Doublesex is essential for masculinization but not feminization in Lygus hesperus.

In most holometabolous insects, sex differentiation occurs via a hierarchical cascade of transcription factors, with doublesex (dsx) regulating genes that control sex-specific traits. Although less is known in hemimetabolous insects, early evidence suggests that substantial differences exist from more evolutionarily advanced insects. Here, we identified and characterized dsx in Lygus hesperus (western tarnished plant bug), a hemipteran pest of many agricultural crops in western North America. The full-length transcript for L. hesperus dsx (Lhdsx) and several variants encode proteins with conserved DNA binding and oligomerization domains. Transcript profiling revealed that Lhdsx is ubiquitously expressed, likely undergoes alternative pre-mRNA splicing, and, unlike several model insects, is sex-biased rather than sex-specific. Embryonic RNA interference (RNAi) of Lhdsx only impacted sex development in adult males, which lacked both internal reproductive organs and external genitalia. No discernible impacts on adult female development or reproductivity were observed. RNAi knockdown of Lhdsx in nymphs likewise only affected adult males, which lacked the characteristic dimorphic coloration but had dramatically elevated vitellogenin transcripts. Gene knockout of Lhdsx by CRISPR/Cas9 editing yielded only females in G0 and strongly biased heterozygous G1 offspring to females with the few surviving males showing severely impaired genital development. These results indicate that L. hesperus male development requires Lhdsx, whereas female development proceeds via a basal pathway that functions independently of dsx. A fundamental understanding of sex differentiation in L. hesperus could be important for future gene-based management strategies of this important agricultural pest.

ß-tubulin functions in spermatogenesis in Lygus hesperus Knight.

Lygus hesperus Knight is an important insect pest of crops across western North America, with field management heavily reliant on the use of chemical insecticides. Because of the evolution of resistance to these insecticides, effective and environmentally benign pest management strategies are needed. Traditional sterile insect technique (SIT) has been successfully employed to manage or eradicate some insect pests but involves introducing irradiated insects with random mutations into field populations. New genetically-driven SIT techniques are a safer alternative, causing fixed mutations that manipulate individual genes in target pests to produce sterile individuals for release. Here, we identified seven ß-tubulin coding genes from L. hesperus and show that Lhßtub2 is critical in male sperm production and fertility. Lhßtub2 is expressed primarily in the male testes and targeting of this gene by RNA interference or gene editing leads to male sterility.

Juvenile hormone regulates the photoperiodic plasticity of elytra coloration in the ladybird Harmonia axyridis.

Many animals, including insects, exhibit plasticity of body colour in response to environmental changes. Varied expression of carotenoids, major cuticle pigments, significantly contributes to body colour flexibility. However, the molecular mechanisms by which environmental cues regulate carotenoid expression remain largely unknown. In this study, we used the ladybird Harmonia axyridis as a model to investigate the photoperiodic-responsive plasticity of elytra coloration and its endocrine regulation. It was found that H. axyridis females under long-day conditions develop elytra that are much redder than those under short-day conditions, resulting from the differential accumulation of carotenoids. Exogenous hormone application and RNAi-mediated gene knockdown indicate that carotenoid deposition was directed through the juvenile hormone (JH) receptor-mediated canonical pathway. Moreover, we characterized an SR-BI/CD36 (SCRB) gene SCRB10 as the carotenoid transporter responding to JH signalling and regulating the elytra coloration plasticity. Taken together, we propose that JH signalling transcriptionally regulates the carotenoid transporter gene for the photoperiodic coloration plasticity of elytra in the beetles, which reveals a novel role of the endocrine system in the regulation of carotenoid-associated animal body coloration under environmental stimuli.

RNAi-Mediated Manipulation of Cuticle Coloration Genes in Lygus hesperus Knight (Hemiptera: Miridae).

