Level of host concealment shape parasitoid community of microlepidopteran species living on hops

Level of host concealment shape parasitoid community of microlepidopteran species living on hops

Abstract

Background: Parasitoid-host interactions are key drivers of insect community structure, with host concealment influencing parasitoid diversity and parasitism rates. However, the effectiveness of different host defense strategies against parasitoids remains insufficiently understood.

Objective: This study examines how host concealment level affects parasitoid communities and parasitism rates in two microlepidopteran species developing on hops (Humulus lupulus L.), Caloptilia fidella and Cosmopterix zieglerella, which employ leaf-rolling and leaf-mining strategies, respectively.

Methods: We combined morphological identification with molecular species delimitation using ITS2 and CO1 markers and applied ASAP and bPTP methods to refine parasitoid taxonomy and detect cryptic species.

Results: Semi-concealed C. fidella larvae in leaf rolls experienced significantly higher parasitism than their mining stages, whereas fully concealed C. zieglerella had lower parasitism rates. Molecular analyses confirmed the idiobiont strategy in Sympiesis acalle, S. sericeicornis, and Elachertus fenestratus, and bPTP proved more sensitive in detecting cryptic species than ASAP.

Significance: These findings demonstrate that semi-concealed hosts face a higher risk of parasitism than fully concealed hosts, suggesting that leaf-mining provides better protection than leaf-rolling in studied hosts. The study also highlights the power of molecular tools in species delimitation, emphasizing their importance for refining parasitoid taxonomy and advancing our understanding of host-parasitoid interactions.

Key words

Parasitoid-host interactions; Species delimitation; Molecular taxonomy; Host defence; Gracillariidae; Cosmopterigidae.

Description of the data and file structure

Dataset Overview

The data for this study were collected to investigate the interactions between two host lepidopteran species, Caloptilia fidella and Cosmopterix zieglerella, and their associated parasitoids. The study focused on parasitoid species composition, bionomy, and parasitism rates. The aims were to (i) reveal how the contrasting defensive strategies of the two hosts affected their susceptibility to parasitism, (ii) identify key parasitoid species and their life histories, and (iii) assess ecological factors that shaped host-parasitoid dynamics.

Funding

This research was supported by the Grant Agency of Charles University (GAUK), project no. 375421 (TH) and grant PRIMUS/24/SCI/015 (PJ). Additional institutional support was provided by the Department of Entomology, National Museum of the Czech Republic under the project IP DRKVO 2024–2028/5.I.a, 00023272 and institutional support MZE-RO0423 (KH).

Author Contribution Statements

Tomáš Hovorka: funding acquisition; methodology; investigation; formal analysis; data curation; validation; visualisation; project administration; writing – original draft. Kamil Holý: conceptualization; resources; writing – review & editing. Cristina Vasilita: writing – review & editing. Lars Krogmann: writing – review & editing. Petr Janšta: funding acquisition; supervision; project administration; formal analysis; data curation; visualisation; writing – original draft.

Conflict of Interests

The authors declare that they have no conflicts of interest related to this work. The research was conducted in accordance with institutional and national ethical guidelines.

Methodology

Sampling and Rearing

Sampling was conducted between 2020 and 2022 across 18 sites in the Czech Republic, Slovakia, Hungary, Romania, and Croatia (Table 1). Whole hop leaves containing 1st or  2nd generation larvae of C. fidella (1st-5th instar) were collected, with a total of 50 leaves per site. For C. zieglerella, leaves with visible mines were collected in as many numbers as available per site (always < 50 leaves per location) where the species was present. Collected leaves containing mines or rolls with host species were transported to the laboratory in separate zip-lock bags for each host and site. Upon arrival, each leaf was carefully inspected and cleaned of any other insect hosts, most commonly (aphids, spider mites, planthopper nymphs and whiteflies). A sufficiently large section of the leaf containing the mine (C. zieglerella) or the corresponding host stage (C. fidella) was then clipped to allow the host species or its parasitoid to complete its life cycle and placed in sterile plastic petri dish (100 mm diameter, 15 mm height). The petri dishes were stored under controlled conditions (20°C, 75% humidity, and a 16:8 h light:dark cycle) in Trigon Plus ST 2 B SMART climate chambers (TRIGON PLUS Ltd., Czech Republic) for 40 days. Every two days until adult insects emerged, the petri dishes were inspected, and the leaves were moistened with a water solution of the fungicide LUNA® (fluopyram 200 g/L and tebuconazole 200 g/L; Bayer AG, Germany) using a laboratory sprayer to prevent mould during rearing. This fungicide has no adverse effects on arthropods (tested by Central Institute for Supervising and Testing in Agriculture, Czech Republic). The specific developmental biology of the host and its parasitoids was recorded. The biology of the parasitoids was determined during their development on the hosts using forceps and dissecting needles to carefully open the leaf shelters, ensuring minimal disruption to their development. Additionally, parasitoids were monitored to determine whether they were koinobionts or idiobionts and classified as either ectoparasitoids or endoparasitoids. Whenever feasible, individual parasitoids were photographed. Direct observation of parasitoid life strategies was supplemented by post-emergence dissection of leaf mines and shelters to analyze remnants of host larvae, pupae, parasitoid cocoons, and the positions of emergence or exit holes. Additionally, the pupae or cocoons of parasitoids were monitored for the potential occurrence of hyperparasitoids. After emergence, the adults of hosts or parasitoids were stored in 96% ethanol at -20°C until DNA isolation.

