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Background Diaprepes abbreviatus is a major pest causing severe root damage and yield loss in over 150 species, with a complex life cycle that hinders effective control. Limited genomic and transcriptomic resources have restricted molecular insights into its biology. De novo transcriptome assembly provides a practical approach for reconstructing the full set of transcripts in organisms without a reference genome, enabling the identification of novel genes and isoforms as well as the study of gene expression patterns. Results We performed RNA-seq across seven developmental stages of D. abbreviatus , generating a high-quality de novo transcriptome assembly comprising 991,860 unique transcripts and 505,007 unigenes with 99.2% completeness based on BUSCO analysis. Gene structure annotation predicted 75,895 coding sequences with an average length of 1,079 bp. Comparative transcriptomic analysis of adult males and females identified 16,082 significantly differentially expressed transcripts, emphasizing distinct sex-biased gene expression patterns. Functional classification revealed major enrichment in metabolism, signal transduction, transport, and developmental processes. Additionally, genes related to mitochondrial function, muscle structure, and detoxification pathways exhibited clear sex-specific expression. Gene Ontology (GO) and KEGG pathway analyses further emphasized enrichment in detoxification, xenobiotic metabolism, and hormonal regulation pathways. We further validated this differential expression data by RT-qPCR analysis. Conclusions We present a high-quality de novo transcriptome assembly of D. abbreviatus with detailed, stage-specific transcript coverage. The identification of key developmentally regulated genes offers valuable insights into the molecular mechanisms underlying their biology. This resource lays the groundwork for future functional studies and the development of targeted pest management strategies, contributing to sustainable control of this economically important pest.

1. Indirect plant defences are well documented for the above‐ground constituents of plants. Although less investigated to date, below‐ground defences that mediate multitrophic interactions are equally important. Entomopathogenic nematodes (Steinernema diaprepesi) are attracted to herbivore‐induced volatiles from Swingle var. (Citrus paradisi×Poncirus trifoliata) when fed upon by root weevil, Diaprepes abbreviatus. 2. We examined the extent to which below‐ground volatiles modify behaviour of nematode species representing various foraging strategies (cruisers versus ambushers) and trophic levels (plant parasites versus entomopathogens). We compared attraction to volatiles of weevil‐infested and non‐infested roots from Swingle citrus rootstock and a parent line of the Swingle hybrid, Poncirus trifoliata (Pt). 3. Swingle weevil‐infested roots attracted more nematodes than non‐infested roots irrespective of nematode foraging strategy and trophic status. The parental line, Pt, attracted all nematode species irrespective of insect herbivory. 4. Dynamic in situ collection and GC-MS analysis of volatiles from soil revealed that Pt roots release attracting cues constitutively. A different non‐hybrid citrus species (sour orange, Citrus aurantium) released nematode attracting cues only in response to larval feeding, similar to responses found in Swingle. Volatile collections from above‐ and below‐ground portions of citrus plants revealed that above‐ground feeding by weevils does not induce production of nematode attracting cues analogous to that induced by root damage, nor does damage by larvae below‐ground induce a similar volatile above ground. 5. Synthesis. Our results suggest that release of nematode attractants by citrus roots occurs broadly and can be constant or herbivore‐induced. The major constituent of this indirect defence is produced by roots and not shoots and in response to below‐ground, but not above‐ground herbivory. Our findings suggest that this cue acts on nematode species broadly, attracting entomopathogenic nematodes that exhibit various foraging strategies. Unexpectedly, we also found that this cue attracts a plant parasitic nematode species. It appears, thus, that release of nematode attracting cues by citrus plants can cause ecological costs. The plants, however, appear to counteract against these costs, because constitutive release was found only in a cultivar that is resistant to phytopathogenic nematodes, while herbivore‐induced release occurred in lines susceptible to pathogenic nematode species.

