RNA interference (RNAi) is a form of gene silencing triggered by double-stranded RNA (dsRNA) that operates in all eukaryotic cells. RNAi has been widely investigated in insects to determine the underlying molecular mechanism, to investigate its role in systemic antiviral defense, and to develop strategies for pest control. When insect cells are infected by viruses, viral dsRNA signatures trigger a local RNAi response to block viral replication and generate virus-derived DNA that confers systemic immunity. RNAi-based insect pest control involves the application of exogenous dsRNA targeting genes essential for insect development or survival, but the efficacy of this approach has limited potency in many pests through a combination of rapid dsRNA degradation, inefficient dsRNA uptake/processing, and ineffective RNAi machinery. This could be addressed by dsRNA screening and evaluation, focusing on dsRNA design and off-target management, as well as dsRNA production and delivery. This review summarizes recent progress to determine the role of RNAi in antiviral defense and as a pest control strategy in insects, addressing gaps between our fundamental understanding of the RNAi mechanism and the exploitation of RNAi-based pest control strategies.

Insecticide resistance in Panonychus citri is a major obstacle to mite control in citrus orchards. Pyrethroid insecticides are continually used to control mites in China, although resistance to pyrethroids has evolved in some populations. Here, the resistance to the pyrethroid fenpropathrin was investigated and 7 out of 8 field-collected populations of P. citri exhibited a high level of resistance, ranging from 171-fold to 15 391-fold higher than the susceptible (SS) comparison strain. Three voltage-gated sodium channel (VGSC) mutations were identified in the tested populations: L1031V, F1747L, and F1751I. Amplicon sequencing was used to evaluate the frequency of these mutations in the 19 field populations. L1031V and F1751I were present in all populations at frequencies of 11.6%–82.1% and 0.5%–31.8%, respectively, whereas the F1747L mutation was only present in 12 populations from Chongqing, Sichuan, Guangxi, and Yunnan provinces. Introduction of these mutations singly or in combination into transgenic flies significantly increased their resistance to fenpropathrin and these flies also exhibited reduced mortality after exposure to the pyrethroids permethrin and β-cypermethrin. Panonychus citri VGSC homology modeling and ligand docking indicate that F1747 and F1751 form direct binding contacts with pyrethroids, which are lost with mutation, whereas L1031 mutation may diminish pyrethroid effects through an allosteric mechanism. Overall, the results provide molecular markers for monitoring pest resistance to pyrethroids and offer new insights into the basis of pyrethroid actions on sodium channels.

The rice stem borer (RSB), Chilo suppressalis, a notorious rice pest in China, has evolved a high resistance level to commonly used insecticides. Tetraniliprole, a new anthranilic diamide insecticide, effectively controls multiple pests, including RSB. However, the potential resistance risk of RSB to tetraniliprole is still unknown. In this study, the tetraniliprole-selection (Tet-R) strain was obtained through 10 continuous generations of selection with tetraniliprole 30% lethal concentration (LC₃₀). The realized heritability (h²) of the Tet-R strain was 0.387, indicating that resistance of RSB to tetraniliprole developed rapidly under the continuous selection of tetraniliprole. The Tet-R strain had a high fitness cost (relative fitness = 0.53). We established the susceptibility baseline of RSB to tetraniliprole (lethal concentration at LC₅₀ = 0.727 mg/L) and investigated the resistance level of 6 field populations to tetraniliprole. All tested strains that had resistance to chlorantraniliprole exhibited moderate- to high-level resistance to tetraniliprole (resistance ratio = 27.7−806.8). Detection of ryanodine receptor (RyR) mutations showed that the Y4667C, Y4667D, I4758M, and Y4891F mutations were present in tested RSB field populations. RyR mutations were responsible for the cross-resistance between tetraniliprole and chlorantraniliprole. Further, the clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-mediated genome-modified flies were used to study the contribution of RyR mutations to tetraniliprole resistance. The order of contribution of a single RyR mutation to tetraniliprole resistance was Y4667D > G4915E > Y4667C ≈ I4758M > Y4891F. In addition, the I4758M and Y4667C double mutations conferred higher tetraniliprole resistance than single Y4667C mutations. These results can guide resistance management practices for diamides in RSB and other arthropods.