Genetic Analysis

DNA was extracted from specimens preserved in 96% ethanol using the Qiagen DNeasy Blood & Tissue Kit with a modified non-destructive protocol based on Cruaud et al. (2019). This protocol was optimized for high DNA yield from small insect specimens while preserving them for morphological identification. Specimens were incubated with lysis buffer and proteinase K at 56°C for 20 hours under gentle vortexing (250rpm). Post-incubation, lysate was transferred to fresh tubes for DNA purification. Elution was performed in two steps using a pre-warmed elution buffer, yielding a final volume of 100 µL with high DNA concentration. Additionally, for high-throughput processing, some samples were extracted using the Xiril AG X100 Automatic Workstation according to Haas et al. (2021) excluding the semi-destructive step as we used the whole specimens. Target gene fragments included CO1 (mitochondrial DNA): LCO1490 (5′-GGT CAA ATC ATA AAG ATA TTG -3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA -3′) (Hebert et al., 2003) and ITS2 (nuclear ribosomal DNA):  ITS2-F (5′-TGT GAA CTG CAG GAC ACA TG -3′) and ITS2-R (5′-AAT GCT TAA ATT TAG GGG GTA -3′) (Campbell et al., 1994). PCR reactions were prepared in 25 µL volumes containing FastGene Optima HotStart Ready Mix (12.5 µL), primers (1 µL), PCR clean H2O (8.5 µL) and template DNA (2 µL). Thermal cycling conditions for CO1 included an initial denaturation at 94°C for 2 minutes, followed by a two-step protocol with annealing temperatures of 45°C for 1 minute (for 5 cycles) and 50°C for 1 minute (for 35 cycles), extension at 72˚C for 1.5 minutes for every cycle and for ITS2 amplification followed a similar conditions but used a single annealing temperature of 53°C for 45 seconds (33 cycles). Successful PCR products were purified using ExoSAP-IT™ (1.5µL of PCR clean H2O, 1.5µL of ExoSAP-IT, 1.5µL of PCR product; cycling conditions - 37°C for 15 minutes and 80°C for 15 minutes) and sequenced bidirectionally using Sanger sequencing (Eurofins Genomics, Germany, Ebersberg).

Sequences were processed in Geneious Prime 2023.2.1 (https://www.geneious.com/). Initially, sequences were categorized by taxonomy (Chalcidoidea or Ichneumonoidea), host (C. fidella or C. zieglerella), and gene (CO1 or ITS2). Forward and reverse of every  sequence were assembled, and BLAST was used to determine the taxonomic identity of individuals. Consensus sequences were generated after successful assembly. Alignments were performed in Geneious using the MAFFT plugin with the E-INS-i strategy for ITS2 and L-INS-i strategy for CO1 (Katoh & Standley, 2013), followed by manual verification and trimming to a uniform length. For CO1 sequences, translations to amino acids were checked for stop codons using Geneious for pseudogenes or misalignments. A concatenated alignment of both genes and hosts was created for Chalcidoidea, while for Ichneumonoidea, only the CO1 alignment was used due to the lack of high-quality ITS2 sequences. BLAST was used to verify the taxonomic identity of individuals.

