Insects

Apis cerana cerana is a subspecies of eastern honeybee, Apis cerana. Nosema ceranae is a widespread fungal parasite of honeybee, causing heavy losses for beekeeping industry all over the world. In this article, total RNA of normal midguts (AcCK1, AcCK2) and N. ceranae-infected midguts of A. c. cerana workers at 7 d and 10 d post inoculation (AcT1, AcT2) were respectively isolated followed by strand-specific cDNA library construction and next-generation RNA sequencing. In tolal, 56270223688, 44860946964, 78991623806, and 92712308296 raw reads were derived from AcCK1, AcCK2, AcT1 and AcT2, respectively. Following strict quality control, 54495191388, 43570608753, 76708161525, and 89467858351 clean reads were obtained, with Q30 value of 95.80%, 95.99%, 96.07% and 96.04%, and GC content of 44.20%, 43.44%, 44.83% and 43.63%, respectively. The raw data were submitted to the NCBI Sequence Read Archive database and connected to BioProject PRJNA562784. These data offers a valuable resource for deep investigation of mechanisms underlying eastern honeybee responding to N. ceranae infection and host-fungal parasite interaction during microsporidiosis. Value of the DataO_LICurrent dataset offers a valuable resource for exploring mRNAs, lncRNAs and circRNAs involved in response of A. c. cerana worker to N. ceranae infection. C_LIO_LIThe accessible data can be used to investigate differential expression pattern and regulatory network of non-coding RNAs in A. c. cerana workers midguts responding to N. ceranae challenge. C_LIO_LIThis data will enable a better understanding of the molecular mechanism regulating eastern honeybee-N. ceranae interaction. C_LI

Apis mellifera ligustica is a subspecies of western honeybee, Apis mellifera. Nosema ceranae is known to cause bee microspodiosis, which seriously affects bee survival and colony productivity. In this article, Nanopore long-read sequencing was used to sequence N. ceranae-infected and un-infected midguts of A. m. ligustica workers at 7 d and 10 d post inoculation (dpi). In total, 5942745, 6664923, 7100161 and 6506665 raw reads were respectively yielded from AmT1, AmT2, AmCK1 and AmCK2, with average lengths of 1148, 1196, 1178 and 1201 bp, and N50 of 1328, 1394, 1347 and 1388 bp. The length distribution of raw reads from AmT1, AmT2, AmCK1 and AmCK2 was ranged from 1 kb to more than 10 kb. Additionally, the distribution of quality score of raw reads from AmT1 and AmT2 was among Q6[~]Q12, while that from AmCK1 and AmCK2 was among Q6[~]Q16. Further, 5745048, 6416987, 6928170, 6353066 clean reads were respectively gained from AmT1, AmT2, AmCK1 and AmCK2, and among them 4172542, 4638289, 5068270 and 4857960 were identified as being full-length. After removing redundant reads, the length distribution of remaining full-length transcripts was among 1 kb[~]8 kb, with the most abundant length of 2 kb. The long-read transcriptome data reported here contributes to a deeper understanding of the molecular regulating N. ceranae-response of A. m. ligustica and host-fungal parasite interaction during microsporidiosis.

Apis cerana cerana is a subspecies of eastern honeybee, Apis cerana. Ascosphaera apis is a widespread fungal pathogen of honeybee, leading to chalkbrood, which results in heavy losses for beekeeping industry. In this article, 4-, 5-, and 6-day-old larval guts of un-infected (AcCK1, AcCK2, and AcCK3) and A. apis-infected A. c. cerana (AcT1, AcT2, and AcT3) were sequenced by next generation sequencing. Totally, 73830148, 96586212, 94552744, 76672564, 90954858, and 83418832 raw reads were respectively produced from AcCK1, AcCK2, AcCK3, AcT1, AcT2, and AcT3. The sequencing depth was enough to detect all expressed genes. After strict quality control, 73775592, 96513798, 94495000, 76593924, 90870608 and 83339288 clean reads were obtained, with a mean GC content of 48.54%. Additionally, average Q20 and Q30 for aforementioned six groups were 98.10% and 94.36%, respectively. Moreover, 45302685, 65872823, 52709987, 49947838, 56476339, and 42657156 clean reads from above-mentioned six groups were mapped to the reference genome of Apis cerana, respectively. In addition, exons were the most abundant regions in reference genome mapped by clean reads, followed by intergenic regions and introns. Our data presented here can be used to identify long non-coding RNAs (lncRNAs), circlular RNAs (circRNAs), mRNAs and their regulatory networks engaged in response of eastern honeybee larvae to A. apis infection, and decipher molecular mechanisms underlying host-pathogen interaction. Value of the DataO_LIThe current data can be used for exploration of mRNAs, lncRNAs, circRNAs and their competing endogenous RNA (ceRNA) networks engaged in response of A. c. cerana larvae to A. apis infection. C_LIO_LIThis data provides a valuable genetic resource for better understanding non-coding RNA-mediated cross-kingdom regulation of eastern honey larvae to A. apis. C_LIO_LIThe accessible data offers novel insights into understanding the molecular mechanism underlying interaction between eastern honeybee larvae and A. apis. C_LI