Insects are the host or vector of diverse viruses including those that infect vertebrates, plants, and fungi. Insect viruses reside inside their insect hosts and are vertically transmitted from parent to offspring. The insect virus–host relationship is intricate, as these viruses can impact various aspects of insect biology, such as development, reproduction, sex ratios, and immunity. Arthropod-borne viruses (arboviruses) that cause substantial global health or agricultural problems can also be vertically transmitted to insect vector progeny. Multiple infections with insect viruses and arboviruses are common in nature. Such coinfections involve complex interactions, including synergism, dependence, and antagonism. Recent studies have shed light on the influence of insect viruses on the competence of insect vectors for arboviruses. In this review, we focus on the biological effects of insect viruses on the transmission of arboviruses by insects. We also discuss the potential mechanisms by which insect viruses affect the ability of hosts to transmit arboviruses, as well as potential strategies for disease control through manipulation of insect viruses. Analyses of the interactions among insect vectors, insect viruses and arboviruses will provide new opportunities for development of innovative strategies to control arbovirus transmission.

Body color polyphenism is common in Diaphorina citri. Previous studies compared physiological characteristics in D. citri, but the ecological and biological significance of its body color polyphenism remains poorly understood. We studied the ecological and molecular effects of stressors related to body color in D. citri. Crowding or low temperature induced a high proportion of gray morphs, which had smaller bodies, lower body weight, and greater susceptibility to the insecticide dinotefuran. We performed transcriptomic and metabolomics analysiis of 2 color morphs in D. citri. Gene expression dynamics revealed that the differentially expressed genes were predominantly involved in energy metabolism, including fatty acid metabolism, amino acid metabolism, and carbohydrate metabolism. Among these genes, plexin, glycosidase, phospholipase, take out, trypsin, and triacylglycerol lipase were differentially expressed in 2 color morphs, and 6 hsps (3 hsp70, hsp83, hsp90, hsp68) were upregulated in gray morphs. The metabolome data showed that blue morphs exhibited a higher abundance of fatty acid and amino acid, whereas the content of carbohydrates was elevated in gray morphs. This study partly explains the body color polyphenism of D. citri and provides insights into the molecular changes of stress response of D. citri.

Microbial communities, derived from food, ambient, and inner, can affect host ecological adaption and evolution. Comparing with most phytophagous arthropods, predators may have more opportunities to develop specific microbiota depending on the level of prey specialization. To explore how diet sources affect host microbial communities and vary across predator species, we considered 3 types of predators from Phytoseiidae (Acari: Mesostigmata): polyphagous (Amblyseius orientalis Ehara, Neoseiulus barkeri Hughes, and Amblyseius swirskii Athias-Henrio), oligophagous (Neoseiulus californicus McGregor), and monophagous (Phytoseiulus persimilis Athias-Henriot) predatory mites. The polyphagous species were fed on 2 types of diets, natural prey and alternative prey. By using 16S rRNA sequencing, we found that diet was the main source of microbiota in predatory mites, while there was no clear pattern affected by prey specialization. Among 3 polyphagous predators, host species had a larger impact than prey on microbial composition. Unlike A. orientalis or N. barkeri which showed consistency in their microbiota, prey switching significantly affected β-diversity of bacterial composition in A. swirskii, with 56% of the microbial alteration. In short, our results confirmed the substantial influence of diet on host microbial construction in predatory species, and highlighted species differences in shaping the microbiota which are not necessarily related to prey specialization.

The ability of mosquitoes to transmit a pathogen is affected, among other factors, by their survival rate, which is partly modulated by their microbiota. Mosquito microbiota is acquired during the larval phase and modified during their development and adult feeding behavior, being highly dependent on environmental factors. Pharmaceutical residues including antibiotics are widespread pollutants potentially being present in mosquito breeding waters likely affecting their microbiota. Here, we used Culex pipiens mosquitoes to assess the impact of antibiotic exposure during the larval and adult stages on the survival rate of adult mosquitoes. Wild-collected larvae were randomly assigned to two treatments: larvae maintained in water supplemented with antibiotics and control larvae. Emerged adults were subsequently assigned to each of two treatments, fed with sugar solution with antibiotics and fed only with sugar solution (controls). Larval exposure to antibiotics significantly increased the survival rate of adult females that received a control diet. In addition, the effect of adult exposure to antibiotics on the survival rate of both male and female mosquitoes depended on the number of days that larvae fed ad libitum in the laboratory before emergence. In particular, shorter larval ad libitum feeding periods reduced the survival rate of antibiotic-treated adult mosquitoes compared with those that emerged after a longer larval feeding period. These differences were not found in control adult mosquitoes. Our results extend the current understanding of the impact of antibiotic exposure of mosquitoes on a key component of vectorial capacity, that is the vector survival rate.

