Insect Science最新文献

Apis mellifera filamentous virus (AmFV) is an emerging DNA virus significantly affecting honey bee health. AmFV infections weaken bee resistance to other pathogens, and can cause tissue lysis and death. Early, accurate detection of AmFV is crucial for timely intervention and preventing large-scale outbreaks. Current AmFV detection relies largely on polymerase chain reaction (PCR)-based methods. To enable rapid field detection of AmFV, we developed a rapid and ultrasensitive detection platform using recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 12a (Cas12a) technology. A CRISPR RNA (crRNA1) specifically targeting the AmFV Bro gene was designed, ensuring no cross-reactivity with other insect DNA viruses or uninfected honey bees. After optimization of the reaction time, the platform generated results within 35 min: 20 min for the RPA reaction and 15 min for CRISPR-mediated cleavage. Two visualization approaches, fluorescence-based and lateral flow dipstick, were used to display the detection results. The detection sensitivity of both approaches was as few as 10 copies of the AmFV genome. Validation with field-collected honey bee samples demonstrated consistency with conventional PCR, revealing widespread latent AmFV infections in the field. Taken together, we successfully developed an RPA-CRISPR/Cas12 platform for rapid, specific, and sensitive detection of AmFV in Apis mellifera and Apis cerana. This platform holds promise as a simple, accurate, and cost-effective tool for point-of-care AmFV diagnosis in the field.

Insects and their associated microbiota have developed a sustained and mutually beneficial relationship, characterized by the influence of the symbiotic microorganisms on the host's physiological processes and fitness parameters. The Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), is one of the world's most ubiquitous, invasive, and harmful agricultural pests. In Tunisia, the medfly is widely distributed across all bioclimatic zones. However, in the absence of surveillance, infestations can escalate drastically, causing damage levels as high as 100%. Our study aimed to characterize the microbiome profile of Tunisian medfly populations from Zaghouan, Tozeur, Siliana, and Bizerte to understand the microbial dynamics implicated in the invasiveness and adaptability potential if SIT is applied. We conducted amplicon sequencing using MiSeq Illumina and a culture-dependent approach. Our findings revealed notable differences in symbiotic communities across regions. For instance, Serratia was prevalent in Tozeur populations, while Klebsiella showed high abundance in Bizerte. The composition of the bacterial communities within the medfly populations was influenced by several factors including the environmental conditions, geographical location, developmental stage, and the sex of the insects. Investigating the intricate relationship between insects and their microbiota is pivotal for understanding their biology and developing effective pest management strategies. Additionally, the isolation of bacteria from adult and larval medflies collected in the Bizerte region revealed the presence of bacterial species that could be utilized as attractants or supplements in larval artificial diets in the case of application of the SIT aiming at producing competitive sterile males.

Trypanosomiasis, transmitted by tsetse flies (Glossina spp.), poses a significant health threat in 36 sub-Saharan African countries. Current control methods targeting tsetse flies, while effective, allow reinfestation. This study investigates paratransgenesis, a novel strategy to engineer symbiotic bacteria in tsetse flies, Sodalis glossinidius, to deliver anti-trypanosome compounds. Disrupting the trypanosome life cycle within the fly and reducing parasite transmission could offer a sustainable solution for trypanosomiasis control. In this context, we tested the effect of cecropin, reported to be lethal for Trypanosoma cruzi (Chagas disease) and TbgTCTP (Translationally Controlled Tumor Protein from Trypanosoma brucei gambiense), previously reported to modulate the growth of bacteria isolated from the fly microbiome, to delay the first peak of parasitemia and the death of trypanosome-infected mice. We have successfully cloned and transfected the genes encoding the two proteins into Sodalis strains. These Sodalis recombinant strains (recSodalisTbgTCTP and recSodaliscecropin) have been then microinjected into the L3 larval stage of Glossina palpalis gambiensis flies. The stability of the cloned genes was checked up to the 20th day after microinjection of recSodalis. The rate of fly emergence from untreated pupae was 95%; it was reduced by nearly 50% due to the mechanical injury caused by microinjection. It decreased to nearly 7% when larvae were injected with recSodalisTbgTCTP, which suggests TCTP could have a lethal impact to larvae development. When challenged with T. brucei gambiense, a slightly lower, but statistically non-significant, infection rate was recorded in flies harboring recSodaliscecropin compared to control flies. The effect of recSodalisTbgTCTP could not be measured due to the very low rate of fly emergence after corresponding treatment of the larvae. The results do not allow to conclude on the effect of cecropin or TCTP, delivered by para-transgenesis into the fly's gut, on the fly infection by the trypanosome. Nevertheless, the results are encouraging insofar as the technical approach works on the couple G. p. gambiensis/T. brucei gambiense. The next step will be to optimize the system and test other targets chosen among the ESPs (Excreted-Secreted Proteins) of the trypanosome secretum, or the differentially expressed genes associated with the sensitivity/resistance of the fly to trypanosome infection.