While the role of herbivore-induced volatiles in plant-herbivore-natural enemy interactions is well documented aboveground, new evidence suggests that belowground volatile emissions can protect plants by attracting entomopathogenic nematodes (EPNs). However, due to methodological limitations, no study has previously detected belowground herbivore-induced volatiles in the field or quantified their impact on attraction of diverse EPN species. Here we show how a belowground herbivore-induced volatile can enhance mortality of agriculturally significant root pests. First, in real time, we identified pregeijerene (1,5-dimethylcyclodeca-1,5,7-triene) from citrus roots 9-12 hours after initiation of larval Diaprepes abbreviatus feeding. This compound was also detected in the root zone of mature citrus trees in the field. Application of collected volatiles from weevil-damaged citrus roots attracted native EPNs and increased mortality of beetle larvae (D. abbreviatus) compared to controls in a citrus orchard. In addition, field applications of isolated pregeijerene caused similar results. Quantitative real-time PCR revealed that pregeijerene increased pest mortality by attracting four species of naturally occurring EPNs in the field. Finally, we tested the generality of this root-zone signal by application of pregeijerene in blueberry fields; mortality of larvae (Galleria mellonella and Anomala orientalis) again increased by attracting naturally occurring populations of an EPN. Thus, this specific belowground signal attracts natural enemies of widespread root pests in distinct agricultural systems and may have broad potential in biological control of root pests.

Diaprepes abbreviatus L. (Coleoptera: Curculionidae) is an occasional root pest of plasticulture strawberry in central Florida, USA. There are few chemical insecticide options for larval D. abbreviatus in strawberry. Therefore, we tested soil-applied aqueous Steinernema riobrave Cabanillas, Raulston and Poinar (Rhabditida: Steinernematidae), which is used for D. abbreviatus control in citrus. When S. riobrave was applied (100 infective juveniles per cm2) to the root zones of plants in a D. abbreviatus-affected area of a commercial strawberry field, less than 12% of plants were severely wilted or dead 17 d after treatment, whereas 23% of plants in control plots were wilted or dead. In research plots, peripheral plants and a central plant in each plot were infested with 4 late-instar D. abbreviatus and treated with 1 or 2 applications of S. riobrave (25 infective juveniles per cm2), 1 application of imidacloprid or water (control). Dead S. riobrave-infected D. abbreviatus larvae were recovered from plots to which S. riobrave was applied, but there was no effect of treatment on numbers of live larvae recovered 1, 2, and 3 wk post-application in the peripheral plants. At the end of the experiment, no live D. abbreviatus larvae were recovered from the central plants or the plants proximal to the central plants in plots treated once or twice with S. riobrave. Plant wilting and death due to larval D. abbreviatus root feeding was minimal (averages of 1–1.4 on a 1–5 rating scale) in plants proximal and distal to the central D. abbreviatus-infested plant. Treatment did not affect plant wilting and death rates in the proximal plants, but death of distal plants occurred only in control plots. Our results showed S. riobrave infected and killed late-instar D. abbreviatus in plasticulture Florida strawberry, and further research should be conducted to optimize S. riobrave applications and develop it into a management strategy for D. abbreviatus.

Plant defense pathways play a critical role in mediating tritrophic interactions between plants, herbivores, and natural enemies. While the impact of plant defense pathway stimulation on natural enemies has been extensively explored aboveground, belowground ramifications of plant defense pathway stimulation are equally important in regulating subterranean pests and still require more attention. Here we investigate the effect of aboveground stimulation of the salicylic acid pathway through foliar application of the elicitor methyl salicylate on belowground recruitment of the entomopathogenic nematode, Steinernema diaprepesi. Also, we implicate a specific root-derived volatile that attracts S. diaprepesi belowground following aboveground plant stimulation by an elicitor. In four-choice olfactometer assays, citrus plants treated with foliar applications of methyl salicylate recruited S. diaprepesi in the absence of weevil feeding as compared with negative controls. Additionally, analysis of root volatile profiles of citrus plants receiving foliar application of methyl salicylate revealed production of d-limonene, which was absent in negative controls. The entomopathogenic nematode S. diaprepesi was recruited to d-limonene in two-choice olfactometer trials. These results reinforce the critical role of plant defense pathways in mediating tritrophic interactions, suggest a broad role for plant defense pathway signaling belowground, and hint at sophisticated plant responses to pest complexes.