Cuticle coloration in insects is a consequence of the accumulation of pigments in a species-specific pattern. Numerous genes are involved in regulating the underlying processes of melanization and sclerotization, and their manipulation can be used to create externally visible markers of successful gene editing. To clarify the roles for many of these genes and examine their suitability as phenotypic markers in Lygus hesperus Knight (western tarnished plant bug), transcriptomic data were screened for sequences exhibiting homology with the Drosophila melanogaster proteins. Complete open reading frames encoding putative homologs for six genes (aaNAT, black, ebony, pale, tan, and yellow) were identified, with two variants for black. Sequence and phylogenetic analyses supported preliminary annotations as cuticle pigmentation genes. In accord with observable difference in color patterning, expression varied for each gene by developmental stage, adult age, body part, and sex. Knockdown by injection of dsRNA for each gene produced varied effects in adults, ranging from the non-detectable (black 1, yellow), to moderate decreases (pale, tan) and increases (black 2, ebony) in darkness, to extreme melanization (aaNAT). Based solely on its expression profile and highly visible phenotype, aaNAT appears to be the best marker for tracking transgenic L. hesperus.

Mining Lygus hesperus (western tarnished plant bug) transcriptomic data for transient receptor potential channels: Expression profiling and functional characterization of a Painless homolog.

The transient receptor potential (TRP) family of cation channels are evolutionarily conserved proteins with critical roles in sensory physiology. Despite extensive studies in model species, knowledge of TRP channel functional diversity and physiological impact remains limited in many non-model insect species. To assess the TRP channel repertoire in a non-model agriculture pest species (Lygus hesperus), publicly available transcriptomic datasets were mined for potential homologs. Among the transcripts identified, 30 are predicted to encompass complete open reading frames that encode proteins representing each of the seven TRP channel subfamilies. Although no homologs were identified for the Pyrexia and Brivido channels, the TRP complement in L. hesperus exceeded the 13-16 channels reported in most insects. This diversity appears to be driven by a combination of alternative splicing, which impacted members of six subfamilies, and gene expansion of the TRPP subfamily. To validate the in silico data and provide more detailed analyses of L. hesperus TRP functionality, the putative Painless homolog was selected for more in depth analysis and its functional role in thermosensation examined in vitro. RT-PCR expression profiling revealed near ubiquitous expression of the Painless transcript throughout nymphal and adult development. Electrophysiological data generated using a Xenopus oocyte recombinant expression system indicated activation parameters for L. hesperus Painless homolog that are consistent with a role in noxious heat (40°-45 °C) thermosensation.

CRISPR-mediated knockout of cardinal and cinnabar eye pigmentation genes in the western tarnished plant bug.

The western tarnished plant bug, Lygus hesperus, is a key hemipteran pest of numerous agricultural, horticultural, and industrial crops in the western United States and Mexico. A lack of genetic tools in L. hesperus hinders progress in functional genomics and in developing innovative pest control methods such as gene drive. Here, using RNA interference (RNAi) against cardinal (LhCd), cinnabar (LhCn), and white (LhW), we showed that knockdown of LhW was lethal to developing embryos, while knockdown of LhCd or LhCn produced bright red eye phenotypes, in contrast to wild-type brown eyes. We further used CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) genome editing to generate germline knockouts of both LhCd (Card) and LhCn (Cinn), producing separate strains of L. hesperus characterized by mutant eye phenotypes. Although the cardinal knockout strain Card exhibited a gradual darkening of the eyes to brown typical of the wild-type line later in nymphal development, we observed bright red eyes throughout all life stages in the cinnabar knockout strain Cinn, making it a viable marker for tracking gene editing in L. hesperus. These results provide evidence that CRISPR/Cas9 gene editing functions in L. hesperus and that eye pigmentation genes are useful for tracking the successful genetic manipulation of this insect.

Nutritional and Physiological Regulation of Glassy-Winged Sharpshooter Oogenesis.

The glassy-winged sharpshooter (Homalodisca vitripennis (Germar); Hemiptera: Cicadellidae: Cicadellinae) is an invasive insect that transmits the plant pathogenic bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadacae). While adult glassy-winged sharpshooter must feed to produce eggs, the role of nutritional status on initiating oogenesis is poorly understood. To determine the effects of glassy-winged sharpshooter nutrition on nymphal development, oogenesis, and fecundity, glassy-winged sharpshooter were reared on cowpea, sunflower, sorghum, and a mixture of the three plant species. Adults emerging from cowpea, sunflower, or plant mixture treatments had shorter development times, attained larger size, and had greater estimated lipid reserves than females reared on sorghum. In choice tests, nymphs avoided sorghum and preferentially fed on cowpea and sunflower. Adult females provisioned with a single plant species during the nymphal stage were provided with either the same host plant species or a mixture of host plant species (cowpea, sunflower, sorghum) for a 9-wk oviposition period, with 37% of females initiating oogenesis. Ovipositing females had greater juvenile hormone and octopamine levels than reproductively inactive females, although topical application of the juvenile hormone analog Methoprene did not promote oogenesis. Across nymphal diets, reproductively active females produced more eggs when held on plant mixtures than on single plant species. In choice tests, adult females were observed most frequently on cowpea, although most eggs were deposited on sorghum, the host least preferred by nymphs. Results suggest that fecundity is largely determined by the quality of the adult diet, although the stimulus that initiates oogenesis does not appear to be related to nutrition.