Comprehensive expression datasets were constructed for Apis mellifera and Apis cerana japonica. Post-oviposition day 6 to day 58 samples of A. mellifera workers (larva to adult); day 9, 10, 12, and 13 samples of A. mellifera queen (larva to pupa); and day 9 to day 18 samples of A. cerana japonica workers (larva to adult) were prepared, and RNA-Seq data were obtained. For A. cerana japonica, reference transcript sequence data, predicted amino acid sequence data, and functional annotation data were generated based on the genome sequence and RNA-Seq data. Using the transcript sequence and RNA-Seq data, comprehensive expression data for all transcripts of A. mellifera and A. cerana japonica were prepared. Hierarchical clustering analyses and the used sample preparation method ensured that both sets of expression data were reliable for use as comprehensive reference expression datasets. Therefore, these data are applicable for honey bee research or for comparative or evolutionary studies on insect species or social insect species at the genetic and molecular levels.

Neonicotinoids, including imidacloprid, harm Apis mellifera in a number of ways, among others by impairing body maintenance and immunity. Energy resources are important to prevent this, particularly, as we hypothesized, in the fat body tissue. But hormesis (adaptative, diaphasic response to a stressor) was also reported in the energy-dependent traits of bees exposed to sublethal doses of imidacloprid. Consequently, concentrations/activities of respiratory and citric cycle compounds (Acetyl-CoA, IMH-2, AKG, succinate, fumarate, NADH2, CoC, COX, and ATP) were compared in the hemolymph and fat bodies of honeybees fed with diets containing 200 ppb (IM-200), 5 ppb (IM-5; sublethal), and 0 ppb of ID. The values of the compounds were higher in the fat body than in the hemolymph, where the variability was higher in hemolymph. The pattern of response to ID was the same in both tissues, but differed between IM-200 and IM-5. The concentrations of the strongly correlated NADH2, ATP and acetyl-CoA decreased both in ID-200 and ID-5, whereas the levels of the remaining compounds decreased in ID-200 but increased in ID-5. Decreased ATP levels in each diet show that the pesticide impairs the fat-body energy metabolism in spite of hormesis in six of the nine respiratory and citric cycle compounds even in the low, residual doses. We believe that sublethal doses of imidacloprid decrease energy demands, as they decreased the levels of ATP, and acetyl-CoA.

Nosema ceranae is a widespread fungal parasite that infects both adult honeybee and honeybee larvae, leading to microsporidiosis, which seriously affects bee health and apicultural industry. In this article, genome sequencing of clean spores of N. ceranae was conducted using third-generation Pacific Biosciences (PacBio) single molecule real time (SMRT) sequencing technology. In total, 152671 subreads were obtained after quality control of raw reads from PacBio SMRT sequencing, with a N50 and average length of 14422 bp and 11310 bp, respectively. Additionally, the length distribution of subreads was from 10000 bp to more than 50000 bp. Nineteen scaffords with a total length of 7354221 bp were assembled, and the N50, N90 and maximum scafford length were 728543 bp, 198795 bp and 1917792 bp, respectively. The GC content was 25.97%. Furthermore, by integration of genes predicted from de novo and homology-based methods, 3112 N. ceranae genes were finally assembled, with a total length of 2730179 bp and mean length of 877.31 bp. In addition, the total length and mean length of exons were 2657637 bp and 854 bp, respectively; and the total length and mean length of introns were 72542 bp and 23.31 bp, respectively. The genome sequencing data documented here will give deep insights into the molecular biology of N. ceranae, facilitate exploration of genes and pathways associated with toxin factors and infection-related factors, and benefit research on comparative genomics and phylogenetic diversity of Nosema species.