Herbivore-induced volatile emissions benefit plant hosts by recruiting natural enemies of herbivorous insects. Such tritrophic interactions have been examined thoroughly in the above-ground terrestrial environment. Recently, similar signals have also been described in the subterranean environment, which may be of equal importance for indirect plant defense. The larvae of the root weevil, Diaprepes abbreviates, are a serious pest of citrus. Infestations can be controlled by the use of entomopathogenic nematodes, yet the interactions between the plant, insect and nematode are poorly understood and remain unpredictable. In bioassays that used a root zone six-arm olfactometer, citrus roots (‘Swingle citrumelo' rootstock) recruited significantly more entomopathogenic nematodes (Steinernema diaprepesi) when infested with root weevil larvae than non-infested roots. Infested plants were more attractive to nematodes than larvae alone. Roots damaged by weevil larvae attracted more nematodes than mechanically damaged roots and sand controls. By dynamic in situ collection and GC-MS analysis of volatiles from soil, we determined that four major terpene compounds were produced by infested plant roots that were not found in samples from non-infested roots or soil that contained only larvae. Solvent extracts of weevil-infested roots attracted more nematodes than extracts of non-infested roots in a two choice sand-column bioassay. These findings suggest that Swingle citrus roots release induced volatiles as an indirect defense in response to herbivore feeding, and that some of these induced volatiles function as attractants for entomopathogenic nematodes.

Diaprepes abbreviatus is an agricultural pest known to affect around 270 plant species across the Caribbean and the United States, posing significant challenges to pest management. Chemical control dominates management, but environmental and health concerns motivate microbiome-informed alternatives. However, limited information exists on the gut anatomy, physicochemical environment, and microbial composition of D. abbreviatus. In this study, we provide the first comprehensive characterization of the gut morphology, pH, and microbiota of adult D. abbreviatus in both females and males collected in Puerto Rico. Using dye-based gut tracing, we identified foregut, midgut, and hindgut or posterior gut compartments, and confirmed the presence of a muscular, sclerotized gizzard. Colorimetric analysis revealed a mildly acidic gut environment (approximately pH 4–5, based on qualitative ranges), consistent across sexes and regions. Shotgun metagenomic sequencing of dissected guts from males and females revealed microbial communities distinct from the leaf samples microbiota. While alpha and beta diversity did not differ significantly between sexes, co-occurrence analyses identified sex-specific correlation patterns among bacterial taxa. Notably, Enterobacter cloacae , Pantoea vagans , Lactococcus lactis , and Pseudomonas monteilii were repeatedly detected across individuals and generated metagenomic datasets, and some were localized to the hindgut, suggesting possible niche specialization. The presence of taxa, such as Enterobacter cloacae , previously reported as symbionts in other phytophagous insects further supports the hypothesis that certain bacteria may contribute to host digestion or adaptation. These findings establish a framework for understanding the gut environment and microbial community of D. abbreviatus , and highlight candidate taxa for future functional studies. More broadly, this work supports further research into the potential roles of gut microbiota in the ecology and management of this pest.

The EFSA Panel on Plant Health performed a pest categorisation of the citrus root weevil Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) for the European Union (EU) following the commodity risk assessment of Ligustrum delavayanum topiary grafted on L. japonicum plants from the UK in which D. abbreviatus was identified as a relevant non‐regulated EU pest which could potentially enter the EU. This species is native to the Caribbean and was introduced to the continental USA in 1964, to Gran Canaria (Spain) in 2014 and to Madeira Island (Portugal) in 2018. It is a polyphagous insect, associated with more than 270 species in 60 plant families. Female D. abbreviatus can lay up to 5,000 eggs in clusters within leaves folded and glued together. Neonate larvae drop off the leaves onto the ground and enter the soil, where they feed on roots for several months. The mature larvae pupate in the soil. After emergence, adults usually stay on the first host plant they encounter and can move long distances on nursery stock. D. abbreviatus is not a regulated pest in the EU. It could enter and spread within the EU via the import and movement of host plants for planting, cut flowers and soil. Some host plants for planting (e.g. Vitis spp., Citrus spp.), and soil are prohibited from entering the EU from countries where this weevil is known to occur. The import of other host plants for planting and cut flowers is subject to phytosanitary certificate and that of soil attached to machinery is regulated. Host availability and climate suitability suggest that the southernmost coastal areas of southern EU MSs would be suitable for establishment of D. abbreviatus. Temporary establishment in greenhouses in other EU territories would be possible. The introduction of D. abbreviatus would likely cause impacts. Measures to prevent entry, spread and impact are available. D. abbreviatus satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

The Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae), was discovered as the cause of early decline and death of citrus trees and ornamental plants along coastal Orange and Los Angeles Counties in 2005 and San Diego County in 2006. We established trials to evaluate the effectiveness of two tactics to manage life stages of Diaprepes root weevil that are found in the soil. The first tactic was soil applications of the pesticides bifenthrin or imidacloprid, with and without entomopathogenic nematodes, to kill larvae. The second tactic was attempting to block adult emergence from the soil using a thick wood-chip mulch layer, a small-meshed landscape fabric, and a dry soil barrier (created by subirrigation). We found that soil treatments with the two insecticides (bifenthrin and imidacloprid) at maximum label rates with and without Steinernema riobrave Cabanillas et al. at 5 million nematodes per tree significantly reduced numbers of soil-borne Diaprepes root weevil larvae in one trial compared with the control. Another entomopathogenic nematode, Heterorhabditis indica Poinar et al. applied in wax moth Galleria mellonella L. cadavers at 86 wax moth cadavers per tree was not effective. Adult emergence was reduced by almost 100% when the landscape fabric was kept intact (2009), but emergence occurred in 2010 when the fabric was torn by grove operations (60% reduction). Adult emergence was reduced to approximately 70% compared with the control in the subirrigation treatment in 2009 when the summer was hot and dry, but emergence occurred in 2010 during periodic rains toward the end of summer. The mulch treatment kept the soil moist in between irrigations resulting in greater adult emergence rates compared with controls during 2009 and 2010. Health ratings of the trees were taken following several years of physical barrier treatments, and trees treated with the landscape fabric were significantly healthier (mean rating 0.9, scale 0 = healthy to 5 = dead) than those in the other treatments (mean rating of 3.6, 4.0, and 2.5 for control, mulch and subirrigation, respectively). Of the physical barrier methods studied, landscape fabric is most effective in reducing Diaprepes root weevil emergence, increases plant health, and is long lasting, but it is the most costly to install and can be damaged during harvest.

Mexico is a center of New World crop origins with many insect species associated with the wild crop relatives, some of which have become pests of cultivated plants. Many of these indigenous pests of Mexico are weevils (Coleoptera: Curculionidae), and some represent potential quarantine pests. In the present work, we present some of the most important species of weevils considered as crop pests, which are presently restricted primarily to Mexico with the potential to invade other regions. We also present species of weevils exotic to Mexico, considered by national quarantine authorities to be possible threats to Mexican agriculture. Potential quarantine weevil pests for avocado include Conotrachelus aguacatae Barber, Conotrachelus perseae Barber, Heilipus lauri Boheman, Heilipus albopictus Champion, and Copturus aguacatae Kissinger (all Coleoptera: Curculionidae). For guava, species include Conotrachelus dimidiatus Champion and Conotrachelus copalensis Salas-Araiza and Romero-Nápoles (both Coleoptera: Curculionidae). Weevil pests reported with quarantine importance for sugar cane are Sphenophorus incurrens Gyllenhal (Coleoptera: Dryophthoridae), Apinocis angustus (Casey), and Apinocis subnudus (Buchanan) (both Coleoptera: Curculionidae). The weevil Rhyssomatus nigerrimus Fåhraeus (Coleoptera: Dryophthoridae) recently has been observed damaging soybeans in northern and southern Mexico. Cactophagus spinolae Gyllenhal (Coleoptera: Dryophthoridae) is a generalist cactus pest. Scyphophorus acupunctatus Gyllenhal (Coleoptera: Dryophthoridae) is a well-known pest of cultivated and wild agaves. Several species of Epicaerus (Coleoptera: Curculionidae) have been reported on various crops in restricted areas including, among others, Epicaerus operculatus (Say) on garlic, and Epicaerus cognatus Sharp (both Coleoptera: Curculionidae) on potato. Amphidees latifrons (Sharp) (Coleoptera: Curculionidae) has been reported from apples in localized regions of northern Coahuila. Weevils exotic to Mexico of quarantine concern are Naupactus cervinus Boheman, Diaprepes abbreviatus (L.) (both Coleoptera: Curculionidae), and Rhynchophorus ferrugineus (Olivier) (Coleoptera: Dryophthoridae).