DOI: 10.5281/zenodo.15576262
title: Dataset for "Defensive compounds of Blissus pulchellus (Hemiptera: Blissidae) as a barrier against infection by entomopathogenic fungi"

description: 1. Dataset language: English

2. Abstract:

Dataset of the present study shows the protective mechanisms of the chinch bug Blissus pulchellus against entomopathogenic fungi and the relationship between the major volatile compounds produced by B. pulchellus and their potential role in enhancing its resilience to disease. Both adults and nymphs exhibited low susceptibility to infection by various fungal strains. The close and continuous contact of conidia with antimicrobial substances on the insect's integument significantly inhibited germination rates. Chemical analyses of body extracts from adults and nymphs revealed both qualitative and quantitative differences in their defensive compound profiles. Our findings suggest that the aldehydes are the primary compounds responsible for fungal inhibition, effectively protecting the insect from infection.

3. Methodological procedures:

3.1. Data file “RawDatasetBioassays” shows five different bioassays listed below:

a) the screening bioassay was performed against adults on grass in cages. Aerial conidia were adjusted to a final concentration of 4 × 107 viable conidia mL-1. Twenty-five adults were transferred to B. humidicola plants grown in pots. Plants and insects were sprayed with a 3 mL conidial suspension (ca. 1 × 105 conidia / cm2) using an airbrush. Treated plants were transferred to a cylindrical plastic cage and kept under controlled conditions  during the entire experimental period. Three independent cages were used for each strain. Negative control groups, sprayed with water plus surfactant, were included. Mortality was assessed after one week, and dead insects found in each cage were collected and placed in wet chambers for an additional five days to confirm the infection.

b) the differential susceptibility of adults and nymphs of B. pulchellus to infection by two fungal strains was also evaluated under laboratory conditions. Cages containing B. humidicola plants were infested with twenty-five adults or fifth-instar nymphs as previously described. Plants with the insects were sprayed with a 3 mL conidial suspension (4 × 107 viable conidia mL-1) of both strains, using the airbrush described above. Five independent cages were used for each of the strains and a negative control group of another five cages was sprayed with water plus surfactant. Adults and nymphs mortalities were assessed after a ten-day period of incubation. The bioassay was repeated on a different date with a different insect generation, and all the conditions and procedures followed those already described.

c) the detrimental effect of defensive compounds produced by B. pulchellus on conidia germination was assessed by their exposure to the extracted compounds. In the first case, crude extracts were obtained by washing living adults in 1000 µL of n-hexane in glass vials. A droplet of each extract was placed on the surface of culture medium, and after complete hexane evaporation, 10 µL of a conidia suspension were inoculated onto the extract layer formed on the medium surface. Pure n-hexane was used as negative control. The plates were sealed and incubated for 18 h. Germination was assessed by direct microscopic observation with germinated conidia identified as those with germ tubes longer than the width of an ungerminated conidium. The bioassay was repeated four times with independent groups of insects.

d) the detrimental effect of defensive compounds produced by B. pulchellus on conidia germination was also assessed by by their direct contact with insect integument. Groups of 20 living adults were immersed in conidia suspensions for 15 seconds. Insects were kept alive in petri dishes for four hours Conidia were retrieved from insect surface by washing the whole group of adults into 200 µL of distilled water plus surfactant  for 1 minute in a vortex. A droplet of 20 µL of these suspensions was placed on culture medium surface and plates were incubated for 18 h. Suspensions of conidia in water without insects was used as negative control. All the conditions and procedures for conidia germination assessment followed those already described. The bioassay was repeated three times with independent groups of insects.

e) the comparative effect of defensive compounds produced by Blissus pulchellus on formulated and non-formulated conidia was determined by the immersion of adult and fifth-instar nymphs in a conidial suspension in water or in an oil-in-water emulsion for 15 seconds and then kept in Petri dishes for 4 h. Conidia adhered to the insect surface were evaluated as previously described. Suspensions of conidia in water and in oil-in-water emulsion without insects were used as negative controls. All the conditions and procedures for conidia germination assessment followed those already described. The bioassay was repeated six times with independent groups of insects.

Analyses of all the bioassays were performed using R Statistical Software. The number of adults killed by the strains in the screening bioassay and the number of germinated conidia in the in vitro experiments were fitted to a generalized linear model (GLM) with binomial distribution (logit-link function). Model selection was performed to choose the best model to fit proportional data using the ‘hnp’ package, considering overdispersion. The selected models underwent analysis of variance (LRT-test). Multiple pairwise comparisons between treatments were performed with estimated marginal means at p < 0.05.

3.2. Data file “RawDatasetCompounds” shows five different bioassays listed below:

Defensive compounds of B. pulchellus were collected with eight or six replicates for each insect stage. The insects were immersed in 2 mL of n-hexane (95%) for 5 minutes and filtered through a Pasteur pipette plugged with glass wool. The resulting extracts were then concentrated using a gentle stream of nitrogen to achieve a final volume of approximately 500 µL. The body extracts were analysed by gas chromatography  using a 30 m × 0.25 mm ID and 0.25 µm film thickness column. The carrier gas was helium. The column effluent was analysed with a flame ionization detector at 270°C. Data were collected with ChemStation. Selected volatile samples were analysed using a gas-chromatograph coupled with an Agilent 5975-MSD mass-spectrometer equipped with a quadrupole analyser, DB-5MS column, and splitless injector with helium as the carrier gas. Ionization was by electron impact. Data were collected and analysed with GC-MS ChemStation 2.1 software. The compounds were identified by comparing the GC retention times and mass spectra fragmentation patterns with those of chemical standards. The compounds were quantified using calibration curves prepared with synthetic solutions of standards. 

To compare the quantities of defensive compounds extracted from insect body the data were analyzed using ANOVA followed by Tukey's post hoc test or using t test (α=0.05).. To determine the composition of defensive compound blends principal component analysis (PCA) was applied to the multivariate data. The statistical analyses were conducted using Paleontological Statistics Software (PAST, version 4.17).