Phylogenetic trees were constructed using the maximum likelihood method in RAxML-HPC2 on XSEDE (8.2.12; Stamatakis, 2006) via the CIPRES server (Miller et al., 2010). The GRTCAT model was used with 1,000 bootstrap replicates (Stamatakis, 2006). Bootstrap percentages (BP) ≥ 70% were considered as strong nodal support. Resulting trees for the concatenated Chalcidoidea dataset and the CO1 alignment for Ichneumonoidea were visualized in FigTree v1.4.4 (Rambaut, 2009). Bayesian analysis was performed in MrBayes v3.2.7 (Ronquist et al., 2012), with input files in NEXUS format set two independent runs with 1,000,000 generations each, saving every 1,000th tree. Parameter files were inspected in Tracer (Rambaut et al., 2018) and the first 25% of trees were discarded as burn-in. Posterior probabilities (PP) ≥ 0.95 were considered as strong support, PP < 0.90 as weak. Final trees were visualized in FigTree, with graphical edits in iTOL (https://itol.embl.de/; Letunic & Bork, 2021).

For species delimitation both gene fragments were used to define species-level entities (OTUs- operational taxonomic units) using two online-available tools. The first method employed was ASAP (Assemble Species by Automatic Partitioning), as described by Puillandre et al. (2021) and Zhang et al. (2022). This method proposes the delimitation of hypothetical species based on genetic distances calculated between DNA sequences. The default settings in the online ASAP interface (available at https://bioinfo.mnhn.fr/abi/public/asap/) were used for the species delimitation analysis. The second method, bPTP, delineated species-level entities using maximum likelihood and the Bayesian implementation of the Poisson Tree Processes (PTP) model for species delimitation (available online at https://species.h-its.org/; Zhang et al., 2013). For the bPTP analysis, the online interface was configured with model settings of 200,000 Markov chain Monte Carlo (MCMC) generations, thinning of 100, and a burn-in of 0.1. ASAP was run on the CO1 and ITS2 sequence alignments in FASTA format without outgroups, while bPTP was run on the phylogenetic trees obtained from these alignments using the maximum likelihood approach.. Additionally, genetic distances across sequences containing CO1 and ITS2 gene fragments were calculated in MEGA 11 using the Kimura-2-parameter (K2P) model (Tamura et al., 2021). Groups of similar sequences were identified using a 2% barcode threshold (Hebert et al., 2003).

Morphological Identification

To facilitate morphological identification, after lysis every specimen was washed two times in water (15 minutes each bath) and stored in 80% EtOH. Drying was performed according to a modified protocol (Heraty & Hawks, 1998) using hexamethyldisilazane (HMDS). The procedure involved sequential immersion in 90% and 95% EtOH for 30 minutes each, followed by two washes in 100% EtOH for 15 minutes each. Specimens were then placed in HMDS for two 30-minute intervals. After removing HMDS, specimens were left to dry. Due to the volatile nature of HMDS, the process was conducted under a fume hood with appropriate protective equipment. Once dried, specimens were mounted on cards. Prepared specimens were sorted and identified using a Leica M205C stereomicroscope (Leica Microsystems). Identification was based on available keys for Ichneumonoidea (e.g., Nixon, 1965; Whitfield & Wagner, 1991) and Chalcidoidea (e.g., Nikol’skaya, 1952; Bouček, 1959; Yoshimoto, 1983; Gibson et al., 1997).

Statistical Analysis

Data were analyzed using the freely available statistical program R (version 4.1.1) with the FSA library. A non-parametric Kruskal-Wallis test was conducted to determine whether parasitism rates differed among the mine, leaf roll, and pupae stages, as the data did not meet the assumptions of normal distribution. To further explore these differences, a post-hoc Dunn test with Bonferroni correction was applied. Additionally, the Wilcoxon test was used to compare parasitization rates between the two generations.

Files and variables

File: Figure_1.tif

Description: Phylogenetic tree of Chalcidoidea parasitoids based on RAxML analysis of concatenated COI and ITS2 sequences. Bootstrap values >70 are indicated at branch nodes. Branch colors correspond to different families, while the background shading of parasitoid species represents their respective hosts. Additionally, colors distinguish the host life stages parasitized, recorded parasitoid bionomy, and species delimitation based on genetic distances using the ASAP and bPTP methods.

File: Figure_2.tif

Description: Overview and associations of observed parasitoids in this study with the studied hosts. The thickness of the lines between species indicates the frequency of parasitoid-host associations, i.e., thicker lines represent more frequent associations. Colored symbols indicate the developmental stages of the host from which the parasitoids emerged.

