Maria Rosa Paiva

Temperature niche shift observed in a Lepidoptera population under allochronic divergence

A process of adaptive divergence for tolerance to high temperatures was identified using a rare model system, consisting of two sympatric populations of a Lepidoptera (Thaumetopoea pityocampa) with different life cycle timings, a ‘mutant’ population with summer larval development, Leiria SP, and the founder natural population, having winter larval development, Leiria WP. A third, allopatric population (Bordeaux WP) was also studied. First and second instar larvae were experimentally exposed to daily‐cycles of heat treatment reaching maximum values of 36, 38, 40 and 42 °C; control groups placed at 25 °C. A lethal temperature effect was only significant at 42 °C, for Leiria SP, whereas all temperatures tested had a significant negative effect upon Leiria WP, thus indicating an upper threshold of survival c.a. 6 °C above that of the WP. Cox regression model, for pooled heat treatments, predicted mortality hazard to increase for Leiria WP (+108%) and Bordeaux WP (+78%) in contrast to Leiria SP; to increase by 24% for each additional °C; and to decrease by 53% from first to second instar larvae. High variability among individuals was observed, a population characteristic that may favour selection and consequent adaptation. Present findings provide an example of ecological differentiation, following a process of allochronic divergence. Results further contribute to a better understanding of the implications of climate change for ecological genetics.

Characterization of the volatile fraction emitted by Pinus spp. by one- and two-dimensional chromatographic techniques with mass spectrometric detection

The chemical composition of the needles of P. pinea, P. pinaster, P. halepensis, P. nigra, P. brutia, P. patula, P. radiata, P. taeda, P. elliotti, P. kesiya, P. sylvestris and P. eldarica was investigated. Headspace solid-phase microextraction and steam distillation extraction were used to collect the volatile fractions. Samples were analyzed using one-dimensional gas chromatography (1D-GC) and comprehensive two-dimensional gas chromatography (GCxGC) associated with a quadrupole and a time-of-flight mass detectors. Results showed that the analytical capabilities of 1D-GC are partially limited by the separation power of the columns. The higher sensibility and the absence of peak skewing of the time-of-flight mass analyzer, with the use of automated peak finding and deconvolution algorithms, allowed for the detection of trace components with qualitative full spectra and the extraction of true mass spectra from coeluting compounds, promoting their reliable identification and thus significantly improving results obtained by 1D-GC/MS, when using a quadrupole mass analyzer. The use of GC x GC resulted in enhanced separation efficiency and increased signal to noise ratio (sensitivity) of the analytes, maximizing mass spectra quality and improving compound detection and identification. This work shows the use of 1D-GC/ToFMS for the analysis of pine needles volatiles, achieving the detection of 177 compounds, that is more than twice the number previously identified by standard 1D-GC/MS. The analysis by GC x GC for the same sample allowed the detection of 212 compounds. The enantioselective GC x GC analysis performed for all the Pinus spp. under study achieved the detection of 422 different compounds. Cross-over phenomena according to operational conditions are highlighted and discussed.

Pine volatiles mediate host selection for oviposition by Thaumetopoea pityocampa (Lep., Notodontidae)

The oviposition behaviour and host selection by females of the pine processionary moth, Thaumetopoea pityocampa, was studied under two conditions: in a field insectary and in a pine stand. An oviposition choice test was conducted in an insectary cage, using artificial Christmas trees (ACTs) baited with extracts from four pine species: Pinus pinea, Pinus pinaster, Pinus halepensis and Pinus brutia, plus a control. Females oviposited significantly more egg batches on the ACT baited with a P. brutia extract, while no oviposition occurred on the control ACT. In a large P. pinea stand, two groups of randomly selected trees were marked and baited respectively with an extract of P. brutia, which was the preferred species under insectary conditions and with a solvent, to act as control. Results showed that oviposition in the field followed an aggregated pattern, fitting a negative binomial distribution and that trees baited with P. brutia extracts, received a significantly larger number of egg‐batches than control trees. In parallel, the volatiles emitted by all pine extracts tested where analysed by gas chromatography and mass spectrometry (GC‐MS), coupled to a time‐of‐flight (TOF) mass analyser after solid phase microextraction (SPME), resulting in the identification of 26 compounds. Comparative chromatograms showed qualitative differences among the pine species used, some compounds being present in one of them only. Findings demonstrate for the first time that: (i) T. pityocampa females discriminate among bouquets extracted from different host pine species and exhibit oviposition preferences; and (ii) olfactory cues play an important role in mediating the selection process.

