Research Article |
Academic editor: Matt Hill
© 2018 Ana O. Farinha, Charlene Durpoix, Susana Valente, Edmundo Sousa, Alain Roques, Manuela Branco.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Farinha AO, Durpoix C, Valente S, Sousa E, Roques A, Branco M (2018) The stone pine, Pinus pinea L., a new highly rewarding host for the invasive Leptoglossus occidentalis. NeoBiota 41: 1-18. https://doi.org/10.3897/neobiota.41.30041
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The invasive seed bug Leptoglossus occidentalis, a species native to Western North America, is of major concern for the producers of stone pine seeds in the Mediterranean countries. The large size of these edible seeds and their nutritive content may represent a pull factor for the seed bug. Cone and seed traits of three main Mediterranean pine species: P. pinea, P. pinaster, and P. halepensis, were evaluated. Preference trials with cone-bearing branches, individual cones and seeds were conducted to test host preference among the three host species.
Considering the kernel size, stone pine seeds provide 4 to 13 times more reward than P. pinaster and P. halepensis seeds, respectively, but also needed a greater effort to be reached as measured by coat thickness. Still, the benefit/cost ratio was higher on P. pinea. Individual seeds and cones of P. pinea were 2 to 3 times more consumed than those of the two other pine species. However, branch preference trials did not reveal any difference in bug visits. Moreover, adults manifested strong group behaviour on branches, frequently dissociating into two persisting groups. The implications of these results for P. pinea producing areas are discussed.
seed feeder, host preference, P. pinea, P. pinaster, P. halepensis
The invasive seed bug, Leptoglossus occidentalis, Heidemann 1910 (Hemiptera: Coreidae), originating from Western North America, was first recorded in Europe in Italy in 1999 (
With the European invasion, this polyphagous insect, which feeds on cones and seeds of many conifer species in its native range (Koerber, 1963), encountered new potential hosts. In Europe, it has been observed feeding on Mountain pine (Pinus mugo), European black pine (P. nigra), Scots pine (P. sylvestris), Maritime pine (P. pinaster), Aleppo pine (P. halepensis) and on Stone pine (P. pinea) (
Although feeding on a wide range of conifers, L. occidentalis seems capable of distinguishing between clones of P. contorta (
Evaluating host preference of this seed feeder under natural conditions is not a straightforward task due to the difficulty in detecting the bug and the absence of visible damage on cone surface. The only study on L. occidentalis feeding preference was carried on under laboratory conditions using individual mature seeds, extracted from cones (
Seeds of P. pinea are large-sized and highly nutritive which can be a pull factor to a seed-eater like L. occidentalis. On the other hand, larger seeds also mean a thicker seed coat which may represent an obstacle for the piercing mouthparts of the bug. We hypothesized that in preference trials using seeds, the bigger individual seeds of P. pinea might visually lead to a host preference towards a higher reward whereas the harder seed coat may constitute an additional cost. Seed volume is a proxy to the seed reward and thickness may represent a proxy to the effort.
Selective behaviour in the field is known to operate at sequential levels. First, individuals select a tree, then a cone and lastly a seed to feed upon (
In this study, our objectives were to evaluate the host preferences of L. occidentalis for branches, cones, and seeds of the three main pine species in the Mediterranean Basin, P. pinaster, P. halepensis and P. pinea. For this purpose, we compared cone volume,
seed weight and volume and seed thickness of the three species, and then tested bug preferences in choice tests.
Three separated choice experiments using different substrates, cone-bearing branches, fresh last year cones and mature seeds, were conducted to evaluate the preference of L. occidentalis adults among Pinus pinea, P. pinaster and P. halepensis. All the experiments were carried out under laboratory conditions. Only adults of L. occidentalis were used since nymphs are apterous and thus not capable of actively choosing the tree or the host where they will feed in natural conditions. All individuals came from a permanent laboratory colony with adults collected in Santarém region, Portugal during the summer of 2015. The colony was supplemented each summer with more adults from the same region to avoid consanguinity thus consisting of individuals with mixed ages. The colony was reared at Centro de Estudos Florestais, Lisbon, Portugal in a climatic room under the following controlled conditions: 21 °C with 60% RH and 14:10 light/dark cycle. Branches and cones from P. pinea were used as food source. Trials began by removing experimental adults from the permanent colony and marking them with an individually coloured and numbered label painted in the thorax. All marked adults were put in a cage with cone-bearing branches and seeds of the three hosts during one week. Individuals were then subjected to a 24-hour starvation period after which the trials began. Adults used to replace dead ones were removed from the permanent colony, marked but were immediately placed in the cages or test boxes.