Molecular and Functional Characterization of Pyrokinin-Like Peptides in the Western Tarnished Plant Bug Lygus hesperus (Hemiptera: Miridae).

The pyrokinin (PK) family of insect neuropeptides, characterized by C termini consisting of either WFGPRLamide (i.e., PK1) or FXPRLamide (i.e., PK2), are encoded on the capa and pk genes. Although implicated in diverse biological functions, characterization of PKs in hemipteran pests has been largely limited to genomic, transcriptomic, and/or peptidomic datasets. The Lygus hesperus (western tarnished plant bug) PK transcript encodes a prepropeptide predicted to yield three PK2 FXPRLamide-like peptides with C-terminal sequences characterized by FQPRSamide (LyghePKa), FAPRLamide (LyghePKb), and a non-amidated YSPRF. The transcript is expressed throughout L. hesperus development with greatest abundance in adult heads. PRXamide-like immunoreactivity, which recognizes both pk- and capa-derived peptides, is localized to cells in the cerebral ganglia, gnathal ganglia/suboesophageal ganglion, thoracic ganglia, and abdominal ganglia. Immunoreactivity in the abdominal ganglia is largely consistent with capa-derived peptide expression, whereas the atypical fourth pair of immunoreactive cells may reflect pk-based expression. In vitro activation of a PK receptor heterologously expressed in cultured insect cells was only observed in response to LyghePKb, while no effects were observed with LyghePKa. Similarly, in vivo pheromonotropic effects were only observed following LyghePKb injections. Comparison of PK2 prepropeptides from multiple hemipterans suggests mirid-specific diversification of the pk gene.

Diapause Termination and Postdiapause in Lygus hesperus (Heteroptera: Miridae).

The western tarnished plant bug, Lygus hesperus Knight, overwinters as a diapausing adult in response to short day lengths. Once environmental conditions are favorable, the bugs revert to an active reproductive state. To determine the impact on life-history traits of diverting resources toward diapause rather than oogenesis during early adulthood, diapausing and nondiapausing L. hesperus females were reared from the same cohorts. Body mass, ovarian maturation, ovipositional activity, and survivorship were monitored starting either at the time of release from diapause-inducing conditions or at adult eclosion for diapausers and nondiapausers, respectively. Females that had gone through 2 wk of diapause were larger and able to mobilize the resources necessary for oogenesis faster than nondiapausers, initiating oogenesis and ovipositing sooner and at a faster initial rate. However, lifetime egg production and average daily rates were similar for both groups. Postdiapausers lived longer than nondiapausers by an average of 19 d, which is five more than the 2-wk period when they were reproductively senescent. Overall, the results indicate that short-term diapause does not have a negative impact on life history. Furthermore, the extra endogenous resources stored during diapause may be able to enhance the alacrity with which the female can take advantage of improved environmental conditions and may prolong life by shielding the females against environmental stressors such as temperature extremes, oxidative agents, or food deficits.

Filling in the gaps: A reevaluation of the Lygus hesperus peptidome using an expanded de novo assembled transcriptome and molecular cloning.

