Transcriptome profiling reveals the developmental regulation of NaCl-treated Forcipomyia taiwana eggs.

BACKGROUND: The biting midge, Forcipomyia taiwana, is one of the most annoying blood-sucking pests in Taiwan. Current chemical control methods only target the adult, not the immature stages (egg to pupa), of F. taiwana. Discovering new or alternative tactics to enhance or replace existing methods are urgently needed to improve the effectiveness of F. taiwana control. The egg is the least understood life stage in this pest species but may offer a novel point of control as addition of NaCl to the egg environment inhibits development. Thus, the objective of this study was to use RNA profiling to better understand the developmental differences between wild-type melanized (black) and NaCl-induced un-melanized (pink), infertile F. taiwana eggs. RESULTS: After de novo assembly with Trinity, 87,415 non-redundant transcripts (Ft-nr) with an N50 of 1099 were obtained. Of these, 26,247 (30%) transcripts were predicted to have long open reading frames (ORFs, defined here as ≥300 nt) and 15,270 (17.5%) transcripts have at least one predicted functional domain. A comparison between two biological replicates each of black and pink egg samples, although limited in sample size, revealed 5898 differentially expressed genes (DEGs; 40.9% of the transcripts with long ORFs) with ≥2-fold difference. Of these, 2030 were annotated to a Gene Ontology biological process and along with gene expression patterns can be separated into 5 clusters. KEGG pathway analysis revealed that 1589 transcripts could be assigned to 18 significantly enriched pathways in 2 main categories (metabolism and environmental information processing). As expected, most (88.32%) of these DEGs were down-regulated in the pink eggs. Surprisingly, the majority of genes associated with the pigmentation GO term were up-regulated in the pink egg samples. However, the two key terminal genes of the melanin synthesis pathway, laccase2 and DCE/yellow, were significantly down-regulated, and further verified by qRT-PCR. CONCLUSION: We have assembled and annotated the first egg transcriptome for F. taiwana, a biting midge. Our results suggest that down-regulation of the laccase2 and DCE/yellow genes might be the mechanism responsible for the NaCl-induced inhibition of melanization of F. taiwana eggs.

Decoding the genome of bloodsucking midge Forcipomyia taiwana (Diptera: Ceratopogonidae): Insights into odorant receptor expansion.

Biting midges, notably those within the Ceratopogonidae family, have long been recognized for their epidemiological significance, both as nuisances and vectors for disease transmission in vertebrates. Despite their impact, genomic insights into these insects, particularly beyond the Culicoides genus, remain limited. In this study, we assembled the Forcipomyia taiwana (Shiraki) genome, comprising 113 scaffolds covering 130.4 Mbps-with the longest scaffold reaching 7.6 Mbps and an N50 value of 2.6 Mbps-marking a pivotal advancement in understanding the genetic architecture of ceratopogonid biting midges. Phylogenomic analyses reveal a shared ancestry between F. taiwana and Culicoides sonorensis Wirth & Jones, dating back approximately 124 million years, and highlight a dynamic history of gene family expansions and contractions within the Ceratopogonidae family. Notably, a substantial expansion of the odorant receptor (OR) gene family was observed, which is crucial for the chemosensory capabilities that govern biting midges' interactions with their environment, including host seeking and oviposition behaviors. The distribution of OR genes across the F. taiwana genome displays notable clusters on scaffolds, indicating localized tandem gene duplication events. Additionally, several collinear regions were identified, hinting at segmental duplications, inversions, and translocations, contributing to the olfactory system's evolutionary complexity. Among the 156 ORs identified in F. taiwana, 134 are biting midge-specific ORs, distributed across three distinct clades, each exhibiting unique motif features that distinguish them from the others. Through weighted gene co-expression network analysis, we correlated distinct gene modules with sex and reproductive status, laying the groundwork for future investigations into the interplay between gene expression and adaptive behaviors in F. taiwana. In conclusion, our study not only highlights the unique olfactory repertoire of ceratopogonid biting midges but also sets the stage for future studies into the genetic underpinnings of their unique biological traits and ecological strategies.