Odorant binding proteins (OBPs) in insects bind to volatile chemical cue and help in their binding to odorant receptors. The odor coding hypothesis states that OBPs may bind with specificity to certain volatiles and aid the insect in various behaviours. Honeybees are eusocial insects with complex behaviour that requires olfactory inputs. Here, we have identified and annotated odorant binding proteins from the genome of the dwarf honey bee, Apis florea using an exhaustive homology-based bioinformatic pipeline and analyzed the evolutionary relationships between the OBP subfamilies. Our study suggests that Minus-C subfamily may have diverged from the Classic subfamily of odorant binding proteins in insects.

Oxidative stress (OX) is a state of imbalance between antioxidants and reactive oxygen species, which are the byproducts of oxidative phosphorylation in the mitochondria. Different landscapes: conventional, organic, and roadside use different pesticides and pest control management practices pest control. The roadside landscape is a control for minimum application of herbicides and management. Two important pollinators, honey bees (Apis mellifera) and small carpenter bees (Ceratina calcarata) are studied here for their abiotic stress from different landscape. It is unknown whether the differences in these landscapes affect the oxidative stress of these two pollinators. Conventional farms offer the most exposure to pesticides, which have been related to increased stress levels. We hypothesized that honey bees and small carpenter bees (SCB) from conventional farms would experience the highest levels of OX. Results from the lipid assay indicated that honey bees and SCB of organic and roadside landscapes experienced the lowest and highest OX levels. The results of the protein carbonyl assay indicated a significant difference found between conventional and organic bees (p < 0.05), based on Tukeys post-hoc test. Previous research has shown that feral bees have a tolerance to high stress levels. The same could be true of honey bees and SCB from roadside landscapes, where the conditions imitate natural habitats. Organic farms use naturally derived pesticides and moderate management practices, likely contributing to their lower levels. Examining how these pollinators are affected by different farm landscapes can reveal the benefits and detriments of their corresponding management practices. This will lead to streamlined practices that are healthier for pollinators.

RNA interference (RNAi) has emerged as an eco-friendly approach to pest management and relies on the processing of exogenous double-stranded RNA (dsRNA). RNAi-based pest management is highly effective in the Colorado potato beetle (Leptinotarsa decemlineata); however, the tissue-specific distribution and processing of exogenous dsRNA following oral uptake remain incompletely understood. In this study, we investigated whether ingested dsRNA reaches the central nervous system (CNS) and is processed into active small interfering RNAs (siRNAs). Adult beetles were fed dsmGFP-coated leaf disks, and RISC-bound small RNAs were isolated from midgut, CNS, and remaining body tissues using a RISC-enrichment approach. Small RNA sequencing revealed abundant 21-nucleotide antisense guide-strand siRNAs in all analysed tissues, with relative proportions following the order midgut > CNS > remaining tissues. Notably, antisense siRNAs of consistent size were detected in CNS samples, indicating that exogenous dsRNA or its processed products can access neural tissue and enter the RNAi silencing machinery. These findings provide strong biochemical evidence that orally taken-up dsRNA is processed into AGO-loaded siRNAs in the L. decemlineata CNS. Together, our results offer a tissue-resolved view of functional RNAi activity in this species and contribute to a mechanistic understanding of systemic dsRNA transport in coleopteran pests. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/711085v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1796858org.highwire.dtl.DTLVardef@1b183ceorg.highwire.dtl.DTLVardef@1447e91org.highwire.dtl.DTLVardef@1d17def_HPS_FORMAT_FIGEXP M_FIG C_FIG

Feeding experiments are standard tools in the pollinator risk assessment. The design (Oomen et al 1992) was developed to test insect growth regulators and herbicides. In recent years there was an update (Luckmann & Schmitzer 2015) on the outline in order to also focus on the advantage of different rates making a dose response design possible where exposure levels are known. Additionally, this design gives the possibility to test different rates for honey bee colonies foraging in the same landscape. The main objective of the experiment presented here was to determine the natural variability of foragers losses of hives fed with a sub-lethal neonicotinoid concentration compared to an untreated control. Other objectives were to see if the neurotoxic exposure results in any observable sub-lethal effects and to find out if losses can be correlated to hive development. This was assessed with traditional methods and a novel, visual monitoring device.