Peptides are the largest and most diverse class of molecules modulating physiology and behavior. Previously, we predicted a peptidome for the western tarnished plant bug, Lygus hesperus, using transcriptomic data produced from whole individuals. A potential limitation of that analysis was the masking of underrepresented genes, in particular tissue-specific transcripts. Here, we reassessed the L. hesperus peptidome using a more comprehensive dataset comprised of the previous transcriptomic data as well as tissue-specific reads produced from heads and accessory glands. This augmented assembly significantly improves coverage depth providing confirmatory transcripts for essentially all of the previously identified families and new transcripts encoding a number of new peptide precursors corresponding to 14 peptide families. Several families not targeted in our initial study were identified in the expanded assembly, including agatoxin-like peptide, CNMamide, neuropeptide-like precursor 1, and periviscerokinin. To increase confidence in the in silico data, open reading frames of a subset of the newly identified transcripts were amplified using RT-PCR and sequence validated. Further PCR-based profiling of the putative L. hesperus agatoxin-like peptide precursor revealed evidence of alternative splicing with near ubiquitous expression across L. hesperus development, suggesting the peptide serves functional roles beyond that of a toxin. The peptides predicted here, in combination with those identified in our earlier study, expand the L. hesperus peptidome to 42 family members and provide an improved platform for initiating molecular and physiological investigations into peptidergic functionality in this non-model agricultural pest.

TRPA1 modulates noxious odor responses in Lygus hesperus.

Lygus hesperus isa key pest of many economically important crops across western North America. Central to many aspects of the lives of these insects is chemical signalling, with identified roles in host plant selection, aggregation and passive mate guarding. The development of novel monitoring and control approaches for this insect will rely on a sound understanding of how these cues are perceived and processed, and their impact on behavior. Towards this end, we investigated allyl isothiocyanate, cinnamaldehyde and citronellal, compounds that are noxious repellents to other insects. We found that L. hesperus avoided areas containing the three compounds and that exposure induced increases in movement velocity and duration in both nymphs and adults. This suggests these compounds may work as repellents. To better understand the underlying physiology of this response, RNA interference by dsRNA injection was used to inhibit the expression of two chemosensory-associated proteins, the odorant receptor co-receptor (Orco) and the transient receptor potential A (TRPA1) channel. While knockdown of Orco did not change the reaction of adult females to citronellal, TRPA1 silencing effectively eliminated the induced increase to movement, suggesting a chemoperceptory role in citronellal detection.

Juvenile Hormone III but Not 20-Hydroxyecdysone Regulates the Embryonic Diapause of Aedes albopictus.

Diapause is an alternative developmental trajectory allowing insects to enter dormancy and persist through predictable periods of seasonally unfavorable conditions. This crucial ecological adaptation defines the geographic and seasonal abundance of many insect pollinators, pests, and vectors. Understanding the hormonal changes by which insects coordinate the perception of external, diapause-inducing cues with the physiological mechanisms that lead to developmental arrest is a long-standing goal in biology. The hormonal regulation of diapause tends to vary by the life stage at which development arrest occurs; for example, diapause is typically regulated by ecdysteroids in larvae and pupae, and by juvenile hormones in adults. However, little is known about the hormonal control of embryonic diapause, particularly in Diptera. To address this fundamental gap, we directly measured 20-hydroxyecdysone (20HE) (via LC-MS/MS) and juvenile hormone III (JH3) (via GC-MS) in diapause and non-diapause eggs of the Asian tiger mosquito, Aedes albopictus. While 20HE abundance did not differ, diapause eggs had lower JH3 abundance than non-diapause eggs. These results are corroborated by transcriptional and manipulative evidence suggesting that reduced JH3 regulates diapause in this medically important mosquito.

Reproductive Development of Lygus hesperus (Hemiptera: Miridae) Adults Under Constant and Variable Temperatures.

As water for agriculture becomes less available in the semi-arid western United States, alternative irrigation strategies such as deficit irrigation may be necessary for continued crop production. Alternative irrigation practices in cotton (Gossypium spp. [Malvales: Valvaceae]) can result in episodic drought stress that alters temperature profiles within the crop canopy. These altered temperatures may influence populations of important pests such as Lygus hesperus Knight. Field studies often associate lower population densities of L. hesperus with limited irrigation. Recent studies of the thermal ecology of L. hesperus egg and nymphal development have demonstrated only subtle effects of the high, variable temperatures typical of moderate drought stress in cotton. However, influences of these conditions on L. hesperus adult reproductive development have not been studied. The reproductive development of L. hesperus adults was examined under constant (±0.2°C) and variable (±8°C) regimes at a low (15°C), moderate (22°C), and high (29°C) daily mean temperatures. No developmental differences were demonstrated between temperature regimes under moderate or high temperatures. At the low temperature, only the times to the occurrence of eggs, filled medial accessory glands, and filling seminal vesicles were shorter under variable regime, compared with the constant temperature. These results suggest that temporary, episodic increases in crop canopy temperatures caused by moderate drought stress are unlikely to impact L. hesperus population growth, and may only promote short-term displacement of adults into adjacent crops with preferable conditions.