Female calling behaviour and male response to the sex pheromone in Thaumetopoea pityocampa (Den. & Schiff.) (Lep., Thaumetopoeidae)

Abstract: Female calling behaviour and male response to natural and synthetic pheromone sources were used for a Portuguese population of the winter pine processionary moth Thaumetopoea pityocampa (Den. & Schiff.). Virgin females 1–5 days old were studied, the mean calling longevity being 3.3 ± 0.7 days. Calling started ≅ 3 h after the onset of scotophase, and 7 h after emergence, lasting, nightly, for 6.4 ± 0.7 h, and was not affected by female age. A drop in air temperatures during the first half of scotophase and an increase in light intensity at dawn apparently determined the onset and termination of calling. Male flight activity began, on average, 3 h before the females started emitting pheromone, and was similarly affected by air temperatures and light intensity. The attractivity of traps baited with synthetic pheromone (TP056A; Yne 11, Z13–16AC) was compared with that of virgin females. Male flight activity was monitored in central and southern Portugal, using pheromone traps, and seen to last between 6 and 10 weeks, with a major peak occurring, in all sites, at the beginning of September.

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus)

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytes in the peripheral blood of the fat snook (Centropomus paralellus) - a fish occurring in Brazil - were investigated. The cytochemical methods were performed to demonstrate four enzymatic reactions - o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (α-NAE), respectively - and two non-enzymatic ones - Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructural study. The cytochemical reactions were positive in neutrophils for MPO, ACP, α-NAE, glycogen and phospholipids; in lymphocytes for ACP and α-NAE; in monocytes for ACP and α-NAE and in thrombocytes for ACP, α-NAE and glycogen. Only neutrophils were positive for MMPs 2 and 9, and none of the cells studied were positive for ALP. Ultrastructurally: 1) neutrophil showed a spherical shape with a spherical, indented or lobulated euchromatic nucleus, and cytoplasm containing granules of varied sizes and mitochondria of varied shapes and sizes. The nucleus/cytoplasm relation and the size of granules suggest neutrophil maturation in peripheral blood; 2) lymphocytes showed partially heterochromatic nucleus and minimal cytoplasm; 3) monocytes had long cytoplasmic projections, an indented nucleus, evident nucleolus and cytoplasm with granules of varied sizes and vacuoles; 4) thrombocytes were predominantly elliptical or roughly spherical in shape, had a partially heterochromatic nucleus and cytoplasm containing electron-dense granules, intricate canalicular system and vacuoles occasionally holding phagocytic material.

Reduction in the pheromone attractant response of Orthotomicus erosus (Woll.) and Ips sexdentatus Boern. (Col., Scolytidae)

Under field conditions, the pheromone attractant responses of the scolytids Orthotomicus erosus and Ips sexdentatus were significantly reduced by the addition of one of the oxidation products of α‐pinene, verbenone, to the attractive volatiles 2‐methyl‐3‐butene‐2‐ol plus ipsdienol. Catches in flight barrier traps also decreased when (S)‐cis‐verbenol was added to the dual bait mixture; the inhibitory action of the terpene alcohol was more noticeable upon the female populations of both species. The observed patterns of behavior might reflect the outcome of a long process of competition among Ips species.