The western honey bee, Apis mellifera, faces a new threat from the spread of parasitic Tropilaelaps (Tropi) mites, specifically T. mercedesae, which adds additional complexity to an apicultural landscape heavily impacted by Varroa destructor. In this study conducted in central Thailand, we investigated the efficacy of two methods of applying formic acid and a thermal remediation technique in controlling Tropi mites and Varroa, focusing our attention on the reproductive stage of the mites which is restricted to capped brood cells. Results revealed that both formic acid treatments (Formic Pro and liquid formic acid) demonstrated an immediate and substantial reduction in live Tropi and Varroa populations, maintaining near-zero levels for the 3- week duration of the study. In contrast, thermal remediation, employing heating pads, exhibited a more gradual decline, achieving an 85.42% reduction in Tropi mites and a 92.33% reduction in Varroa mites by week three. Notably, heat-treated colonies experienced an unexpected resurgence in mite populations during week two. The findings contribute valuable insights into potential strategies for mitigating the threat of Tropi mites and highlight the urgency of further research to safeguard global honey bee populations.

In the era of global warming, much attention has been paid to the possibility that high temperature may influence diverse insects not only directly but also indirectly via effects on their symbiotic microorganisms. The stinkbug Plautia stali develops a midgut symbiotic organ that harbors a specific bacterial symbiont indispensable for its growth and survival. Being maintainable in laboratory and tractable experimentally, P. stali is recently highlighted as a model system to investigate the mechanisms underpinning insect-microbe symbiotic interactions. In this study, we reared newly-emerged adult insects of P. stali under different temperature conditions for 8 days and monitored how their symbiotic organs and symbiotic bacteria are affected. While all insects survived at temperatures from 25{degrees}C to 37{degrees}C, some insects died at 38{degrees}C, 39{degrees}C and 40{degrees}C, wherein mortality rates increased as temperature elevated. While the symbiotic organs of the normal insects exhibited vivid yellow color, the symbiotic organs of the insects reared at 35{degrees}C or higher frequently exhibited abnormal colors such as pale yellow, yellowish white, or white, the extent of which became more severe as temperature elevated. Symbiont quantification revealed that, while the symbiont titers were almost constant for 8 days at 25{degrees}C and 30{degrees}C, the symbiont titers on the 8th day drastically declined to 1/100 at 35 and 1/10000 at 37{degrees}C and 39{degrees}C. Based on these results, we propose that rearing at 37{degrees}C for a week is a recommended treatment regime by which the symbiont is effectively suppressed with minimal damage to the host insect.

Fipronil, a non-competitive antagonist of {gamma}-aminobutyric acid receptors (GABA-Rs), inhibits the flow of chloride ions in the nerve cells, leading to the insect death. In a previous work, the volatolome of bees chronically exposed to this insecticide (0.5 and 1 g/L) was analyzed and characterize the Apis mellifera metabolic responses. Two Volatile Organic Compounds or VOCs (1-octen-3-ol and 2,6-dimethylcyclohexanol) produced by bee were highlighted. We assumed that these VOCs could act as modulators of the GABA-R to counteract the effect of fipronil. The toxicity of these VOCs was tested on emerging bees for concentrations ranging from 0 to 3.4 g/L. Survival and the sucrose consumption were recorded during 21 days of chronic exposure. 1-octen-3-ol and 2,6-dimethylcyclohexanol did not significantly affect the proportion of survival at the end of the experiment, whatever their concentration. 1-octen-3-ol (0.5, 1, 1.7 and 3.4 g/L) was chosen to assess its effect in the case of co-exposure with fipronil (0.5 and 1 g/L). A beneficial effect on survival at 21 days was observed with an average improvement in survival rate for the co-exposed groups. This positive effect was not related to the VOC concentration and could be a direct effect (interaction with GABA-R) or a hormetic effect (global improvement of bee fitness).

