible involvement of the twoRickettsiaspecies in the feeding habits ofM. pygmaeus, we first showed that the microbiome of the insect is dominated by these three symbionts, and later described the distribution pattern of the twoRickettsiaspecies in its digestive system. Although bothRickettsiaspecies were located in certain gut bacteriocyes, in caeca and in Malpighian tubules of both sexes, each species has a unique cellular occupancy pattern and specific distribution along digestive system compartments. Infrequently, both species were found in a cell. In females, bothRickettsiaspecies were detected in the germarium, the apical end of the ovarioles within the ovaries, but not in oocytes. Although the cause for theseRickettsiadistribution patterns is yet unknown, it is likely linked to host nutrition while feeding on prey or plants.

Fruit flies in the genus Bactrocera are among the most destructive insect pests of fruits and vegetables throughout the world. A number of studies have identified volatiles from fruit flies, but few reports have demonstrated behavioral effects or sensitivities of fly antennae to these compounds. We applied a recently developed method of automated headspace analysis using SPME (Solid Phase Microextraction) fibers and GC MS (gas chromatography mass spectrometry), termed SSGA, to reveal volatiles specific to each sex of B. zonata that are emitted in a diel periodicity. The volatiles released primarily at dusk were identified by GC MS and chemical syntheses as several spiroacetals, pyrazines, and ethyl esters. Solvent extraction of male rectal glands or airborne collections from each sex, followed by GC MS, showed that certain of the volatiles increase or decrease in quantity sex-specifically with age of the flies. Electroantennographic (EAG) analysis of dose-response indicates differences in sensitivities of male and female antenna to the various volatiles. Our study provides a comprehensive analysis of the volatile chemicals produced and released by B. zonata and their antennal responses. The possible pheromone and semiochemical roles of the various volatiles released by each sex and the difficulties of establishing behavioral functions are discussed.

Lipids have a key role in a variety of physiological functions in insects including energy, reproduction, growth and development. Whereas most of the required fatty acids can be synthesized endogenously, omega-3 and omega-6 polyunsaturated fatty acids (PUFA) are essential fatty acids that must be acquired through nutrition. Honey bees (Apis mellifera) obtain lipids from pollen, but different pollens vary in nutritional composition, including of PUFAs. Low floral diversity and abundance may expose bees to nutritional stress. We tested the effect of total lipids concentration and their omega-6:3 ratio on aspects of honey bee physiology: brood development, adult longevity and body fatty acids composition. All three parameters were affected by dietary lipid concentration and omega-6:3 ratio. Higher lipid concentration in diet increased brood production, and high omega-6:3 ratio increased mortality rate and decreased brood rearing. Fatty acid analysis of the bees showed that the amount of lipids and the omega-6:3 ratio in their body generally reflected the composition of the diet on which they fed. Consistent with previous findings of the importance of a balanced omega-6:3 ratio diet for learning performance, we found that such a balanced PUFA diet, with above threshold total lipid composition, is also necessary for maintaining proper colony development.

The metabolic adaptations by which phloem-feeding insects counteract plant defense compounds are poorly known. Two-component plant defenses, such as glucosinolates, consist of a glucosylated protoxin that is activated by a glycoside hydrolase upon plant damage. Phloem-feeding herbivores are not generally believed to be negatively impacted by two-component defenses due to their slender piercing-sucking mouthparts, which minimize plant damage. However, here we document that glucosinolates are indeed activated during feeding by the whiteflyBemisia tabaci. This phloem feeder was also found to detoxify the majority of the glucosinolates it ingests by the stereoselective addition of glucose moieties, which prevents hydrolytic activation of these defense compounds. Glucosylation of glucosinolates inB. tabaciwas accomplished via a transglucosidation mechanism, and two glycoside hydrolase family 13 (GH13) enzymes were shown to catalyze these reactions. This detoxification reaction was also found in a range of other phloem-feeding herbivores.

The glutathione S-transferase (GST) family plays an important role in the adaptation of herbivorous insects to new host plants and other environmental constrains. The family codes for enzymes that neutralize reactive oxygen species and phytotoxins through the conjugation of reduced glutathione. Here, we studied the molecular evolution of the GST family in Bemisia tabaci, a complex of >35 sibling species, differing in their geographic and host ranges. We tested if some enzymes evolved different functionality, by comparing their sequences in six species, representing five of the six major genetic clades in the complex. Comparisons of the non-synonymous to synonymous substitution ratios detected positive selection events in 11 codons of 5 cytosolic GSTs. Ten of them are located in the periphery of the GST dimer, suggesting a putative involvement in interactions with other proteins. Modeling the tertiary structure of orthologous enzymes, identified additional 19 mutations in 9 GSTs, likely affecting the enzymes' functionality. Most of the mutation eventswere found in the environmentally responsive classes Delta and Sigma, indicating a slightly different delta/sigma tool box in each species. At a broader genomic perspective, our analyses indicated a significant expansion of the Delta GST class in B. tabaci and a general association between the diet breadth of hemipteran species and their total number of GST genes. We raise the possibility that at least some of the identified changes improve the fitness of the B. tabaci species carrying them, leading to their better adaptation to specific environments.