Hoverflies are essential to ecosystems, with adults serving as important pollinators and larvae preying on plant-feeding insects or recycling nutrients. Species like Sphaerophoria rueppellii are used in biocontrol programs to target aphid pests. To enhance these programs, markers can be used in a mark-release-recapture (MRR) method to track hoverfly feeding and oviposition sites. Effective markers must be persistent and not harm the hoverflies' vital functions. This study evaluated three marking methods for S. rueppellii: rubidium (RbCl), fluorescein, and fluorescent dust. Laboratory experiments assessed the effects of these markers on female hoverfly fecundity, mating behavior and marking persistence. Results showed no significant differences in egg-laying or survival time between marked and unmarked females. Rubidium and fluorescein did not affect mating behavior, but dust-treated females mated significantly less than untreated females. In terms of marking persistence, rubidium and fluorescent dusts remained detectable throughout the hoverflies' adult lifespan, while fluorescein markings faded within 24 h. Fluorescent dusts were easy-to-use, durable, and cost-effective, but careful application and further study are needed to avoid potential effects on insect activity and mating ability. Fluorescein showed no adverse effects on insect biology, was economical and quick to apply, but had short persistence, making it unsuitable for long-term field studies. Rubidium was harmless to insects and detectable for long periods, but its detection required financial investment, time, and specialized equipment. This research provides valuable insights into the potential of hoverflies as biocontrol agents and offers new tools for their effective management in agricultural settings.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a serious invasive pest, which has attracted concern regarding the effectiveness of environmental bioinsecticide as a substitution for synthetic insecticide in controlling its damage to numerous agricultural crops in recent years. Hence, laboratory and field experiments have investigated insecticidal toxicity in S. frugiperda by 5 insecticides and have determined the activity of acetylcholinesterase (AChE) and detoxifying enzymes, namely mixed function oxidase (MFO), carboxylesterase (CarE), and glutathione S-transferase (GST) on 3rd instar larvae at 25% lethal concentration (LC₂₅) to explore detoxification mechanisms. The results showed that the most effective insecticides were 3.0% emamectin benzoate (0.024 mg/L), 60 g/L spinetoram (0.282 mg/L), and 1.3% matrine (0.380 mg/L) at 48 h depending on LC₅₀ values. The field efficiencies of emamectin benzoate and spinetoram were over 80% at 24 h and 90% at 72 h, indicating that both insecticides had acute and long-lasting toxicity on S. frugiperda; matrine had extended-release toxicity with 90% field efficiency at 72 h. Emamectin benzoate and spinetoram activated the activities of AChE, MFO and GST, and spinetoram decreased in CarE activity; cyantraniliprole induced an increase of 4 detoxifying enzymes; spinosad increased the activities of AChE, MFO, and CarE but not GST. Furthermore, matrine had an inhibiting effect on AChE and acceleration on CarE and GST. Overall results obviously depicted that semi-synthetic insecticide spinetoram and bioinsecticide matrine were recommended to control S. frugiperda with effective and long-lasting toxicity. Moreover, this study will provide basic information for sustainable control of S. frugiperda under field conditions in China.

Silkworm silk gland cells undergo multiple rounds of endoreplication, a process in which the genome is duplicated without cell division, leading to cellular polyploidization. This results in the accumulation of genomic DNA, serving as the foundation for rapid silk proteins synthesis. For the first time, we report a previously uncharacterized gene, SGDAcn, in the silkworm silk gland that clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9-mediated SGDAcn knockout in the posterior silk gland increased cell size and enhanced silk production. SGDAcn knockout facilitated the progression of endoreplication by upregulating the expression of various cyclin genes and promoting energy metabolism, leading to a substantial increase in fibroin gene expression and its transcription factor Dimm, as well as the stimulation of ribosome biogenesis for messenger RNA translation and enhancement of eukaryotic translation initiation factors for protein synthesis. Our findings demonstrate that SGDAcn influences endoreplication, cell growth, and nucleolus size through SGDAcn-EGFR/PI3K/AKT and SGDAcn-NF-κB signaling pathways. Overall, SGDAcn acts as a negative regulator of silk gland development, affecting cell size and protein synthesis, thus modulating silk production. These mechanisms might be conserved in cell growth and progression, making an attractive target for genetic editing to improve silk yield in silkworms and potentially in mammalian cell growth regulation.