Evaluation of the Potential Entomopathogenic Fungi Purpureocillium lilacinum and Fusarium verticillioides for Biological Control of Forcipomyia taiwana (Shiraki).

Forcipomyia taiwana (Diptera: Ceratopogonidae) is a nuisance blood-sucking pest to humans in Taiwan. An F. taiwana bite causes itching and redness and usually causes serious harassment to human outdoor activity. In terms of F. taiwana control, chemical pesticides are ineffective. Therefore, other efforts are needed. Fungal mycosis in the larvae, pupae, and emerging F. taiwana adults was found during the rearing of F. taiwana. In this study, six fungal isolates were isolated from infected cadavers and subjected to molecular identification. In addition, their biocontrol potential was evaluated against different life stages of F. taiwana. Based on the pathogenicity screening, two fungal isolates, NCHU-NPUST-175 and -178, which caused higher mortality on the fourth instar larvae of F. taiwana, were selected for virulence tests against different life stages of F. taiwana larvae. The results of the phylogenetic analysis indicated that the NCHU-NPUST-175 and -178 belonged to Purpureocillium lilacinum and Fusarium verticillioides, respectively. Bioassay against different life stages of F. taiwana with different spore concentrations (5 × 105 to 5 × 107 conidia/mL) revealed a dose-dependent effect on larvae for both fungal isolates, while only 38% and 50% mortality was found in highest concentration (5 × 107 conidia/mL) at fourth instar larvae by Pl-NCHU-NPUST-175 and Fv-NCHU-NPUST-178, respectively. Moreover, reductions in egg-hatching rate and adult emergence rate were found, when the last stage of F. taiwana was inoculated with both fungal isolates, indicating the ovicidal potential and the impact of entomopathogenic fungi on the development of F. taiwana. In conclusion, Pl-NCHU-NPUST-175 and Fv-NCHU-NPUST-178 showed larvicidal activity, ovicidal activity, and impact on adult emergence on F. taiwana.

Evaluation of Repellent Effectiveness of Polyvinyl Alcohol/Eucalyptus globules Nanofibrous Membranes against Forcipomyia taiwana.

This study aims to develop nanofibrous membranes where Eucalyptus globules oil (EGO) is wrapped in polyvinyl alcohol (PVA). The EGO-based nanofibrous membranes are then evaluated for the protection against Forcipomyia taiwana (F. taiwana). In the first stage, the PVA solutions are formulated with different concentrations and are measured for viscosity and electrical conductivity. In the next stage, PVA solution and EGO are blended at different ratios and electrospun into PVA/EGO nanofibrous membranes (i.e., EGO-based repellent). In this study, a PVA concentration of 14 wt% has a positive influence on fiber formation. Furthermore, the finest nanofibers of 291 nm are presented when the voltage is 15 kV. The repellent efficacy can reach 80% in a 60-min release when the repellent is composed of a PVA/oil ratio of 90/10. To sum up, the nanofibrous membranes of essential oil exhibit good repellent efficacy against F. taiwana and significant slow-release effect, instead of adversely affecting the cell viability.

Establishing and maintaining colonies of Forcipomyia taiwana in the laboratory.

Successful colonies of the biting midge Forcipomyia taiwana (Shiraki) were established and maintained in the laboratory by feeding blood with an artificial blood-feeding apparatus, rearing larvae on a soil substrate employing algae liquid, and setting suitable mating cages. The feeding rates of F. taiwana fed on pig blood (69.9%) and artificial blood (72.7%) were not significantly different from those fed on human blood (67.0%). The mean numbers of adults produced by females fed on the artificial blood and the human blood were 32.0 and 33.0, respectively. The algae liquid, Chlorella vulgaris, was suitable for rearing larvae, with larval hatching rate, pupation rate, and emergence rate of midges fed with artificial blood and human blood meal cohorts observed as 76.0%-88.8%, 98.2%-96.4%, and 98.0-94.3%, respectively. Swarming and copulation occurred 1 h before and 2 h after the lights were turned on (07:00-10:00). The average female mating rates were approximately 50-60%, and males were observed to mate with multiple females.