Author summary Harmful insect populations can be eliminated for a lack of females if they are made to produce mostly male offspring. There are genes that occur naturally that make males produce mostly sons and, although we don't know exactly how they work, this appears to coincide with damage to the X-chromosome during the production of sperm. Recently, we showed in a mosquito species that such sex-biasing genes could also be constructed artificially from first principles. To better understand if this works in other species too, we designed and built male-biasing genes of two types in the fruit fly and determined what is needed to for a shift towards males. We show how different ways of cutting the X-chromosome DNA at different times with CRISPR, results in distinct outcomes and started to ask what cellular processes are involved in this. These models will help us to design such genes for the control of insect species that transmit disease or threaten crops. Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion-''X-shredding'' and ``X-poisoning''-and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Our results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when we directed Cas9 cleavage to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species.

Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are a superfamily of small phloem-feeding insects. They rely on their primary endosymbionts ``Candidatus Portiera aleyrodidarum'' to produce essential amino acids not present in their diet. Portiera has been codiverging with whiteflies since their origin and therefore reflects its host's evolutionary history. Like in most primary endosymbionts, the genome of Portiera stays stable across the Aleyrodidae superfamily after millions of years of codivergence. However, Portiera of the whitefly Bemisia tabaci has lost the ancestral genome order, reflecting a rare event in the endosymbiont evolution: the appearance of genome instability. To gain a better understanding of Portiera genome evolution, identify the time point in which genome instability appeared and contribute to the reconstruction of whitefly phylogeny, we developed a new phylogenetic framework. It targeted five Portiera genes and determined the presence of the DNA polymerase proofreading subunit (dnaQ) gene, previously associated with genome instability, and two alternative gene rearrangements. Our results indicated that Portiera gene sequences provide a robust tool for studying intergenera phylogenetic relationships in whiteflies. Using these new framework, we found that whitefly species from the Singhiella, Aleurolobus, and Bemisia genera form a monophyletic tribe, the Aleurolobini, and that their Portiera exhibit genome instability. This instability likely arose once in the common ancestor of the Aleurolobini tribe (at least 70 Ma), drawing a link between the appearance of genome instability in Portiera and the switch from multibacteriocyte to a single-bacteriocyte mode of inheritance in this tribe.

Rangelands are a dominant anthropogenic land use and a main driver of natural habitat loss worldwide. Land sharing, the integration of agricultural production and biodiversity conservation, may provide a platform for managing rangelands to fulfill multiple ecosystem services. However, livestock grazing can greatly affect biodiversity and little is known about its effects on providers of focal ecosystem services, such as pollinators. We investigated the effect of cattle grazing on bee communities and their foraging and nesting resources in Mediterranean rangelands. Specifically, we explored the effect of moderate cattle grazing on flowering plant abundance, species richness and composition, the diversity of nesting substrates, and consequently, the possible effects on wild bee and honey bee foraging activity, species diversity, and community composition. We conducted field research in the Mediterranean rangelands of Israel during the main bee activity season, in the spring of 2012 and 2013, comparing paired cattle-grazed and ungrazed areas. The availability of floral and nesting resources for bees was unaffected or positively affected by grazing. Similarly, wild bee abundance, species richness, and composition were not affected by grazing, but were instead shaped by spatiotemporal factors. Nor was honey bee activity level impaired by grazing. The foraging preferences of bees, as well as flower species composition and peak bloom differed between grazed and ungrazed areas. Therefore, in our studied rangelands, grazing had its main effect on the foraging choices of honey bees and wild bees, rather than on their abundance and diversity. Moreover, our results indicate the potentially important role of ungrazed patches in increasing nectar and pollen diversity and availability in rangelands for both honey bees and wild bees in the spring. Hence, maintaining a mosaic of moderately grazed and ungrazed patches is expected to provide the greatest benefits for wild bee conservation and honey bee activity in Mediterranean rangelands. Our findings support the notion of rangeland sharing by cattle and bees in Mediterranean ecosystems under moderate grazing intensities, mimicking the coexistence of honey bees, wild bees, and cattle in Mediterranean ecosystems on an evolutionary timescale.

