Lina Nunes

Colony interactions in Reticulitermes grassei population assessed by molecular genetic methods

Abstract.The cryptic habits of subterranean termites impair studies of colony delimitation and the organization of foraging. Whereas feeding sites can be identified and the movements of foragers between them can be assessed using traditional mark-release techniques, the assignment of the termites found at feeding sites to their parent colonies remains problematical. Thus the extent and overlap of individual colony foraging territories are unknown. We used microsatellite markers to delineate colonies of R. grassei and compared the results with data from a mark-release-recapture study (Nobre et al., 2007) carried out in the same population and over the same period of time. The majority of colonies exhibited only a single-feeding site, but presented, even at a local scale, a high degree of variability in colony structure and no evidence of inbreeding. F-statistics for some colonies were consistent with pleometrosis. The population as a whole contained individual colonies that were separately identified as Mendelian, mixed family or pleometrotic families. Furthermore, microsatellite data suggest that what was represented by the MRR technique as a single foraging group could be drawn from more than one colony. Colony delineation is discussed as a tool for understanding overall population structure and termite feeding strategies.

Antifungal activity of Bacillus subtilis 355 against wood-surface contaminant fungi

A strain of Bacillus subtilis was examined for antifungal activity against phytopathogenic and wood-surface contaminant fungi. The bacterium was grown in five culture media with different incubation times in order to study cell development, sporulation, and the production of metabolites with antifungal activity. The anti-sapstain and anti-mould activity of the bacterium grown in yeast extract glucose broth (YGB) medium in wood was also evaluated. In YGB, the bacterium inhibited the growth of several fungi and displayed a broader spectrum of activity than in the other media tested. A relationship between bacterial spore production and the formation of metabolites with antifungal activity was detected. YGB medium displayed effective control in wood block tests. YGB medium was extracted with solvents of increasing polarity and the dry residues were applied to silicagel plates, resolved with the appropriate solvent and sprayed with different solutions, detecting the presence, of amines, and higher alcohols. The bioautographic method revealed the presence of at least two active compounds against the blue-stain fungus Cladosporium cucumerinum.

Distribution and genetic variation of Reticulitermes (Isoptera: Rhinotermitidae) in Portugal

The genus Reticulitermes has a Holartic distribution and is widely represented in southern Europe. It is established in Portugal, across the full range of ambient environmental conditions, using different types of wood resources, as available, and nesting ubiquitously in many types of soil. Sequencing of part of the mtDNA COII gene revealed that all the specimens sampled on a north–south transect of the country were Reticulitermes grassei (considered synonymous with R. lucifugus grassei). Nevertheless, an association between the genetic and the geographical distances of sampled specimens was shown, leading to the conclusion that discrete termite populations have differentiated in situ. The geographic component of the genetic polymorphism should be the subject for further research.

First records of urban invasive Cryptotermes brevis (Isoptera: Kalotermitidae) in continental Spain and Portugal

Occurrence of the West Indian drywood termite, Cryptotermes brevis, is registered for the first time as established populations in Barcelona (Spain) and also additional information is given regarding a recent record from Lisbon (Portugal). This serious invasive termite pest was detected in five buildings in Barcelona, Spain and one building in Lisbon, Portugal.

Tunnel geometry of the subterranean termite Reticulitermes grassei (Isoptera: Rhinotermitidae) in response to sand bulk density and the presence of food

The cryptic habits of subterranean termites restricts detailed analysis of their foraging patterns in situ, but the process is evidently dominated by tunnel constructions connecting the nest with woody resources discovered within the territory of each colony. In this study, tunnel formation and orientation were studied experimentally in the termite Reticulitermes grassei (Clément), using 2‐dimensional laboratory foraging arenas containing fine sand as the substratum. The building of exploratory tunnels over a 10‐day period and the geometry of the resulting network are described. Fractal analysis showed that tunnel geometry had a fractal dimension, regardless of the total length tunnelled whether foragers encountered the food source or not. The bulk density of the sand in the arenas affected the distances tunnelled, with higher density reducing construction, but did not affect tunnel geometry. Tunnels were not discernibly orientated with respect to the positioning of the food source, even in a situation where termites had failed to find the food source at a distance of less than 50 mm, suggesting that volatiles from wood are not attractants.

Estimation of foraging territories of Reticulitermes grassei through mark–release–recapture

Subterranean termites have highly cryptic life habits and their foraging activities are as a rule confined to below‐ground level gallery systems. Mark–release–recapture (MRR) using fat‐soluble histological dyes is a candidate method for the study of foraging dynamics and territories, but has not hitherto been standardized experimentally. A wide range of dye types and concentrations is reported in the literature. In this study, six potential dyes were evaluated at different concentrations for marking workers of Reticulitermes grassei (Clément) (Isoptera: Rhinotermitidae), under laboratory and simulated field conditions. Neutral red (1% wt/wt) was considered the most effective while showing acceptably low toxicity. In a subsequent field trial using dye impregnated into a wooden bait, the MMR procedure was used to map the foraging territory perimeter of a single colony. Assumptions inherent in the interpretation of MMR data are reviewed. To map the foraging territory perimeter by this method, two theoretical approaches are defined (a conservative and a non‐conservative hypothesis). We show that the approach adopted may affect the estimate obtained by as much as 100%. Results are discussed in the context of the ecology and behaviour of subterranean termite colonies.