Cone-bearing branches used in trials were collected in different locations for each of the pine species: stone pine branches were collected in Monsanto, Lisboa (38°43.09'N; 09°12.41'W) in a natural pure stand of adult trees; maritime pine branches were collected in Sobreda, Almada (38°38.06'N; 09°12.66'W) in an urban patchy stand; finally, branches of aleppo pine were collected in the university campus, Lisbon (38°72.80'N; 09°12.66'W). Cones / seeds used in preference trials were from branches / cones from the same locations as above with the exception of stone pine seeds which came from a pure, grafted stand in Santarém region, Portugal (39°6.50'N; 08°21.91'W) and maritime pine seeds in the two-choice trail which came from Setúbal region, Portugal (38°34.82'N; 09°11.09'W).
The volume of a sample of the cones used in the experiments was measured by displacement of water in a graduated cylinder (n=6 for P. pinea and P. pinaster and n=12 for P. halepensis).
Respecting mature seeds, all that were used in preference trials were weighted at the beginning and at the end of the experiments. At the end of the trials, all seeds from the three pine species were opened, and the volume of the kernels showing no feeding damage were measured by displacement of water in a graduated cylinder with a sensitivity of 0.25 ml. Due to the very small size of P. pinaster and P. halepensis, these seeds were measured in batches of 20 seeds and then the individual volume was extrapolated. The thickness of the seed coat for each host species was measured on the images collected by the Scanning Electron Microscope (SEM) using Image J software. The coats of three seeds per host were photographed in SEM with 20 measures taken in each photo.
Choice experiments were conducted in large cages (100×50×40) cm made up of a wooden frame and mesh walls. Preference among the three host species, P. pinea, P. pinaster and P. halepensis was tested in pairs by offering two potted branches of different plant species per cage to ten adults. All branches used in the trials bear cones at all development stages (1st and 2nd year for P. pinaster and P. halepensis and 1st, 2nd and 3rd year for P. pinea). The number of last-year cones (2nd year for P. pinaster and P. halepensis and 3rd year for P. pinea) in the tested branches was the same for the pair P. pinaster - P. pinea (ranging from 1 to 2 cones each), but not for the pairs P. pinaster - P. halepensis and P. pinea - P. halepensis in which the number of P. halepensis cones varied between 2 and 5 due to their smaller size. Young conelets (1st cones for P. pinaster and P. halepensis and 1st and 2nd cones for P. pinea) varied in number in all three species between 1 and 4. The experiment was replicated three times, on 21–22 April, 28–29 April and 5–6 May 2016. Branches for each experiment were collected at the end of the afternoon of the day before the start of the experiment and kept in the refrigerator until then. Ten adult bugs were assigned to each of the three big cages. Cage number 1 had three males and 7 females and cages number 2 and 3 had four males and 6 females. All adults were individually marked in the thorax with a colour and number. The group of insects of each cage remained constant in all three trials varying only the host pairs to be tested. Between trials all individuals were kept together in a single big cage in the laboratory under controlled conditions and with branches from all the three hosts. For each trial, the ten adults were introduced into the cages by placing them one by one, within a two minutes interval, at the centre of the cage, between the two potted branches. Individual bug’s behaviour and localization was recorded after that, at 1-h intervals from 8 a.m. until 6 p.m. for two days.
Three separate laboratory trials, with 3 to 5 days length each, were conducted from April to September 2017, using a video camera BRINNO TLC200 Pro. In each trial, two video cameras each videotaping two plastic boxes (23×20×19) cm simultaneously, were set. Each box contained a small branch of P. pinea, a petri dish with wet cotton to keep moisture and small aluminium cases filled with sand to place the cone. In this way, the insect was not allowed to hide underneath the cones. Similarly as in the branch preference trials, the bugs were individually marked with a coloured label. One cone of P. pinea, one of P. pinaster, two of P. halepensis and three adults of L. occidentalis were then added to each box. All cones were from last year of development which corresponds to the 3rd year in P. pinea and 2nd year in P. pinaster and P. halepensis. Experiments were carried on in a room under control conditions (26 °C, 60 % RH, 16:8 L:D) from 20–24 April, 2–6 May and 12–14 September of 2017. The videotaping was done using the time lapse function with one picture taken every two minutes, and played back at a speed of one frame per second. Videos were analysed with the program VLC media player 2.2.6 Umbrella for windows (https://www.videolan.org/vlc/index.html). Both the number of times each bug started feeding on a cone (frequencies), and the duration of the feeding was recorded. Feeding was assumed to have occurred whenever stylet insertion was observed.
Two laboratory trials were carried out to evaluate bug preferences for individual seeds of the Mediterranean pines. A two-choice test compared P. pinea and P. pinaster whereas a three-choice test included the three species. The first trial lasted three weeks whilst the second one lasted four weeks. The experiments were carried out using small plastic boxes (20×15×10) cm with a perforated lid for gas exchange in a climatic chamber under controlled conditions (21 °C, 60 % RH, 16:8 L:D).
