Research Article |
Corresponding author: Hana Konvičková ( hpatzenhauerova@gmail.com ) Academic editor: Petr Janšta
© 2024 Hana Konvičková, Václav John, Martin Konvička, Michal Rindoš, Jan Hrček.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Konvičková H, John V, Konvička M, Rindoš M, Hrček J (2024) High hymenopteran parasitoid infestation rates in Czech populations of the Euphydryas aurinia butterfly inferred using a new molecular marker. Journal of Hymenoptera Research 97: 29-42. https://doi.org/10.3897/jhr.97.113231
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We apply a molecular approach to quantify the level of hymenopteran parasitoids infestation in the larvae of the marsh fritillary (Euphydryas aurinia), a declining butterfly species, in western Bohemia, Czech Republic, in two subsequent years. We used the novel primer HymR157 in combination with known universal 28SD1F to establish a PCR detection system which amplifies hymenopteran parasitoids, but not the lepidopteran host. In the 14 sampled E. aurinia colonies, the infestation rates per individuum were 33.3% and 40.2%; whereas per sampled larval colony, these were on average 38.5% (range 0–100) and 40.1% (0–78). The per-colony infestation rates correlated with the numbers of larval webs censused per colony the year prior to sampling the parasitoids, pointing to a time lag in parasitoid infestation rates. The levels of the hymenopteran parasitoid prevalence are thus relatively high, supporting the importance of parasitoids for the population dynamics of the threatened host. The detection primers we developed can detect a range of hymenopteran parasitoids on other butterfly hosts.
butterfly ecology, Braconidae, Lepidoptera, Marsh Fritillary, molecular detection, Nymphalidae, population dynamics
Hymenopteran parasitoids are one of the most diverse groups of animals in terrestrial ecosystems and play a key role in the natural regulation of their host populations (
The Marsh Fritillary, Euphydryas aurinia (Rottemburg, 1775) is an EU-protected butterfly, declining in many European countries (
Like many other insects, E. aurinia hosts numerous hymenopteran parasitoids (
Diverse methods were used so far to study the parasitoids of E. aurinia, and related butterflies, ranging from field counts of hymenopteran cocoons (
DNA-based methods are increasingly used in studies of parasitoid-hosts interactions (
In this paper, we quantify Hymenoptera parasitoids infestation rates in a selection of the Czech Republic populations of Euphydryas aurinia and relate the infestation rates to the stage of the butterfly population cycle. Additionally, we document utility of our primers’ combination for rapid Hymenoptera infestation assessment in butterflies.
We sampled E. aurinia caterpillars in western Bohemia (Fig.
While sampling the caterpillars, we recorded the following: Julian date, to account for infestation changes during larval period; longest and shortest dimension of the larval web (cm); sward height, i.e., visually estimated height of surrounding vegetation in 2.5 metre radius circles around each larval web sampled; host plant density, expressed as the number of Succisa flowerheads in the circle; and webs density, expressed as the number of larval webs in the circle.
The material was stored in 96% ethanol, the DNA was extracted using the Tissue Genomic DNA Mini Kit (GenAid Biotech, Taiwan) following the manufacturer’s protocol.
We targeted a part of the 28S D1 region. We used primer 28SD1F (GGGGAGGAAAAGAAACTAAC; Larsen et al. 1992) in combination with a new primer HymR157 (TGGCCCCATTCAAGATGG) with a resulting product of 164–167 bp. For the primer design, we assembled a library of target sequences (Hymenoptera parasitoids) and non-target sequences (Lepidoptera) from sequences available in GenBank (primarily PopSet 300390962,
Each in vitro PCR reaction contained 6.5 µl of Combi PPP Mastermix (Top-Bio, Czech Republic), 4.5 µl of H2O, 0.5 µl of both reverse and forward primer, and 1 µl of DNA template. The cycling conditions of PCR were as follows: 94 °C of initial denaturation (5 mins), 30 cycles at 94 °C denaturation (40 secs), 50 °C annealing (30 secs), and 72 °C elongation (1 min), with the final elongation at 72 °C (5 mins). The presence/absence of PCR products was checked using agarose electrophoresis (1.5% gel, 150V, 30 mins). The samples with a band on the gel were assumed as positive; i.e., individuals infested by parasitoids (Fig.