A review of invasive alien species impacts on eucalypt stands and citrus orchards ecosystem services: Towards an integrated management approach

Multidisciplinary knowledge on the impact caused by invasive alien species (IAS) on ecosystems is crucial for guiding policy makers in the adoption of sustainable management measures. This research was focused on insect IAS impacts on two managed ecosystems: eucalypt plantations and citrus orchards. It begins with an identification of the wide range of ecosystem services (ES) and disservices provided by each of these managed ecosystems, according to the methodology proposed by the Millennium Ecosystem Assessment. Subsequently, a comprehensive review of studies that promoted the identification and valuation of direct and indirect impacts IAS impacts on these ecosystems was performed. From the synthesis of previous findings, an integrative management framework is advanced. This links the identification of ES, drivers of change and development of IAS management strategies by means of assessment processes that account for multiple dimensions of ES values. The article concludes with a discussion on the challenges underpinning assessment and valuation approaches that inform the design of inclusive strategies and interventions to tackle IAS impacts.

Water stress affects Tomicus destruens host pine preference and performance during the shoot feeding phase

Abstract• The effect of water stress on host selection and performance during the shoot feeding phase, by the Mediterranean pine shoot beetle Tomicus destruens, was studied.• Young potted Pinus pinaster plants were subjected to two water supply regimes: (i) well watered; (ii) 4–5 months of moderate drought stress. Plant volatile organic compound (VOCs) emissions were collected by SPME and analysed by GC/MS.• In tests with paired plants, the number of holes and tunnels excavated by beetles in well watered pines was significantly larger than in stressed plants, whereas in no-choice tests no differences occurred.• Beetle survival was significantly higher on well watered, than on stressed pines (p = 0.006), while fat contents also increased (p = 0.09).• Plants could be assigned to treatments based on VOCs emissions, stressed pines emitting more myrcene and less β-pinene than well watered ones.• It is concluded that during the shoot feeding phase, T. destruens preferentially attacks non-stressed plants, in detriment of stressed ones, upon which a higher fitness is attained.• The role of pines VOCs emissions under moderate drought stress, in the host selection and colonization process by the pine shoot beetle, is discussed.

Arthropod diversity sheltered in Thaumetopoea pityocampa (Lepidoptera: Notodontidae) larval nests

Abstract.  1 In pine ecosystems, the role of the larval nests of the Mediterranean defoliator Thaumetopoea pityocampa as shelter of other arthropods, was studied for the first time. In Portugal, Pinus stands which differed in understorey plant diversity and level of attack by T. pityocampa were compared. 2 The arthropod fauna found sheltering inside the nests consisted of 60 species, representing 12 foraging types. Both arthropod richness and abundance were positively correlated with nest size, expressed as larval biomass. Arthropod richness was further positively correlated with understorey plant diversity, while no correlation was detected between arthropod richness and nest density. 3 Spiders accounted for up to 50% of the species richness, while 80% of the individuals collected were either juveniles, or females with brood, implying that T. pityocampa nests are used for overwintering and brood care. Seventy‐six per cent of the Araneae were nocturnal ground foragers and specialised cryptic hunters, demonstrating the occurrence of indirect interactions among species belonging to different communities, namely ground vegetation layers and forest pine coppice. 4 It was concluded that, in Mediterranean pine ecosystems, T. pityocampa nests (i) create habitat diversification and contribute to improve overwintering survival of a wide range of arthropod species; (ii) play a functional role in the food web of pine ecosystems, by promoting indirectly mediated interactions among species from different communities; and (iii) do not contribute to create habitat for symtopic phytophagous arthropods, since contrary to literature references for different types of shelters, 98% of the associated species belonged to foraging types other than T. pityocampa.

Interactions between Orthotomicus erosus (Woll.) (Col., Scolytidae) and the Argentine ant Linepithema humile (Mayr) (Hym., Formicidae)

Interactions occurring between Orthotomicus erosus, an economically important pine bark beetle in Portugal, and the aggressive Argentine ant Linepithema humile were investigated. Tri-trophic “experimental enclosures” consisting of Pinus pinaster logs, O. erosus adults and L. humile workers were set up in the field and in the laboratory. Interactions between ants and scolytids were observed and quantified in terms of the bark beetle’s success in colonizing the logs. The presence of L. humile did not influence the ability of O. erosus to colonize the logs, apparently due to the difficulties met by the ants in handling the bark beetles and to their inability to injure them. Thus, although abundant in many pine stands in Portugal and in spite of its aggressiveness, L. humile does not seem to affect populations of O. erosus.