The two-choice trials were carried out at INRA Orléans, France, and the three-choice one at Centro de Estudos Florestais Lisbon, Portugal.
In the two-choice experiments boxes containing both P. pinea and P. pinaster seeds (nseeds=5 and nseeds=12, respectively) were set (nbox =2). No-choice, control experiments were conducted using boxes with only P. pinea seeds (nseeds=10 per box; nbox=6) and boxes only with P. pinaster seeds (nseeds=24 per box; nbox=2). The experiments were carried out in February and March of 2015 with four adults per box.
The three-choice experiment was performed during January and February of 2017. Twelve boxes, each with ten seeds per pine species, acted as replicates. Each box had three adults.
All boxes included for water supply and as a substrate for resting and laying eggs, a twig of P. nigra in the two-choice and of P. pinea in the three-choice trial. Previous trials using boxes have shown that insect mortality increases greatly when there is no fresh twig inside (personal observation). The use of different pine species was dependent on conifer availability near the laboratory where the experiments took place. In addition to the twig, a petri dish with wet cotton to keep moisture and foam to support the seeds were also added to each box. Control boxes with no bugs were present in both trials. The sex of the adult specimens was not considered since previous studies found no significant differences in the consumption of conifer seeds between sexes (
The size of the mature seeds, the thickness of the seed coat and the volume of the cones all had a normal distribution. The analysis was made using a one-way ANOVA, with three levels corresponding to the three-host species (P. pinea, P. pinaster and P. halepensis). Post-hoc pairwise comparisons were made using the Student-Newman-Keuls (SNK) test. In both the cone and branch preference trials we used Generalized Linear Models (GLM) with repeated measures (each bug was an individual with repeated observations). To compare frequencies among cones and branches, we used GLM with a Poisson distribution, log link function, and Wald Chi-Square test. In the branch preference trials, we performed the analysis for the overall data for each pine species combination, pulling the three cages, as well as per cage. Finally, we used GLM with normal distribution and log link function to analyse differences in the feeding duration time in the cone trials. Again, each bug was considered an individual with repeated measures. For the seed preference trials, to compare frequencies among seeds, we used GLM with a Poisson distribution, log link function, and Wald Chi-Square test. In the two-choice seed trial, we compared: i) the mean number of seeds consumed between choice and non-choice tests for each pine species; ii) the mean number of seeds consumed between pine species on both choice and iii) on non-choice tests. In the three-choice trial, we analysed the differences in the mean number of seeds consumed between the three pines species with boxes considered as repeated measures. We further compared the seed weight consumed and the percentage of kernel consumed per host and box in both seed trials using GLM with normal distribution and log link function. Boxes with no consumption were removed from the analysis.
All statistical analyses were performed using SPSS, version 24.0 (IBM Corp., Armonk, New York) with a statistical significance level of 0.05.
The volume of last-year cones differed significantly among pine species (F=92.38; df=2; p<0.001), with the volume of P. pinea cones being two and three times larger than those of P. pinaster and P. halepensis, respectively. The weight of mature coated seeds also differed significantly among species (F=10387.92; df=2; p<0.001) as well as the kernel volume (F= 1526.33; df=2; p<0.001), and coat thickness (F= 4681.251; df=2; p<0.001: Table
Cone and seed average measures (± SE) of the three host species. Different letters within a column indicate significant differences between the values per host species after ANOVA tests (p-value=0.05) followed by SNK test.
Host species | Cone volume (cm3) | Mature seed weight (g) | Kernel volume (KV) (mm3) | Coat thickness (CT) (mm) | KV: CT |
---|---|---|---|---|---|
P. pinea | 90.0 ± 7.6a | 0.867 ± 0.008a | 202 ± 17.0a | 1.438 ± 0.030a | 140.5 |
P. pinaster | 43.0 ± 1.7b | 0.062 ± 0.001b | 50 ± 11.0b | 0.380 ± 0.009b | 131.6 |
P. halepensis | 27.3 ± 7.7c | 0.022 ± 0.000c | 15 ± 1.0c | 0.117 ± 0.003c | 128.2 |
No host preference was detected in either of the three host pine choice combinations on the frequencies of visits per bug (P. halepensis x P. pinea: Wald Chi2=2.485, p=0.115; P. pinaster x P. pinea: Wald Chi2=0.005, p=0.943; P. halepensis x P. pinaster: Wald Chi2=0.008, p=0.927). Overall, 70% of the individuals remained on the same host species during the trial period (48h) with the few changes happening on the first day. When each cage was consider separately, a significant preference was observed for one branch or the other, whereas preferred host species may differ from one trial to the other for the same host species combination (Figure
Branch preference trials. Bugs distribution per host in each cage and for each host pair comparison in the preference trials using potted branches. Numbers with asterisk on the x axis correspond to male bugs. The absence of bars means that individuals were never observed on the branches during the trial, but remained on the floor or on the walls of the cage.