Silicon is absorbed by plant roots as silicic acid. The acid moves with the transpiration stream to the shoot, and mineralizes as silica. In grasses, leaf epidermal cells called silica cells deposit silica in most of their volume using an unknown biological factor. Using bioinformatics tools, we identified a previously uncharacterized protein in Sorghum bicolor, which we named Siliplant1 (Slp1). Slp1 is a basic protein with seven repeat units rich in proline, lysine, and glutamic acid. We found Slp1 RNA in sorghum immature leaf and immature inflorescence. In leaves, transcription was highest just before the active silicification zone (ASZ). There, Slp1 was localized specifically to developing silica cells, packed inside vesicles and scattered throughout the cytoplasm or near the cell boundary. These vesicles fused with the membrane, releasing their content in the apoplastic space. A short peptide that is repeated five times in Slp1 precipitated silica in vitro at a biologically relevant silicic acid concentration. Transient overexpression of Slp1 in sorghum resulted in ectopic silica deposition in all leaf epidermal cell types. Our results show that Slp1 precipitates silica in sorghum silica cells.

Fruit flies in the genus Bactrocera are among the most destructive insect pests of fruits and vegetables throughout the world. A number of studies have identified volatiles from fruit flies, but few reports have demonstrated behavioral effects or sensitivities of fly antennae to these compounds. We applied a recently developed method of automated headspace analysis using SPME (Solid Phase Microextraction) fibers and GC–MS (gas chromatography mass spectrometry), termed SSGA, to reveal volatiles specific to each sex of B. zonata that are emitted in a diel periodicity. The volatiles released primarily at dusk were identified by GC–MS and chemical syntheses as several spiroacetals, pyrazines, and ethyl esters. Solvent extraction of male rectal glands or airborne collections from each sex, followed by GC–MS, showed that certain of the volatiles increase or decrease in quantity sex-specifically with age of the flies. Electroantennographic (EAG) analysis of dose-response indicates differences in sensitivities of male and female antenna to the various volatiles. Our study provides a comprehensive analysis of the volatile chemicals produced and released by B. zonata and their antennal responses. The possible pheromone and semiochemical roles of the various volatiles released by each sex and the difficulties of establishing behavioral functions are discussed. © 2019 Elsevier Ltd

Philornis downsi Dodge and Aitken (Diptera: Muscidae) is an avian parasitic fly that has invaded the Galapagos archipelago and exerts an onerous burden on populations of endemic land birds. As part of an ongoing effort to develop tools for the integrated management of this fly, our objective was to determine its long-and short-range responses to bacterial and fungal cues associated with adult P. downsi. We hypothesized that the bacterial and fungal communities would elicit attraction at distance through volatiles, and appetitive responses upon contact. Accordingly, we amplified bacteria from guts of adult field-caught flies and from bird feces, and yeasts from fermenting papaya juice (a known attractant of P. downsi), on selective growth media, and assayed the response of flies to these microbes or their exudates. In the field, we baited traps with bacteria or yeast and monitored adult fly attraction. In the laboratory, we used the proboscis extension response (PER) to determine the sensitivity of males and females to tarsal contact with bacteria or yeast. Long range trapping efforts yielded two female flies over 112 trap-nights (attracted by bacteria from bird feces and from the gut of adult flies). In the laboratory, tarsal contact with stimuli from gut bacteria elicited significantly more responses than did yeast stimuli. We discuss the significance of these findings in context with other studies in the field and identify targets for future work. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.

Plants of Eruca sativa Mill. (Brassicaceae) from desert and Mediterranean populations in Israel differ in flower color and size. In the desert habitat, the population has higher abundance of flowers with cream color and longer petals, whereas in the Mediterranean habitat, the population has higher abundance of flowers with yellow and shorter petals. Choice experiments with honey bee foragers (Apis mellifera Linn., Apidae, Hymenoptera), the main pollinator in the natural habitat in Israel, confirmed that they are more attracted to the yellow flower morph than to the cream one. A proboscis extension response test indicated that honey bees are able to discriminate between flower scents of the desert and Mediterranean populations. Considering the advantage of plants of the yellow morph in attracting pollinators, we further tested in a common garden experiment whether these possess higher fitness than plants of the desert population. Indeed, a significant association was found between flower color and fruit set, and seed mass. In general, our results provide evidence for ecotypic differentiation between populations imposed by pollinators. The advantage of the yellow color morph in attracting pollinators may explain its dominance among plants of the Mediterranean population. We discuss why the cream color morph may be dominant in the desert habitat, considering the possibility of different pollinators, tradeoffs between traits, or pleiotropy. © 2019 The Author(s).

