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Carabids as biocontrol agents for slugs in Oregon and Ireland - a novel and interdisciplinary approach to determine key malacophagous species and beneficial management options

Periodic Reporting for period 1 - CaraSlug (Carabids as biocontrol agents for slugs in Oregon and Ireland - a novel and interdisciplinary approach to determine key malacophagous species and beneficial management options)

Reporting period: 2017-10-01 to 2019-09-30

Agricultural pests such as slugs are major contributors to financial losses in the farming sector and the use of pesticides, which is currently the mainstay of control, is an unsustainable management option due to their toxicity to non-target species and consumers but also because of their cost and variable efficacy. Pest control using natural enemies poses a more sustainable approach. Ground beetles are the most significant natural enemies of slugs in agroecosystems and several studies have shown that slugs are a major part of their diet. Most ground beetle species do not exclusively eat slugs, and the presence of alternative food sources is crucial for their survival during slug-free periods. However, alternative prey can also impact the consumption rate of the target pest species by being preferentially consumed. Additionally, factors such as field margin composition / structure or production practices such as tillage can have a substantial influence on the abundance and community composition of farmland invertebrates.
This project aims to assess the potential of ground beetles as biocontrol agents against slugs and other pests in grass seed fields in western Oregon and grass fields in Ireland and to evaluate the influence of tillage and/or field margin structure on the abundance of slugs and other agricultural pests, ground beetles and invertebrates which could serve as possible alternative prey for the beetles. The control potential is evaluated by examining the abundance of ground beetles in agricultural fields at times when slugs cause most damage and through molecular gut-content analysis using high-throughput sequencing (to assess the breadth of their diet) as well as real-time PCR (to screen specifically for slugs, caterpillars and crane fly larvae and to get an approximate quantification of their predation).
This research generates valuable knowledge aimed at working towards a more environmentally friendly approach to farming by reducing the use of toxic molluscicides.
Field study
10 fields in the Willamette Valley, Oregon, were sampled bi-monthly between April 2018 and June 2019 using refuge traps for slugs and caterpillars and dry pitfall traps for carabids and other invertebrates. 5 fields were not tilled before planting in fall 2017 but were tilled 2018 (‘experimental fields’), while the other 5 fields (‘control fields’) were tilled both years.
After sampling, carabids were identified to species and other invertebrates to at least order level, slugs and caterpillars were weighed and identified to species level. Grass height and slug damage were recorded once a month and a soil sample was taken to determine soil moisture.
DNA was extracted from the most abundant slug, carabid and other invertebrate species and sequences of partial COI gene were obtained to build up a reference library.

Carabid gut content analysis
879 guts (crop plus proventriculus) were dissected from 9 carabid and 1 rove beetle species which were selected as they were either very abundant during the study or large in size (>10mm) and from the larvae of two carabid and one rove beetle species that were commonly collected during fieldwork. The sex, size and number of eggs of each beetle was noted, as well as the condition of the gut (empty, very little food, moderately full, full, very full). DNA was extracted using the QIAGEN Blood & Tissue Kit.

The primers employed for qPCR screening for Gastropoda, Lepidoptera and Tipulidae DNA were tested for amplification success with a range of target and non-target species using both qPCR and conventional PCR. The half-life detectability of gastropod DNA in N. brevicollis was also tested. All samples were run on Applied Bioscience QuantStudio 3 or Step-One Plus real-time PCR systems using SYBR green PCR Mastermix.

The Next-Generation sequencing (NGS) primer set was tested on several species of slugs, lepidoptera and aphids and successfully amplified all except for one slug species. Gut extractions were pooled by species and sampling date, and after a PCR with the NGS primers plus Illumina adapter overhangs, a 96 sample plate was submitted for 2x300bp sequencing on Illumina MiSeq v3.

Feeding trials were carried out to assess whether N. brevicollis was capable of predating on live slugs/eggs or whether the positive results were more likely due to scavenging.

Main results
1. Activity patterns of the most common carabid species N. brevicollis overlap with the fall and spring juvenile life stages of D. reticulatum, those of two other common carabid species (Agonum muelleri and Poecilus laetulus) overlap with the spring juvenile life stages of the slugs.
2. Slug numbers did not significantly change in the spring 2019 compared to 2018 in the experimental fields (Wilcoxon signed-rank test (WSRT), p=0.094) or the control fields (WSRT, p=0.056)
3. Two carabid species, N. brevicollis and A. longula showed a significant decrease in abundance (WSRT, p<0.001) in the spring 2019 compared to spring 2018 in the experimental fields, while N. brevicollis abundances were significantly increased (WSRT, p<0.001) in the control fields in spring 2019.
4. N. brevicollis was the only carabid that tested positive for gastropod DNA (3.9% of all tested individuals). 6.4% of N. brevicollis tested positive for Lepidoptera DNA and 3.2% for Tipulidae. 1.9% of A. muelleri tested positive for each of Lepidoptera and Tipulidae DNA; 5.9% of C. cancellatum tested positive for Lepidoptera DNA; 1% P. laetulus and 7% P. melanarius tested positive for Tipulidae DNA.
5. N. brevicollis did not feed on the eggs or live slugs in the feeding trials.
The original proposal focused solely on slugs as pests in grass seed fields, however, it was noted during sampling that large quantities of caterpillars and cranefly larvae also congregated underneath the blanket traps. As these, these can also cause serious damage to their grass crops so I am including them in my study. Additionally, I have collected life history data on carabids in my study such as sex, size and number of eggs in gravid females which will be published. Another paper I am preparing with the data I have collected will be about N. brevicollis as an exotic and possibly invasive species in Oregon. Expected results until the end of the project will include multivariate statistical analysis of my field data with the goal of elucidating key factors that benefit beneficial carabid species in the fields. Beneficial carabids will have been identified through the outcomes of the molecular gut content analyses (indicating so far that N. brevicollis and C. cancellatum might have biocontrol potential) and feeding experiments. I will also gather results on carabid and slug activity density and community composition from extensively and intensively managed farmland in the West of Ireland which are part of the FARM-ECOS project (a collaboration between Teagasc, NUI Galway, Sligo IT, Trinity College Dublin and Dublin City University). This collaboration will generate critical mass for the CaraSlug project.
Additionally, I will have prepared and delivered a module on Biocontrol and Integrated Pest Management which will be accessible online.
The outcomes of this study and the module I am preparing will promote the use of beneficial arthropods as an alternative to toxic pesticides in invertebrate pest control in agriculture, paving the way towards a more sustainable future in agriculture.
Calosoma cancellatum and spiders caught in pitfall trap
Dissected Nebria brevicollis
Nebria brevicollis feeding on slug in feeding trial