Foraging Experiences Durably Modulate Honey Bees' Sucrose Responsiveness and Antennal Lobe Biogenic Amine Levels.

Foraging exposes organisms to rewarding and aversive events, providing a selective advantage for maximizing the former while minimizing the latter. Honey bees (Apis mellifera) associate environmental stimuli with appetitive or aversive experiences, forming preferences for scents, locations, and visual cues. Preference formation is influenced by inter-individual variation in sensitivity to rewarding and aversive stimuli, which can be modulated by pharmacological manipulation of biogenic amines. We propose that foraging experiences act on biogenic amine pathways to induce enduring changes to stimulus responsiveness. To simulate varied foraging conditions, freely-moving bees were housed in cages where feeders offered combinations of sucrose solution, floral scents, and aversive electric shock. Transient effects were excluded by providing bees with neutral conditions for three days prior to all subsequent assays. Sucrose responsiveness was reduced in bees that had foraged for scented rather than unscented sucrose under benign conditions. This was not the case under aversive foraging conditions, suggesting an adaptive tuning process which maximizes preference for high quality, non-aversive floral sites. Foraging conditions also influenced antennal lobe octopamine and serotonin, neuromodulators involved in stimulus responsiveness and foraging site evaluation. Our results suggest that individuals' foraging experiences durably modify neurochemistry and shape future foraging behaviour.

Development and Survival of Lygus hesperus (Hemiptera: Miridae) Nymphs Under Constant and Variable Temperatures.

Thermal environments of the arid western United States are often harsh compared with the ranges of temperatures favorable for development and survival of crop insect pests. In cotton [Gossypium spp. (Malvales: Malvaceae)], new irrigation practices such as deficit irrigation may impact populations of pest and beneficial arthropods by temporarily altering temperature profiles within the plant canopy. Most information regarding the temperature-dependent development and survival of an important cotton pest, the western tarnished plant bug (Lygus hesperus Knight), is derived from constant temperature studies. We examined the development and survival of L. hesperus nymphs under constant (±0.2°C) and variable (±8°C) temperature regimes at daily mean temperatures of 15, 22, and 29°C. Under the low temperature (15°C), stadium lengths and duration of the nymphal stage were shorter when temperatures were variable compared with a constant temperature. No differences in development times were observed between regimes at the medium temperature (22°C). Except for the first stadium, development times under the high variable temperature regime were longer compared with the high constant regime (29°C). Nymph survival was unaffected by temperature regime except at the lowest temperature, where daily thermal fluctuations substantially improved survival compared with the constant conditions. These results suggest that temporarily increased crop canopy temperatures caused by altered irrigation schemes are unlikely to substantially reduce the growth of L. hesperus populations. However, enhanced nymphal development and survival under low variable temperatures likely contribute to the survival of overwintering L. hesperus in the absence of acute, low-temperature mortality.

Individual differences in learning and biogenic amine levels influence the behavioural division between foraging honeybee scouts and recruits.

Animals must effectively balance the time they spend exploring the environment for new resources and exploiting them. One way that social animals accomplish this balance is by allocating these two tasks to different individuals. In honeybees, foraging is divided between scouts, which tend to explore the landscape for novel resources, and recruits, which tend to exploit these resources. Exploring the variation in cognitive and physiological mechanisms of foraging behaviour will provide a deeper understanding of how the division of labour is regulated in social insect societies. Here, we uncover how honeybee foraging behaviour may be shaped by predispositions in performance of latent inhibition (LI), which is a form of non-associative learning by which individuals learn to ignore familiar information. We compared LI between scouts and recruits, hypothesizing that differences in learning would correlate with differences in foraging behaviour. Scouts seek out and encounter many new odours while locating novel resources, while recruits continuously forage from the same resource, even as its quality degrades. We found that scouts show stronger LI than recruits, possibly reflecting their need to discriminate forage quality. We also found that scouts have significantly elevated tyramine compared to recruits. Furthermore, after associative odour training, recruits have significantly diminished octopamine in their brains compared to scouts. These results suggest that individual variation in learning behaviour shapes the phenotypic behavioural differences between different types of honeybee foragers. These differences in turn have important consequences for how honeybee colonies interact with their environment. Uncovering the proximate mechanisms that influence individual variation in foraging behaviour is crucial for understanding the ecological context in which societies evolve.