File: Figure_5.tif

Description: Parasitoids emerged from Caloptilia fidella (CF), Cosmopterix zieglerella (CZI) and Pholetesor circumscriptus (PC). A. Acrolyta rufocincta (PC), B. Gelis agilis (PC), C., D. Sympiesis dolichogaster (PC), E. S. acalle (CF), F. S. sericeicornis (CF), G., H., I. Pupa, larva and adult of Chrysocharis purpureus (CF), J. Egg near paralized caterpillar of CF, K. Pnigalio sp. (CF, CZI), L. larva of Elachertus fenestratus in mine of CZI, M. E. fenestratus emerged from mine of CZI. 

File: Figure_3.tif

Description: Life cycle of two microlepidoptera living on hops. A–E. Caloptilia fidella: A. Mining larva within a leaf mine located between the veins at the leaf axil. B. Leaf mine and a leaf roll with visible silk threads spun by the larva. C. Silvery silk cocoon with remnants of the larval pupa on the underside of the leaf. E–H. Cosmopterix zieglerella: E. Characteristic leaf mine on hop leaves, which later develops into a broader, flattened shape. F. Young larva. G. Final instar larva before pupation. H. Adult moth. 

File: Figure_4.tif

Description: Parasitoids of the subfamily Microgastrinae reared from hosts and identified to species. A. Cocoon with polar threads and an opened top, typical for the parasitoid Pholetesor circumscriptus (visible in the bottom left corner is an emergence hole chewed by the parasitoid in the leaf roll of the host Caloptilia fidella). B. Lateral habitus of P. circumscriptus (female) reared from C. fidella. C. Characteristic silk cocoon of the parasitoid Microgaster novicia located inside the mine of the host Cosmopterix zieglerella. D. Lateral habitus of M. novicia (male) reared from the host C. zieglerella.

File: Table_1.xlsx

Description: Observed parasitoids of Caloptilia fidella and Cosmopterix zieglerella recorded in this study. This table presents all parasitoid species recorded in our study, based solely on our observations of Caloptilia fidella (CF) and Cosmopterix zieglerella (CZI). The table includes information on their life strategy, host association, and developmental characteristics. Abbreviations used in the table: M = mine, LR = leaf roll, CCF = cocoon of C. fidella, CCB = cocoon of the braconid P. circumscriptus, SOL = solitary, IDIO = idiobiont, ECTO = ectoparasitoid, ENDO = endoparasitoid, KOION = koinobiont, H = hyperparasitoid, P = primary parasitoid, P/H = primary or secondary parasitoid, PC = Pholetesor circumscriptus, ? = uncertain or undetected life strategy in our research. The background color in the table represents the host association: yellow shading corresponds to CF, green shading corresponds to CZI, and white cells indicate hyperparasitoids.

File: Table_2.xlsx

Description: Total and average parasitism rates of individual hosts recorded in this study.

Supplementary files

File: Figure_S1_SuppInfo.pdf

Description: Phylogenetic tree of Chalcidoidea parasitoids based on MrBayese analysis of concatenated COI and ITS2 sequences.

File: Figure_S2_SuppInfo.tiff

Description: Phylogenetic tree of Ichneumonoidea parasitoids based on RAxML analysis of COI sequences. Bootstrap values >70 are indicated at branch nodes. Branch colors correspond to different families, while the background shading of parasitoid species represents their respective hosts. Additionally, colors distinguish the delimitation based on genetic distances using the ASAP and bPTP methods.

File: Figure_S3_SuppInfo.pdf

Description: Phylogenetic tree of Ichneumonoidea parasitoids based on RAxML analysis of COI.

File: Figure__S4_SuppInfo.pdf

Description: Phylogenetic tree of Ichneumonoidea parasitoids based on MrBayese analysis of COI.

Sequence datasets

File: Sequence_list_1_CO1_Chalcidoidea.fasta

Description: List of CO1 gene sequences in parasitoids of Chalcidoidea.

File: Sequence_list_2_CO1_Ichneumonoidea.fasta

Description: List of CO1 gene sequences in parasitoids of Ichneumonoidea.

File: Sequence_list_3_ITS2_Chalcidoidea.fasta

Description: List of ITS2 gene sequences in parasitoids of Chalcidoidea.