Neuropeptides are crucial in regulation of a rich variety of developmental, physiological, and behavioral functions throughout the life cycle of insects. Using an integrated approach of multiomics, we identified neuropeptide precursors in the greater wax moth Galleria mellonella, which is a harmful pest of honeybee hives with a worldwide distribution. Here, a total of 63 and 67 neuropeptide precursors were predicted and annotated in the G. mellonella genome and transcriptome, in which 40 neuropeptide precursors were confirmed in the G. mellonella peptidome. Interestingly, we identified 12 neuropeptide precursor genes present in G. mellonella but absent in honeybees, which may be potential novel pesticide target sites. Honeybee hives were contaminated with heavy metals such as lead, enabling its bioaccumulation in G. mellonella bodies through the food chain, we performed transcriptome sequencing to analyze the effects of Pb stress on the mRNA expression level of G. mellonella neuropeptide precursors. After treatment by Pb, the expression of neuropeptide F1 was found to be significantly downregulated, implying that this neuropeptide might be associated with responding to the heavy metal stress in G. mellonella. This study comprehensively identified neuropeptide precursors in G. mellonella, and discussed the effects of heavy metals on insect neuropeptides, with the example of G. mellonella. The results are valuable for future elucidation of how neuropeptides regulate physiological functions in G. mellonella and contribute to our understanding of the insect's environmental plasticity and identify potential new biomarkers to assess heavy metal toxicity in insects.

Insect flight is a complex trait involved in different behaviors, from the search for sexual partners, food, or breeding sites. Many studies have postulated the adaptive advantages of certain morphological traits in relation to increased flight capacity, such as low values of wing loading or high values of wing:thorax ratio and wing–aspect ratio. However, few studies have evaluated the relationship between variables related to flight and morphological traits in Drosophila. This work aimed to study morphological traits in males and females of two pairs of sibling species: Drosophila buzzatii Patterson and Wheeler-Drosophila koeferae Fontdevila and Wasserman, and Drosophila melanogaster Meigen-Drosophila simulans Sturtevant, and to analyze its relationship with flight. We detected the highest proportion of flight time in D. koepferae and D. simulans compared to D. buzzatii and D. melanogaster, respectively. Our results also revealed sexual dimorphism, with males exhibiting a higher proportion of flight time than females. Surprisingly, we did not find a general pattern to explain the relationship between morphology and the proportion of flight time because associations varied depending upon the analyses (considering all groups together or each sex-species combination separately). Moreover, these associations explained a low percentage of variation, suggesting that other nonmorphological components related to flight, such as physiological variables, should be taken into account. This work allowed us to show the variability and complexity of an aspect of flight, suggesting that the adaptive role of the morphological traits studied might have been overestimated.

Apoptosis is an important process for organism development that functions to eliminate cell damage, maintain homeostasis, and remove obsolete tissues during morphogenesis. In mammals, apoptosis is accompanied by the release of cytochrome C (Cyt-c) from mitochondria to the cytoplasm. However, whether this process is conserved in the fruit fly, Drosophila melanogaster, remains controversial. In this study, we discovered that during the degradation of Drosophila salivary gland, the transcription of mitochondria apoptosis factors (MAPFs), Cyt-c, and death-associated APAF1-related killer (Dark) encoding genes are all upregulated antecedent to initiator and effector caspases encoding genes. The proteins Cyt-c and the active caspase 3 appear gradually in the cytoplasm during salivary gland degradation. Meanwhile, the Cyt-c protein colocates with mito-GFP, the marker indicating cytoplasmic mitochondria, and the change in mitochondrial membrane potential coincides with the appearance of Cyt-c in the cytoplasm. Moreover, impeding or promoting 20E-induced transcription factor E93 suppresses or enhances the staining of Cyt-c and the active caspase 3 in the cytoplasm of salivary gland, and accordingly decreases or increases the mitochondrial membrane potential, respectively. Our research provides evidence that cytoplasmic Cyt-c appears before apoptosis during Drosophila salivary gland degradation, shedding light on partial conserved mechanism in apoptosis between insects and mammals.