RNA interference-mediated knockdown of eye coloration genes in the western tarnished plant bug (Lygus hesperus Knight).

Insect eye coloration arises from the accumulation of various pigments. A number of genes that function in the biosynthesis (vermilion, cinnabar, and cardinal) and importation (karmoisin, white, scarlet, and brown) of these pigments, and their precursors, have been identified in diverse species and used as markers for transgenesis and gene editing. To examine their suitability as visible markers in Lygus hesperus Knight (western tarnished plant bug), transcriptomic data were screened for sequences exhibiting homology with the Drosophila melanogaster proteins. Complete open reading frames encoding putative homologs for all seven genes were identified. Bioinformatic-based sequence and phylogenetic analyses supported initial annotations as eye coloration genes. Consistent with their proposed role, each of the genes was expressed in adult heads as well as throughout nymphal and adult development. Adult eyes of those injected with double-stranded RNAs (dsRNAs) for karmoisin, vermilion, cinnabar, cardinal, and scarlet were characterized by a red band along the medial margin extending from the rostral terminus to the antenna. In contrast, eyes of insects injected with dsRNAs for both white and brown were a uniform light brown. White knockdown also produced cuticular and behavioral defects. Based on its expression profile and robust visible phenotype, cardinal would likely prove to be the most suitable marker for developing gene editing methods in Lygus species.

Molecular evolution of juvenile hormone esterase-like proteins in a socially exchanged fluid.

Socially exchanged fluids are a direct means by which an organism can influence conspecifics. It was recently shown that when workers of the carpenter ant Camponotus floridanus feed larval offspring via trophallaxis, they transfer Juvenile Hormone III (JH), a key developmental regulator, as well as paralogs of JH esterase (JHE), an enzyme that catalyzes the hydrolysis of JH. Here we combine proteomic, phylogenetic and selection analyses to investigate the evolution of this esterase subfamily. We show that Camponotus JHE-like proteins have undergone multiple duplications, experienced positive selection, and changed tissue localization to become abundantly and selectively present in trophallactic fluid. The Camponotus trophallactic esterases have maintained their catalytic triads and contain a number of positively-selected amino acid changes distributed throughout the protein, which possibly reflect an adaptation to the highly acidic trophallactic fluid of formicine ants. To determine whether these esterases might regulate larval development, we fed workers with a JHE-specific pharmacological inhibitor to introduce it into the trophallactic network. This inhibitor increased the likelihood of pupation of the larvae reared by these workers, similar to the influence of food supplementation with JH. Together, these findings suggest that JHE-like proteins have evolved a new role in the inter-individual regulation of larval development in the Camponotus genus.

Drosophila Kruppel homolog 1 represses lipolysis through interaction with dFOXO.

Transcriptional coordination is a vital process contributing to metabolic homeostasis. As one of the key nodes in the metabolic network, the forkhead transcription factor FOXO has been shown to interact with diverse transcription co-factors and integrate signals from multiple pathways to control metabolism, oxidative stress response, and cell cycle. Recently, insulin/FOXO signaling has been implicated in the regulation of insect development via the interaction with insect hormones, such as ecdysone and juvenile hormone. In this study, we identified an interaction between Drosophila FOXO (dFOXO) and the zinc finger transcription factor Kruppel homolog 1 (Kr-h1), one of the key players in juvenile hormone signaling. We found that Kr-h1 mutants show delayed larval development and altered lipid metabolism, in particular induced lipolysis upon starvation. Notably, Kr-h1 physically and genetically interacts with dFOXO in vitro and in vivo to regulate the transcriptional activation of insulin receptor (InR) and adipose lipase brummer (bmm). The transcriptional co-regulation by Kr-h1 and dFOXO may represent a broad mechanism by which Kruppel-like factors integrate with insulin signaling to maintain metabolic homeostasis and coordinate organism growth.