O_LIHemiptera and Thysanoptera insects pose persistent threats to agricultural production. Conventional management strategies involve the release of chemical or plastic agents, causing adverse environmental and global health issues. Notably, Nicotiana benthamiana, a globally utilized model plant, exhibits remarkable lethal effects and attraction towards these pests. C_LIO_LIIn this study, we explored the potential of using N. benthamiana for Hemiptera and Thysanoptera pest control in the laboratory and field. Through net cover and three field assays over two years, we demonstrated the efficacy and benefits of using N. benthamiana as a field-deployed pest control dead-end trap. C_LIO_LIN. benthamiana demonstrated nearly 100% lethality to whiteflies, aphids, and thrips, with emitted volatiles attracting these insects. Field trials showed that potted and planted N. benthamiana blocks and traps whiteflies and thrips from several Solanaceae and Cucurbitaceae crops effectively, comparable to common commercial yellow and blue sticky boards. Moreover, N. benthamiana in the field exhibits robust growth in commercial greenhouses without negatively impacting crop growth, natural enemies, and pollinators. C_LIO_LIOur study introduces an innovative, easily implementable, and sustainable approach for controlling Hemiptera and Thysanoptera pests. Moreover, it unveils the novel utility of N. benthamiana in field-based pest management. C_LI

Immunosuppression and premature senescence are main risks of exposure to toxic compounds that might define individual longevity and the fate of natural animal populations. However, these sublethal effects have not been studied in insects that are of fundamental importance for human economy and well-being such as dung beetles. We exposed adult dung beetles Euoniticellus intermedius to ivermectin, a commonly used antiparasitic drug in cattle that is excreted in dung, and measured immune activity through phenoloxidase (PO) and its zymogen, prophenoloxidase (proPO), in males and females of different ages. We predicted that both ivermectin exposure and age would reduce immune activity, with the negative effect of ivermectin being more pronounced in older individuals, revealing immunosenescence. Despite PO and proPO activities decreased with age and ivermectin exposure, ivermectin effects on these immune mechanisms were constant across ages, revealing no immunosenescence caused by ivermectin. Whereas males suffered a reduction in PO and proPO with ivermectin and age, female proPO was not affected by ivermectin or age. This reveals a strategy of self-care adopted by females to prioritize immune function when facing stressful conditions. Among sublethal effects caused by ivermectin, immunosuppression might be a main physiological driver of population declines in contaminated environments.

As critical pollinators of agricultural crops, honey bees (Apis mellifera) play a vital role in food production. Therefore, it is imperative to investigate and develop new methods to control Varroa destructor, a devastating parasitic mite of honey bee colonies that weakens bees and spreads deadly diseases that lead to colony loss. Here, we adapted existing methods to investigate the role of ABCB1 transporters in mitigating the toxicity of amitraz, a widely used miticide approved for use in honey bee colonies, demonstrating that a pharmacological inhibitor can synergistically increase amitraz toxicity compared to the equivalent dose of amitraz alone. Evaluations performed on the mites used in one of the described experiments revealed a high proportion of the test subject mites (88.5%) possessed the amitraz resistant genotype, indicating that even in resistant mites, inhibiting ABCB1 transporters can increase amitraz efficacy. This promising finding may be useful in developing powerful synergists that can be used to increase the efficacy of new and existent miticides.

The mass-rearing of black soldier fly (Hermetia illucens) larvae (BSFL) is a promising solution for converting organic waste into high-quality insect protein, but preventing larval escape from open-top rearing containers remains a major management challenge. Conventional escape-control methods are often unreliable or impractical. To address this, we developed and evaluated a novel physical barrier, the anti-climbing tape, featuring regularly arranged macroscale protrusions designed to disrupt larval locomotion on vertical surfaces. We conducted a series of experiments to examine the design parameters of the anti-climbing tapes, including the gap size between protrusions and the number of protrusion rows. Our results demonstrate that the anti-climbing tape prevents escape via a dual mechanism: (1) physical obstruction, in which gaps narrower than the larval body width block larvae from passing through, and (2) adhesion reduction, in which the elevated protrusion array decreases the effective contact area for wet adhesion while increasing gravitational torque acting on the larval body. The effectiveness of these mechanisms was dependent on larval size. A design featuring 0.5-mm gaps and a 15-row protrusion array completely prevented the escape of later-instar larvae (>10 mm) in a 20-day large-scale trial, whereas the method was less effective for smaller larvae. In conclusion, the anti-climbing tape provides a robust and chemical-free approach to BSFL escape in mass rearing. To ensure reliable performance, its design parameters, both gap size and array width must be optimised to suppress the mechanical and adhesive components of larval climbing according to the target larval size. Conflict of interestS. Jang and M. Shimoda are inventors on a Japanese patent application (No. 2022-172252, filed November 27, 2022) related to the method described in this manuscript. FundingThis study was supported by Korea-Japan Joint Government Scholarship Program for the Students in Science and Engineering Departments, the Korean Scholarship Foundation, and the University of Tokyo Foundations Support Fund for International Students.