For almost a decade, natural populations of the South American fruit fly have been targeted for control through Sterile Insect Technique projects. To ensure a sustainable supply of competitive sterile flies for this approach, it is essential to understand the effects of domestication when strains of this pest are initially brought into the laboratory to establish colonies as well as the changes occurring after multiple generations of adaptation to conditions used for mass rearing. Using one colony established from a wild population of the Brazil-1 morphotype (WIL) and two from laboratory colonies in Brazil known as the Piracicaba (PL) and Vacaria (VL) strains, this study evaluated genetic diversity in samples from 10 generations after domestication and maintenance under semimass rearing conditions. Another aim of this study was to analyze changes in the genetic makeup of the colonies of the two laboratory strains after refreshment. Eight microsatellite markers were used for the genetic analyses. Results indicated a moderate but significant amount of genetic differentiation between the WIL population and the two laboratory strains. Results also showed that levels of genetic diversity in both the VL and PL strains were maintained at similar levels over a period of more than two years of rearing. Additionally, results suggest that successful creation of admixture via refreshment is more likely to be beneficial in relatively short-term domesticated colonies, and that performing refreshment approximately every six to eight generations could be beneficial to maintain the genetic diversity of A. fraterculus colonies under laboratory mass rearing conditions.

The Asian citrus psyllid (ACP), Diaphorina citri, serves as the primary vector for Candidatus Liberibacter asiaticus (CLas), the pathogen responsible for citrus Huanglongbing (HLB). D. citri modulates the expression of its key proteins in response to CLas infection. Previous research has revealed that CLas infection significantly alters the expression levels of E3 ubiquitin ligases in D. citri; however, the specific functions of these E3 ligases remain largely uncharacterized. In this study, a total of 11 E3 ubiquitin ligases were identified from the proteomics database of D. citri, among which E3 ubiquitin ligase RNF115 was significantly upregulated following CLas infection. RING finger protein 115 (RNF115) consists of 156 amino acids and contains a RING finger domain at its N-terminus. Silencing RNF115 via RNA interference (RNAi) and injecting the inhibitor disulfiram, which targets RNF115, significantly increased CLas bacterial content in D. citri. In contrast, injection of recombinant RNF115 protein markedly inhibited CLas bacterial proliferation. Furthermore, interaction between RNF115 and D. citri histone H1 was confirmed using yeast 2-hybrid assay, pull-down experiments and molecular docking analysis. Knockdown of histone H1 via RNAi significantly reduced CLas bacterial content, whereas injection of recombinant histone H1 protein led to an increase in CLas content within D. citri. These findings suggest that CLas infection may induce an upregulation of RNF115 expression in D. citri, leading to subsequent interactions with histone H1 that facilitate the ubiquitination of histone H1, ultimately resulting in reduced expression levels and inhibiting CLas proliferation within D. citri.

Insects have evolved a diversity of regulatory mechanisms to determine their sex. Understanding the molecular regulation mechanisms of insect sex determination is of great significance in revealing the general law of insect sex determination and providing potential routes for the genetic manipulation of pest species. Although the sex determination cascade and doublesex (dsx) gene functions have been well described in some holometabolous insects, little is known about this cascade in hemimetabolous insects. In this study, we identified the dsx homolog in Gryllus bimaculatus, which belongs to the Orthoptera order and is an important model for developmental and evolutionary biology. We found that Gbdsx has two alternative splicing isoforms (male-specific Gbdsx^M and non-sex-specific Gbdsx^C). Using RNAi-mediated knock-down of Gbdsx^M in 6th-instar nymphs resulted in adult male forewings showing feminized vein development and abnormal external genitalia. CRISPR/Cas9 knockout of Gbdsx in embryos resulted in adult males becoming pseudofemales, with feminized forewings and abnormal external and internal genitalia. Additionally, the pseudofemales created by Gbdsx knockout demonstrated normal courtship trends and aggressive behavior but no actual mating behavior. However, the knockout and knock-down of Gbdsx in female crickets does not affect their sexual traits or fertility. Our results suggest that Gbdsx^M plays a critical role in the development of male cricket sexual traits, courtship and mating behavior, which furthers our understanding of sex determination in hemimetabolous insects.

Spiders are important predatory enemies that control a range of insect pests in the rice ecosystem. m-Aminophenylacetylene (m-A) has been demonstrated to induce maternal care behaviors in Pardosa pseudoannulata, protecting young spiders and promoting population growth. Here the roles of m-A on other reproduction stages were evaluated by injecting m-A into virgin and postreproductive female spiders. When injected into virgin females, m-A can prolong the first pulli-carrying stage of the females after mating and breeding offsprings. In postreproductive females, m-A injection shortened the eggsac-carrying stages, especially in the second and third stages. Transcriptomic analysis showed that differentially expressed genes between the control female and m-A-injected female were enriched in the arachidonic acid (AA) metabolism pathway. Real-time quantitative PCR (qPCR) confirmed the upregulation of cyclooxygenase-II (COX-II), prostaglandin E synthase 2 (PGES2), and prostaglandin E synthase 3 (PGES3) genes involved in PGE₂ (prostaglandin E₂) synthesis. In a reverse validation experiment, the sequential injection of m-A followed by a COX inhibitor aspirin restored the shortened eggsac-carrying stage to normal level. The findings indicated that m-A enhanced PGs synthesis, leading to a reduced eggsac-carrying stage. The eggsac-carrying stage can be fatal when female spiders encounter their enemies, so the shortening of the stage by m-A would reduce the potential threat. These findings are significant for developing environmentally friendly control strategies.