Electrophoresis gels used to assess whether the primers used can discriminate lepidopteran hosts and hymenopteran parasitoids a various adult hymenopteran parasitoids (PCRs are positive) and positive (P) and negative (N) samples of E. aurinia b four species of adult butterflies; PCRs are negative. The adult specimens of Hymenoptera and Lepidoptera were identified by M. Rindoš, M. Konvička, and Z. Faltýnek Fric.
To test the utility of the primer used, we also carried out control reactions which contained DNA extracted from various adult hymenopteran parasitoids and butterflies (Fig.
Positive results were recalculated to infestation levels per larval web (1/0 factor, infested or not) and per site (% of larvae sampled). To relate the per-web infestation level to larval web properties, we carried out logistic regressions (binomial error distribution; in R 4.2.3,
To relate the per site percentual infestation to larval counts at the sites, we used data from annual monitoring of the sites, ongoing since 2001 (
Out of 210 (year 2019) and 358 (year 2020) E. aurinia caterpillars assayed for hymenopteran DNA, we obtained 70 and 144 positive results, respectively; i.e., the total infestation rates were 33.3% and 40.2% per individuum. On a per-site basis, this translates to mean±SD / median / range 38.5±29.89 / 40 / 0–100 per cent in 2019, and 40.1±26.51 / 50 / 0–77.5 per cent in 2020.
The sequences of the positive samples were 124–126 bp long. The most similar sequences in GenBank according to nblast algorithm are those of Cotesia glomerata (Linnaeus, 1758), with the query identity 95.2–96.03%. The next similar sequences did not even reach a match of 93%.
According to the logistic regressions, none of the recorded properties of larval webs were related to infestation of the web (Table
Logistic regressions relating field-measured properties of larval webs to infestation of the sampled larvae.
Model | Predictor mean±SD/median/range | Coefficient | Residual deviance | Residual DF | AIC |
---|---|---|---|---|---|
Null 1 a) | 223.2 | 160 | 225.2 | ||
Longest dimension | 4.3±2.68/4/1–13 | 0.038 | 222.9 | 159 | 226.9 |
Shortest dimension | 7.6±12.76/12/3–40 | -0.001 | 146.5 | 159 | 227.2 |
Null 2 a) | 181.1 | 194 | 237.7 | ||
Julian date | 240±4.3/238/235–248 | 0.052 | 178.9 | 193 | 239.0 |
Sward height | 42±23.1/4/5–100 | 0.010 | 178.8 | 193 | 239.8 |
Host plant density | 76±49.5/60/5–200 | 0.004 | 179.6 | 193 | 237.7 |
Webs density | 1.7±1.98/1/0–9 | -0.095 | 179.6 | 193 | 236.9 |
At the level of individual colonies, the infestation rates were highly variable (Fig.
Per-site hymenopteran parasitoids infestation rates in colonies of the butterfly Euphydryas aurinia in two consecutive years (2019–20), with information of caterpillar web counts in the respective colonies in 2018–2021 (above), and illustration of the relationship between hymenopteran parasitoids infestation rates and E. aurinia caterpillar web counts in the previous year (below).
Because the absolute values of web counts highly varied among the sites (Fig.
The novel combination of primers 28SD1F (
Although the role of parasitoids on population fluctuations of E. aurinia, and related species, had been proposed almost a century ago (
It also should be noted that our approach did not distinguish parasitoids from hyperparasitoids; i.e., the insects developing within parasitoids and thus killing them (
With all the limitations, we found that the infestation rate per site positively correlated with per-site caterpillar webs’ numbers of the previous year. This is fully expected if the parasitoids need a rich resource supply (i.e., high host density) to multiply in a butterfly colony, depleting the hosts’ numbers in the process (
The DNA-based method detected high hymenopteran parasitoids’ infestation rates in colonies of the declining butterfly, Euphydryas aurinia. These rates, however, widely varied among the butterfly colonies and between two study years, likely interfering with, and possibly driving, the inter-annual butterfly abundance changes within colonies, as well as the metapopulation dynamics of the butterfly, described in detail by
Part of the development of the primers took place during the stay of JH in the lab of Graham Stone, University of Edinburgh. The work was funded by the Technology Agency of the Czech Republic (SS01010526). JH was supported by Czech Science Foundation grant no. 20-30690S.
List of sampled colonies of E. aurinia
Data type: xlsx
Explanation note: Spreadsheet with information on all 97 E. aurinia colonies monitored in western Czech Republic in 2001–2021, with caterpillar webs counts for individual years. The fourteen colonies sampled for hymenopteran parasitoids are indicated in red.