Huanglongbing (HLB, or citrus greening disease) affects all citrus varieties world-wide. In the USA, Asia, and South America the causal agent is "Candidatus Liberibacter asiaticus" (CLas), a phloem-limited, uncultured, alphaproteobacterium. The hemipteran insect vector, Diaphorina citri (Asian citrus psyllid) acquires and transmits CLas in a circulative, propagative manner. In addition to CLas, D. citri hosts multiple symbiotic bacterial species including Wolbachia (wDi). In D. citri, wDi has been sequenced and studied but specific roles in D. citri biology are unknown. Using well established quantitative PCR methods we measured CLas titer in D. citri collected from four groves in central Florida with distinct HLB management strategies and tested whether CLas and wDi titer were correlated in a sub-set of these insects. Grove site had the largest effect on CLas titer. Sex had no effect on CLas titer, while higher wDi titer was correlated with non-infected insects. Our results suggest that more directed follow-up research is necessary and important to clarify whether field management tactics influence CLas titer in D. citri and to better understand gene-by-environment interactions among D. citri, wDi and CLas. Now that millions of trees in Florida have been treated with injectable formulations of oxytetracycline, which is likely to decrease bacterial populations in D. citri, this study may represent the last biologically meaningful snapshot of grove-level vector-pathogen ecology in the state during the HLB epidemic.

Observing honeybee (Apis mellifera) brood dynamics and host-parasite interactions, especially within sealed cells, is vital for colony health research but traditionally relies on disruptive or destructive methods. Here we present BroodScan, a monitoring system employing a modified consumer-grade Charge-Coupled Device (CCD) flatbed scanner for continuous, high-resolution basal imaging of brood cells directly within an active beehive. Our experiments, conducted over a 35-day period, demonstrate BroodScans capacity to track the complete honeybee brood development cycle from egg to adult emergence, yielding quantitative data such as an average egg stage duration of 72.93 {+/-} 2.04 hours (n=58). Notably, the system provided detailed, time-lapse visual documentation of the complete intra-cellular reproductive cycle of the parasitic mite Varroa destructor within sealed worker cells, including the progression from foundress mite to multiple distinct offspring stages and their maturation. BroodScan offers a valuable, non-invasive tool that overcomes key limitations of previous techniques, opening new avenues for research into honeybee biology, colony health, and the impacts of diverse stressors like Varroa destructor, thereby promising to enhance our understanding and stewardship of these vital pollinators.

Appropriate design and selection of PCR primers plays a critical role in determining the sensitivity and specificity of a metabarcoding assay. Despite several studies applying metabarcoding to insect pest surveillance, the diagnostic performance of the short "mini-barcodes" required by high-throughput sequencing platforms has not been established across the broader taxonomic diversity of invasive insects. We address this by computationally evaluating the diagnostic sensitivity and predicted amplification bias for 68 published and novel cytochrome c oxidase subunit 1 (COI) primers on a curated database of 110,676 insect species, including 2,625 registered on global invasive species lists. We find that mini-barcodes between 125-257 bp can provide comparable resolution to the full-length barcode for both invasive insect pests and the broader Insecta, conditional upon the subregion of COI targeted and the genetic similarity threshold used to identify species. Taxa that could not be identified by any barcode lengths were phylogenetically clustered within problem groups, many arising through taxonomic inconsistencies rather than insufficient diagnostic information within the barcode itself. Substantial variation in predicted PCR bias was seen across published primers, with those including 4-5 degenerate nucleotide bases showing almost no mismatch to major insect orders. While not completely universal, a single COI mini-barcode can successfully differentiate the majority of pest and non-pest insects from their congenerics, even at the small amplicon size imposed by 2 x 150 bp sequencing. We provide a ranked summary of high-performing primers and discuss the bioinformatic steps required to curate reliable reference databases for metabarcoding studies.