Unlike European species, the potential of Nearctic syrphids as biological control agents is still poorly studied. However, the American hoverfly, Eupeodes americanus (Wiedemann), has recently demonstrated promising traits as a biocontrol agent, notably against the foxglove aphid, Aulacorthum solani Kaltenbach, on pepper. The present study aims to extend our knowledge of the American hoverfly by evaluating its potential as a biocontrol agent in a banker plant system against the melon aphid, Aphis gossypii Glover, in a greenhouse cucumber crop. The preimaginal development and voracity of E. americanus were compared when preying upon the focal prey/pest (A. gossypii) or the banker prey (bird cherry-oat aphid, Rhopalosiphum padi L.) by daily observations of larvae from egg to adult. Preimaginal development time, survival rate, and occurrence of deformation were similar on both prey species. The weight of third instar and pupae, however, was higher for larvae that fed on the banker prey. The ad libitum voracity of the syrphid larvae was generally very high and did not significantly differ between prey species, except for the third-instar larvae which consumed more focal prey. Results suggest that a banker plant system involving the bird cherry-oat aphid may be a promising tactic for utilizing E. americanus for melon aphid biocontrol.

C-type lectins (CTLs) are a family of proteins that contain 1 or more carbohydrate-recognition domains (CRDs) and bind to a broad repertoire of ligands in the presence of calcium ions. CTLs play important roles in innate immune defenses against microorganisms by acting as pattern-recognition receptors (PRRs) for invading pathogens, such as bacteria, viruses, and parasites. After binding to pathogen-associated ligands, CTLs mediate immune responses, such as agglutination, phagocytosis, and the activation of phenol oxidase progenitors, thereby clearing pathogens. CTLs are an evolutionarily conserved family found in almost all vertebrates and invertebrates. Medical arthropods can acquire and transmit a range pathogens through various approaches, such as bloodsucking, lancing, and parasitism, thus infecting humans and animals with related diseases, some of which can be life-threatening. Recent studies have shown that lectins are important components of the arthropod immune system and are essential for the immune responses of arthropods to arthropod-borne pathogens. This article reviews the current understanding of the structure, function, and signaling pathways involved in CTLs derived from important medical arthropods.

Monochamus alternatus is the primary carrier of pine wood nematodes, which pose a serious threat to Pinus spp. in many countries. Newly emerging M. alternatus adults feed on heathy host pines, while matured adults transfer to stressed host pines for mating and oviposition. Several odorant-binding proteins (OBPs) of M. alternatus have been proved to aid in the complex process of host location. To clarify the corresponding relations between OBPs and pine volatiles, more OBPs need to be studied. In this research, MaltOBP19 showed a specific expression in the antennae and mouthparts of M. alternatus, and it was marked in 4 types of antenna sensilla by immunolocalization. Fluorescence binding assays demonstrated the high binding affinity of MaltOBP19 with camphene and myrcene in vitro. In Y-tube olfactory experiments, M. alternatus adults were attracted by camphene and RNAi of OBP19 via microinjection significantly decreased their attraction index. Myrcene induced phobotaxis, but RNAi had no significant effect on this behavior. Further, we found that ingesting dsOBP19 produced by a bacteria-expressed system with a newly constructed vector could lead to the knockdown of MaltOBP19. These results suggest that MaltOBP19 may play a role in the process of host conversion via the recognition of camphene, which has been identified to be strongly released in stressed host pines. In addition, it is proved that knockdown of OBP can be achieved by oral administration of bacteria-expressed double-stranded RNA in M. alternatus adults, providing a new perspective in the control of M. alternatus.

The aboveground oligolectic bee, Heriades truncorum, is a particularly good model for studying the impact of pesticides on sexual communication, since some aspects of its mating behavior have previously been described. We have tested (1) the interference of the pesticide flupyradifurone on male precopulatory behavior and male mating partner preferences, (2) the way that the pesticide interferes in male quality assessment by the female, and (3) the effects of the pesticide on the chemical compounds in the female cuticle. We exposed bees of both sexes to a sublethal concentration of flupyradifurone. Various behaviors were registered in a mating arena with two females (one unexposed and one exposed) and one male (either unexposed or exposed). Unexposed males were quicker to attempt to mate. Treatment also impacted precopulatory behavior and male quality assessment by females. Males approached unexposed females more quickly than insecticide-exposed ones. Females exposed to insecticide produced lower amounts of some cuticular hydrocarbons (sex pheromone candidates) and appeared less choosy than unexposed females. Our findings suggest that insecticide exposure affects sexual communication, playing a role both in male preference and in male quality assessment by the female.

