Microscopic Identification of Anatomical Elements and Chemical Analysis of Secondary Nests of Vespa velutina nigrithorax du Buyson

SIMPLE SUMMARY: Vespa velutina nigrithorax, one of the eleven subspecies of Vespa velutina, has become a matter of concern due to its invasive behavior after its accidental introduction into France in 2004. This Vespa species, also known as the yellow-legged hornet, builds large nests, the structure and chemical composition of which are of interest to know and understand in order to estimate the areas that these hornets may find acceptable for establishing new colonies. Its presence causes serious negative impacts on ecosystems and on pollinating species, especially on honeybees. As the presence of this type of hornet is very recent in Europe, there are large gaps in our knowledge about its adaptability and control measures. The main objective of this study is to identify the composition of the materials used by the Asian hornet in the construction of secondary nest envelopes. The samples that were collected from each nest were analyzed using two microscopic devices in non-disaggregated and disaggregated material. The same samples were also chemically analyzed by means of X-ray diffraction and SEM/EDS (scanning electron microscopy). It was noticed that the nests were formed by the agglomeration of small particles that are spatially organized in brown and beige strips. The microscopic analysis showed the presence of cellular elements of agricultural origin, woody material from forest species, and leaves, almost all of which were lignocellulose materials. The results achieved from chemical analysis led to the conclusion that the main chemical constituent of the nests is cellulose. As for the elemental analysis, about 99% of the nests were composed of C and O and low amounts of other microelements (Na, Al, Si, K, and Ca). ABSTRACT: Vespa velutina accidentally arrived in Europe (France) in 2004, and rapidly expanded throughout the entire country. Its presence in mainland Portugal was first noticed in 2011. Being an invasive species with no natural predators in the region to control it, it has caused enormous environmental and economic damage, particularly on Apis mellifera (honeybee) colonies. Although there is already some research on this species’ biology, little is known about its adaption to European ecological conditions, specifically in terms of nest building. This type of hornet builds a primary nest in the spring to start a colony. During the summer, they build a secondary nest to develop the main colony. These secondary nests are ovoid-shaped and range in size from 18.7 cm to 45.0 cm in diameter and from 19.2 cm to 65.0 cm in length, attaining their highest development in late summer. The external appearance of these nests is characterized by alternating stripes that are beige and brown in color. The main objective of this study is to identify the composition and the origin of the materials that are used by Vespa velutina nigrithorax to build the outer envelope of these secondary nests. This information could be very interesting and will not only increase our knowledge on the biology of the species in regions far from its original area, but will also be relevant for the future implementation of new policies to control this invasive species by means biological control. Several samples were taken from each nest and were observed under different optical magnifying devices. In the second stage, their chemical composition was analyzed by X-ray diffraction (XRD) and scanning electron microscopy (SEM/EDS). It was noticed that almost all of the materials used in the nests’ construction were lignocellulose from woody materials from both softwood (gymnosperm) and hardwood (angiosperm) forest species as well from leaves and small particles of agricultural origin (grasses). The beige strips were formed almost exclusively from woody softwood cells, while the brown strips were composed of hardwood cells, leaf tissues, and grasses. Chemically, it was noticed that this material mainly consisted of cellulose, with more than 99% being composed of C and O and very little mineral material from elements such as Na, Al, Si, K, and Ca. The achieved results allow us to state that in the construction of these secondary nests, these hornets only used organic materials that are then probably agglomerated through their mouths.