Toxicity of pine resin derivatives to subterranean termites (Isoptera: Rhinotermitidae)

In the search of alternatives for conventional fungicides and insecticides, the use of natural plant products for the protection of wood is one of the most appealing fields of work. Dehydroabietic acid, the main compound of dismutated rosin, a renewable raw material from Portuguese forests, and some of its derivatives, were recognized as having bactericidal, fungicidal or fungistatic properties that could eventually be used for wood protection. The work presented here is part of a project aimed at developing this possibility and to extend that action to insects, namely termites, as diterpene acids have been reported to be antifeedants against aphids and sawflies and several other similar products have been studied for the same end‐use. Pine resin and eight of its derivatives were bio‐assayed to test for termiticidal activity and to assess the LC50 of the compounds, whenever toxic activity was recorded. A comparison was made with the activity of an established termiticide, boron. There was a high degree of variation between the products tested: resin, resinate, resinol, dehydroabietic acid and trans‐deisopropyldehydroabietanol showed negligible activity; dehydroabietanol and cis‐deisopropyldehydroabietanol showed activity at higher concentrations; two mixtures of cis‐/trans‐deisopropyldehydroabietanol had a promising performance in terms of anti‐termite activity and will be further evaluated. This paper presents results that will contribute to the development of new termiticides from a renewable resource and with better behaviour towards the environment.

Survey of subterranean termites (Isoptera: Rhinotermitidae) in a managed silvicultural plantation in Portugal, using a line-intersection method (LIS).

Subterranean termites (Reticulitermes grassei) were surveyed over successive seasons in a managed eucalyptus plantation in southeastern Portugal for 26 months. Termite activity in seven diameter categories of lying dead wood was investigated by a modified line intersection method (LIS). Each item sampled was inspected and assessed for termite attack and for general (i.e. fungal) decay status using standard protocols. Line intersection is quantitative to the extent that it can link foraging and decay parameters to woody biovolume. It was found that termites selected items with larger diameter, the observed trend showing an exponential character with greater termite attack as diameter increased. Attack by termites was positively associated with prior decay by fungi. A clear positive relationship was shown between rainfall and total woody biovolume containing live termites, underlining the importance of moisture for termite activity. Subterranean termites appeared to be important wood decomposers in the woodland studied, with an average of 30% of lying dead wood branches showing signs of termite attack.

First Record of Reticulitermes flavipes (Isoptera: Rhinotermitidae) from Terceira Island (Azores, Portugal)

Reticuliterm.es Holmgren is a Holarctic genus of subterranean termites indigenous to North America, the Mediterranean and Black Sea re gions, and eastern Asia (Emerson 1971). It is unequivocally the most important economic ter mite genus to North America. Although Reticuli termes spp. constitute the dominant complex of subterranean termite pests of structures in most of these areas (Edwards & Mill 1986), records of emigrations to non-indigenous locations are remarkably less common compared to pest spe cies of Coptotermes Wasmann and Cryptoterm.es Banks (Gay 1967). The involvement of military operations as a progenitor for anthropogenic sourcing of exotic insect establishments is well known. For example, the Formosan subterra nean termite, Coptotermes formosanus Shiraki, was brought to the continental USA after World War II via military retrograde from the Pacific Theater (La Fage 1987), and associated with military ports receiving and storing equipment (Spink 1967; Scott & Scott 1996; Austin 2006). Another important subterranean species, Retic ulitermes flavipes (Kollar), has been identified in several areas throughout the world (Austin et. al. 2005a, 2005b; Su et al. 2006), where introduc tions to both hemispheres have been reported. Due to its establishment capability, R. flavipes occupies the largest geographic distribution of any subterranean termite within the genus. Establishment and subsequent spread of R. fla vipes throughout the Palearctic—recently con firmed by its establishment in northern Italy— also implicates human aided dispersal (Ghesini et al. 2010; Ghesini et al. 2011) from areas where earlier introductions are known (e.g., southwest ern France and northern Germany). We herein report the discovery of established R. flavipes populations on Terceira Island, Azores, Portugal (Fig. 1A, IB). The sample was subjected to mtDNA testing according to meth ods outlined in Austin et al. (2005a), and the GenBank submission is available as accession number DQ001964. The samples were provided as part of an ongoing survey of the phylogeog raphy of Reticulitermes throughout the world. Samples were procured by PAVB and TGM near the Lajes Air Force Base (38.75413-27.06872) (see Fig. 1C). The samples were vouchered in RHSs collection at the University of Florida, Fort Lauderdale Research and Education Cen

Dietary-driven variation effects on the symbiotic flagellate protist communities of the subterranean termite Reticulitermes grassei Clément

The ability of subterranean termites to digest lignocellulose relies not only on their digestive tract physiology, but also on the symbiotic relationships established with flagellate protists and bacteria. The objective of this work was to test the possible effect of different cellulose‐based diets on the community structure (species richness and other diversity metrics) of the flagellate protists of the subterranean termite Reticulitermes grassei. Termites belonging to the same colony were subjected to six different diets (natural diet, maritime pine wood, European beech, thermally modified European beech, cellulose powder and starvation), and their flagellate protist community was evaluated after the trials.