Black soldier fly larvae (Hermetia illucens) receive growing interest as a potential alternative animal feed source. These insects may be exposed to insecticide residues in the rearing substrate. This study aimed to investigate the effects of six different pyrethroid and organophosphate insecticides on this insect species’ performance. The toxicity of two “model” substances for each of these classes (cypermethrin; pirimiphos-methyl) was quantified, with and without the synergist piperonyl butoxide (PBO). Critical effect doses corresponding to 10% yield (CED10) for cypermethrin (0.4 mg/kg) and pirimiphos-methyl (4.8 mg/kg) were determined. The addition of PBO to cypermethrin enhanced its relative potency with a factor 2.6. These data were compared against the relative toxicity of two analogue substances in each class (permethrin, deltamethrin; chlorpyrifos-methyl, malathion). Results suggest that exposure to concentrations complying with legal limits can cause significant reductions in yield. Exposure to multiple substances at lower concentrations resulted in negative additive and synergistic effects. Of the tested substances, deltamethrin was most toxic, causing 94% yield at 0.5 mg/kg. Analytical results suggested that transfer of tested substances to the larval biomass was substance- and concentration-specific, but appeared to be correlated to reduced yields and the presence of PBO. Transfer of organophosphates was overall low (<2%), but ranged from 8% to 75% for pyrethroids. Due to very low limits in insect biomass (∼0.01 mg/kg), high transfer may result in noncompliance. It is recommended that rearing companies implement lower contractual thresholds, and that policymakers consider adjusting legally allowed maximum residue levels in insect feed.

Diaphorina citri is a global citrus pest. As a vector insect, it can transmit the causative agents of citrus huanglongbing, causing irreversible losses to the citrus industry. The acquisition of genomic information can provide a molecular genetic basis for effective control of D. citri. Here, the DNBSEQ™, Oxford Nanopore Technologies, and Hi-C technologies are applied to generate a high-quality chromosome-level genome of D. citri. The genome size of D. citri was 523.78 Mb with a scaffold N50 of 47.05 Mb distributed on 13 chromosomes. A total of 250.64 Mb (47.85%) repeat sequences and 24 048 protein-coding genes were predicted. Genome resequencing of female and male individuals indicated that the sex chromosome system of D. citri is XO. Phylogenetic analysis demonstrated that D. citri and Pachypsylla venusta, which separated from their most recent common ancestor about 336.62 million years ago, were the most closely related. Additionally, we identified genes potentially involved in detoxification metabolism, pathogen transmission, and honeydew secretion for further investigation. The high-quality genome provides an important reference for developing effective management strategies of D. citri.

Termite queens and kings live longer than nonreproductive workers. Several molecular mechanisms contributing to their long lifespan have been investigated; however, the underlying biochemical explanation remains unclear. Coenzyme Q (CoQ), a component of the mitochondrial electron transport chain, plays an essential role in the lipophilic antioxidant defense system. Its beneficial effects on health and longevity have been well studied in several organisms. Herein, we demonstrated that long-lived termite queens have significantly higher levels of the lipophilic antioxidant CoQ₁₀ than workers. Liquid chromatography analysis revealed that the levels of the reduced form of CoQ₁₀ were 4 fold higher in the queen's body than in the worker's body. In addition, queens showed 7 fold higher levels of vitamin E, which plays a role in antilipid peroxidation along with CoQ, than workers. Furthermore, the oral administration of CoQ₁₀ to termites increased the CoQ₁₀ redox state in the body and their survival rate under oxidative stress. These findings suggest that CoQ₁₀ acts as an efficient lipophilic antioxidant along with vitamin E in long-lived termite queens. This study provides essential biochemical and evolutionary insights into the relationship between CoQ₁₀ concentrations and termite lifespan extension.

The Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae), a globally distributed storage pest, relies on odors that are emitted from stored foods to select a suitable substrate for oviposition. However, the molecular mechanism underlying the chemical communication between P. interpunctella and its host remains elusive. In this study, 130 chemosensory genes were identified from the transcriptomes of 7 P. interpunctella tissues, and the quantitative expression levels of all 56 P. interpunctella odorant receptor genes (PintORs) were validated using real-time quantitative polymerase chain reaction. The functional characteristics of 5 PintORs with female antennae-biased expression were investigated using 2-electrode voltage clamp recordings in Xenopus laevis oocytes. PintOR23 was found to be specifically tuned to acetophenone. Acetophenone could elicit a significant electrophysiological response and only attracted mated females when compared with males and virgin females. In addition, molecular docking predicted that the hydrogen bonding sites, TRP-335 and ALA-167, might play key roles in the binding of PintOR23 to acetophenone. Our study provides valuable insights into the olfactory mechanism of oviposition substrate detection and localization in P. interpunctella and points toward the possibility of developing eco-friendly odorant agents to control pests of stored products.

In moths, pheromone receptors (PRs) are crucial for intraspecific sexual communication between males and females. Moth PRs are considered as an ideal model for studying the evolution of insect PRs, and a large number of PRs have been identified and functionally characterized in different moth species. Moth PRs were initially thought to fall into a single monophyletic clade in the odorant receptor (OR) family, but recent studies have shown that ORs in another lineage also bind type-I sex pheromones, which indicates that type-I PRs have multiple independent origins in the Lepidoptera. In this study, we investigated whether ORs of the pest moth Spodoptera frugiperda belonging to clades closely related to this novel PR lineage may also have the capacity to bind type-I pheromones and serve as male PRs. Among the 7 ORs tested, only 1 (SfruOR23) exhibited a male-biased expression pattern. Importantly, in vitro functional characterization showed that SfruOR23 could bind several type-I sex pheromone compounds with Z-9-tetradecenal (Z9-14:Ald), a minor component found in female sex pheromone glands, as the optimal ligand. In addition, SfruOR23 also showed weak responses to plant volatile organic compounds. Altogether, we characterized an S. frugiperda PR positioned in a lineage closely related to the novel PR clade, indicating that the type-I PR lineage can be extended in moths.

Due to rapidly developed resistance, pest management relies less on pyrethroids to control economically damaging infestations of the tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois) in cotton fields of Mississippi. Yet, pyrethroid resistance remains prevalent in TPB populations. This study assessed the resistance levels in adult TPB to six common pyrethroids and acephate. Resistant TBPs were collected from wild host plants in late October after harvest in the Mississippi Delta region of the United States. Based on LC₅₀ values, the field-resistant TPBs displayed higher resistance to permethrin, esfenvalerate, and bifenthrin (approximately 30 fold) and moderate resistance to λ-cyhalothrin, β-cyfluthrin, ζ-cypermethrin, and acephate (approximately 15 fold). Further investigations showed that the inhibitors of three detoxification enzyme, triphenyl phosphate (TPP), diethyl maleate (DEM), and piperonyl butoxide (PBO) had synergistic effects on permethrin, λ-cyhalothrin, and bifenthrin in resistant TPBs. Furthermore, elevated esterase, GST, and P450 activities were significantly expressed in field-resistant TPBs. Additionally, GST and esterase were reduced after 48 h exposure to certain pyrethroids at LC₅₀ dose. The synergistic and biochemical assays consistently indicated that P450 and esterase were involved in pyrethroid detoxification in TPBs. This study provides valuable information for the continued use of pyrethroids and acephate in controlling TPBs in cotton fields in the Mississippi Delta region of the United States.

Bumblebees are important pollinators in agricultural ecosystems, but their abundance is declining globally. There is an urgent need to protect bumblebee health and their pollination services. Bumblebees possess specialized gut microbiota with potential to be used as probiotics to help defend at-risk bumblebee populations. However, evidence for probiotic benefits on bumblebees is lacking. Here, we evaluated how supplementation with Lactobacillus melliventris isolated from bumblebee gut affected the colony development of Bombus terrestris. This native strain colonized robustly and persisted long-term in bumblebees, leading to a significantly higher quality of offspring. Subsequently, the tyrosine pathway was upregulated in the brain and fat body, while the Wnt and mTOR pathways of the gut were downregulated. Notably, the field experiment in the greenhouse revealed the supplementation of L. melliventris led to a 2.5-fold increase in the bumblebee survival rate and a more than 10% increase in the number of flowers visited, indicating a better health condition and pollination ability in field conditions. Our study represents a first screening for the potential use of the native gut member, L. melliventris, as probiotic strains in hive supplement for bumblebee breeding, which may be a practical approach to improve immunity and hive health.