In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest Ceratitis capitata (Mediterranean fruit fly, or Medfly), we identified a Y-linked gene, Maleness-on-the-Y (MoY), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of MoY in XY embryos causes feminization, whereas overexpression of MoY in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Ychromosome can be transmitted by XY females. MoY is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests. Copyright © 2019 The Authors.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit crop production worldwide. Based on genetic and phenotypic properties, A. citrulli strains are divided into two major groups: group I strains have been generally isolated from melon and other non-watermelon cucurbits, while group II strains are closely associated with watermelon. In a previous study, we reported the genome of the group I model strain, M6. At that time, the M6 genome was sequenced by MiSeq Illumina technology, with reads assembled into 139 contigs. Here, we report the assembly of the M6 genome following sequencing with PacBio technology. This approach not only allowed full assembly of the M6 genome, but it also revealed the occurrence of a ∼53 kb plasmid. The M6 plasmid, named pACM6, was further confirmed by plasmid extraction, Southern-blot analysis of restricted fragments and obtention of M6-derivative cured strains. pACM6 occurs at low copy numbers (average of ∼4.1 ± 1.3 chromosome equivalents) in A. citrulli M6 and contains 63 open reading frames (ORFs), most of which (55.6%) encoding hypothetical proteins. The plasmid contains several genes encoding type IV secretion components, and typical plasmid-borne genes involved in plasmid maintenance, replication and transfer. The plasmid also carries an operon encoding homologs of a Fic-VbhA toxin-antitoxin (TA) module. Transcriptome data from A. citrulli M6 revealed that, under the tested conditions, the genes encoding the components of this TA system are among the highest expressed genes in pACM6. Whether this TA module plays a role in pACM6 maintenance is still to be determined. Leaf infiltration and seed transmission assays revealed that, under tested conditions, the loss of pACM6 did not affect the virulence of A. citrulli M6. We also show that pACM6 or similar plasmids are present in several group I strains, but absent in all tested group II strains of A. citrulli. Copyright © 2019 Yang, Santos Garcia, Pérez Montaño, da Silva, Zhao, Jiménez Guerrero, Rosenberg, Chen, Plaschkes, Morin, Walcott and Burdman.

Chitin is a linear polysaccharide of N-acetylglucosamine, which is highly abundant in nature and mainly produced by marine crustaceans. Chitosan is obtained by hydrolytic deacetylation. Both polysaccharides are renewable resources, simply and cost-effectively extracted from waste material of fish industry, mainly crab and shrimp shells. Research over the past five decades has revealed that chitosan, in particular, possesses unique and useful characteristics such as chemical versatility, polyelectrolyte properties, gel- and film-forming ability, high adsorption capacity, antimicrobial and antioxidative properties, low toxicity, and biocompatibility and biodegradability features. A plethora of chemical chitosan derivatives have been synthesized yielding improved materials with suggested or effective applications in water treatment, biosensor engineering, agriculture, food processing and storage, textile additives, cosmetics fabrication, and in veterinary and human medicine. The number of studies in this research field has exploded particularly during the last two decades. Here, we review recent advances in utilizing chitosan and chitosan derivatives in different technical, agricultural, and biomedical fields.

Foraging decisions of social animals occur in the context of social groups, and thus may be subject to considerations of not only an individual's nutritional state and nutrient input, but those of the social group in which they live. In eusocial insects, which live in colonies containing workers that forage for food that is mostly consumed by others, foraging decisions that reflect colony needs may also be considered at both the colony and individual level. If colony energy balance is perturbed, is the counteracting response occurring on the group level (a change in division of labor) or on the individual level (a change in individual foraging choices)? To address this, colony and individual level foraging behaviors were observed in two species of eusocial bees: the highly social honey bee Apis mellifera and the primitively eusocial bumble bee Bombus terrestris. After manipulations of protein (P) and carbohydrate (C) stores in colonies of both species, there were changes in multiple different behavioral responses including colony level (number of foragers, allocation to nectar and pollen foraging, nutrient mass foraged) and individual level (P and C concentration preference and loading during foraging). These results suggest both honey bee and bumble bee colonies balance nutrient needs through a combination of both colony level shifts in foraging allocation, as well as slight modulation of individual nutrient preferences. This study also uncovered colony level differences between the two bee species; honey bees balanced P intake while bumble bees balanced C intake. These patterns may reflect differences in life history traits such as perenniality and hoarding, traits that are developed in more highly social species. Overall, this study highlights the importance of considering both group and individual level behavioral responses in foraging decisions in social animals.