Size-dependent behavioral and antennal responses to doses of (+)-isopinocamphone and 1,8-cineole mixed with pheromone: a potential host selection strategy in female Ips typographus L.

Context:
Ips typographus, a major pest of Norway spruce (Picea abies) in Europe, is experiencing more frequent outbreaks due to climate change. These outbreaks involve shifts in population dynamics and phenotypic traits, influencing beetle responses to olfactory cues from stressed host trees.

Aims:
The study examines the size-dependent behavioral and antennal responses of female I. typographus to two host selection–deciding volatiles with contrasting ecological roles: 1,8-cineole, which inhibits attraction to unsuitable trees, and (+)-isopinocamphone, a pheromone synergist. Size-linked morphological and olfactory adaptations may influence females' ability to select suitable host trees for reproduction.

Methods:
In field trap experiments conducted in 2019 and 2022, the body size of I. typographus females caught in response to different doses of (+)-isopinocamphone or 1,8-cineole in combination with pheromone was compared. Female Ips typographus were sorted based on body length, the size of the antennal club was measured, and size-dependent antennal responses to these volatiles were analyzed using electroantennography.

Results:
Larger females were more attracted to (+)-isopinocamphone in combination with pheromone in the field, showed stronger antennal detection, and had proportionally larger antennal clubs than smaller females. In contrast, smaller females were less repelled by 1,8-cineole added to pheromone, but, in contradiction, antennally detected it more strongly than larger females despite having smaller antennal clubs.

Conclusion: The total body length significantly influences semiochemical detection in I. typographus females. (+)-isopinocamphone was detected more effectively by larger females, implying an advantage in the selection of suitable host trees. In contrast, the discrepancy between behavioral and antennal responses to 1,8-cineole in smaller females suggests involvement of not only peripheral detection but also central nervous processing of olfactory signals driving behavior. This adaptation may enable smaller females to reduce competition with large ones by selecting less suitable trees. These findings provide new insights into the ecological relationship between beetle morphology and olfactory cues, with implications for tree–bark beetle interactions.

Methods
1) Study Location and years:

The experimental areas: Norway spruce forest, the natural habitat of Ips typographus, at 600 m a.s.l. within the Forest CZU property, Kostelec nad Černými lesy, Czech Republic.

2019 site: Coordinates 49°56′02″N, 14°52′21″E.
2022 site: Coordinates 49°55′57″N, 14°55′13″E.

2) Time Frame and field experiments:

       Experiments ran from June 3 to July 28 in both 2019 and 2022.

2.1 Trap Type:

Cross-vane Ecotraps (Fytofarm, Slovak Republic) were used for the experiments.

2.2 Trap Placement:

Traps were positioned 15 meters apart, 1.5 meters above the ground, and approximately 30 meters from the forest edge.

2.3 Experimental Design (2019):

Seven traps were used.

Six traps were baited with a pheromone and three different doses (low, medium, high) of either 1,8-cineole or (+)-isopinocamphone.
One control trap was baited with pheromone alone.

2.4  Experimental Design (2022):

Two experimental blocks were established:

One block tested 1,8-cineole dose.
The second block tested (+)-isopinocamphone dose.
Each block included a pheromone control trap to ensure reliable comparisons.
Experimental setup are detailed in Moliterno et al. (2023). 

Field effects of oxygenated monoterpenes and estragole combined with pheromone on attraction of Ips typographus and its natural enemies. Front. For. Glob. Change 6:1292581. doi: 10.3389/ffgc.2023.1292581

2.5 Morphological Measurements i. Insects

Study Subject: Ips typographus (F0 generation) emerged from naturally infested Norway spruce logs (n = 12; ~50 x 28 cm).
**Logs: **Tree logs were collected in Kostelec nad Černými Lesy during June–July 2024.

Selection Criteria: Only female insects (~3 days old) were selected after sex sorting for further measurements and experiments.

Size Groups Considered:

Large-sized females: Body length ≥ 4.80 mm (n = 30).

Small-sized females: Body length ≤ 4.70 mm (n = 30).

ii. Dose-Responses to Pheromones, 1,8-Cineole, and (+)-Isopinocamphone in Laboratory Experiments

Subjects Tested: Only female insects.

Size Groups:

Large-sized females: Body length ≥ 4.80 mm (n = 10).

Small-sized females: Body length ≤ 4.70 mm (n = 10).

Exclusion: The analysis did not include damaged insects (broken or missing elytra).

2.6 Statistical Analysis

Preliminary Tests:

Data normality and variance homogeneity were assessed using the Shapiro-Wilk test and Levene’s test.

Dependent and Independent Variables:

Dependent Variable: Body length.

Independent Variable: Doses of tested compounds.

Comparative Analysis:

Generalized Linear Model (GLM).
The model employed a Gamma distribution with a log link function.

Post hoc comparisons were conducted with the Tukey test, followed by a multiplicity correction using the Bonferroni method.

Isometric vs. Allometric Relationships:

Assessed via the Standardized Major Axis (SMA) method for antennal clubs of large and small females;

Electroantennography Analysis:

Compared responses of large and small females (n = 10 each) using the exact Wilcoxon rank-sum test;

Software and alpha value:

Analyses were performed in RStudio (version 4.1.1).
Significance level (α) was set at 0.05.

Additional Information:

For further details, please refer to the related article.