Workers’ task specialization and division of labor are critical features of social insects’ ecological success. It has been proposed that the division of labor relies on response threshold models: individuals varying their sensitivity (and responsiveness) to biologically relevant stimuli and performing a specific task when a stimulus exceeds an internal threshold. In this work, we study carbohydrate and protein responsiveness and their relation to worker task specialization in Vespula germanica, an invasive social wasp. The sucrose and peptone responsiveness of two different subcastes, preforagers and foragers, was determined by stimulating the antenna of the wasps with increasing concentrations of the solution and quantifying whether each concentration elicited a licking response. We studied responsiveness in five different ways: (1) response threshold, (2) concentration 50 (concentration to which at least 50% of wasps responded), (3) maximum response, (4) mean scores and (5) median scores. Our results suggest that V. germanica foragers are more sensitive to sucrose (lower thresholds) than preforager workers. However, we found no differences for peptone thresholds (i.e., a protein resource). Nonetheless, this is the first study to investigate response thresholds for protein resources. The intercaste variation in sucrose responsiveness shown in our work contributes to the existing knowledge about response threshold theory as a mechanism for task specialization observed in V. germanica.

Pollination networks are increasingly used to model the complexity of interactions between pollinators and flowering plants in communities. Different methods exist to sample these interactions, with direct observations of plant–pollinator contacts in the field being by far the most common. Although the identification of pollen carried by pollinators allows uncovering interactions and increasing sample sizes, the methods used to build pollen-transport networks are variable and their effect on network structure remains unclear. To understand how interaction sampling influences the structure of networks, we analyzed the pollen found on wild bees from eight communities across Mallorca Island and investigated the differences in pollen loads between bee body parts (scopa vs. body) and sexes. We then assessed how these differences, as well as the uncovered interactions not detected in the field, influenced the structure of wild bee–plant networks. We identified a higher quantity and diversity of pollen in the scopa than in the rest of the female body, but these differences did not lead to differences in structure between plant-pollination (excluding scopa pollen) and bee-feeding interaction (including scopa pollen) networks. However, networks built with pollen data were richer in plant species and interactions and showed lower modularity and specialization (H₂'), and higher nestedness than visitation networks based on field observations. Female interactions with plants were stronger compared to those of males, although not richer. Accordingly, females were more generalist (low d’) and tended to be more central in interaction networks, indicating their more key role structuring pollination networks in comparison to males. Our study highlights the importance of palynological data to increase the resolution of networks, as well as to understand important ecological questions such as the differences between plant-pollination and bee-feeding interaction networks, and the role of sexes in pollination.

Mosquitoes are of great medical significance as vectors of many deadly diseases. Mitogenomes have been widely used in phylogenetic studies, but mitogenome knowledge within the family Culicidae is limited, and Culicidae phylogeny is far from resolved. In this study, we surveyed the mitogenomes of 149 Culicidae species, including 7 newly sequenced species. Comparative analysis of 149 mosquito mitogenomes shows gene composition and order to be identical to that of an ancestral insect, and the AT bias, length variation, and codon usage are all consistent with that of other reported Dipteran mitogenomes. Phylogenetic analyses based on the DNA sequences of the 13 protein-coding genes from the 149 species robustly support the monophyly of the subfamily Anophelinae and the tribes Aedini, Culicini, Mansoniini, Sabethini, and Toxorhynchitini. To resolve ambiguous relationships between clades within the subfamily Culicinae, we performed topological tests and show that Aedini is a sister to Culicini and that Uranotaeniini is a sister to (Mansoniini + (Toxorhynchitini + Sabethini)). In addition, we estimated divergence times using a Bayesian relaxation clock based on the sequence data and 3 fossil calibration points. The results show mosquitoes diverged during the Early Jurassic with massive Culicinae radiations during the Cretaceous, coincident with the emergence of angiosperms and the burst of mammals and birds. Overall, this study, which uses the largest number of Culicidae mitogenomes sequenced to date, comprehensively reveals the mitogenome characteristics and mitogenome-based phylogeny and divergence times of Culicidae, providing information for further studies on the mitogenome, phylogeny, evolution, and taxonomic revision of Culicidae.