As a general trend, we observed that the ten individuals from each cage dissociated into one or two fix groups in the three trials (Figure
We found no differences between the three trials and so results were analysed together. The adults were observed visiting and feeding more frequently on P. pinea cones in comparison with cones from the other two species (visiting: Wald Chi2 =17.42; p<0.001; feeding: Wald Chi2 =15.31; p<0.001). Per feeding meal, the adults also spent more time feeding on P. pinea cones in comparison with other cones (Wald Chi2 =12.05; p=0.002) (Table
Bug behaviour averages (± SE) in the cone preference trials. Different letters within a column indicate significant differences between the values per host species after GLM tests (p-value=0.05).
Host species | Visiting frequency | Feeding frequency | Time spent per feeding meal (minutes) |
---|---|---|---|
P. pinea | 13.4 ± 4.2a | 5.5 ± 1.2a | 131.9 ± 25.9a |
P. pinaster | 5.1 ± 1.2b | 2.0 ± 0.6b | 53.2 ± 12.0b |
P. halepensis | 3.7 ± 1.5b | 1.1 ± 0.5b | 62.5 ± 13.3b |
Bug preference between seeds of P. pinea and P. pinaster
For four weeks, the four individuals always ate two, out of ten, seeds of P. pinea per box, either if it was offered alone (non-choice tests) or mixed with P. pinaster seeds (choice tests) (Table
Seed consumption in choice and non-choice trials. Average number (± SE) of consumed seeds per box, kernel weight consumed per box and bug and percentage of the kernel that was consumed in each of the seed preference trials. Different letters within a trial indicate significant differences between the values per host species after GLM tests (p-value=0.05).
Type of trial | Host (s) | Seeds (total) | number of seeds consumed | kernel consumed (mg) | kernel consumed (%) |
---|---|---|---|---|---|
two-choice | P. pinea | 5 | 2.0 ± 0.0 | 11.71± 2.53a | 97.12 ± 15.01a |
P. pinaster | 12 | 0.5 ± 0.5 | 1.28 ± 3.58b | 5.77 ± 21.23b | |
non-choice | P. pinea | 10 | 2.5 ± 0.7 | 21.03± 2.21 | – |
P. pinaster | 24 | 5.5 ± 1.7 | 4.13 ± 2.12 | – | |
three-choice | P. pinea | 10 | 2.7 ± 1.2 | 23.91 ± 1.40a | 92.30 ± 10.62a |
P. pinaster | 10 | 3.0 ± 1.8 | 3.69 ± 1.76b | 29.14 ± 10.61b | |
P. halepensis | 10 | 5.3 ± 2.7 | 4.51 ± 1.55b | 39.73 ± 12.26b |
Testing bug preference among seeds of P. pinea , P. pinaster , and P. halepensis
Seed consumption was observed on 8 out of the 12 boxes (Figure
Seed three-choice trial. A sample of seeds consumed by L. occidentalis on the three-choice trial. Each row corresponds to a different host pine: (from top to bottom) P. pinea, P. pinaster and P. halepensis, with seeds arranged in each row from the less (left) to the more damaged (right). Photographs taken by Canon 1100 D. The marks on the scale correspond to 1mm. (Photos were taken by Charlene Durpoix).
The impact of an invasive species must be assessed at different levels from the individual to the ecosystem processes level (
Under natural conditions, L. occidentalis has been shown to select a host in a multi-level process. At first, the bug selects a tree, then a cone and finally a seed where to feed upon (
With all that has been said in mind, we hypothesized that branch selection by this bug, a polyphagous insect that feeds on growing plant parts, should rely mostly on vigour (e.g., increased resources, higher food quality, and lack of induced defensive compounds) and cone size and not so much on plant species and their chemical profiles.
In our experimental trials using potted branches with cones, the seed bug showed no clear preference between the three host species but rather a preference for one of the two branches on each trial. The plant vigour hypothesis (
Preference studies on a related species, L. phyllopus (L.), which is also polyphagous, have evidenced that nutritional and host quality issues (wild vs cultivated plants) are more determinant in host plant selection rather than plant species (
The branch preference trial also revealed an overall trend for L. occidentalis to form two groups of individuals per cage, one in each plant. In general, the composition of the two groups remained similar in each cage in all three trials. During the time between trials, all insects (n=30, 10 from each cage) were placed together in a single cage but when they were replaced one by one again in the test cage they regrouped in the same way as in the very first trial. Furthermore, once the individuals had chosen one of the plants, they remained there, in 70% of the cases, throughout the experiment. Group dynamics and not an individual host selection is, thus, a more suitable explanation for the results obtained. The gregarious behaviour of this insect has already been proposed by other authors (
Preference for a host species was further tested exposing cones to adults in trials using video cameras. The use of video recording can be very informative on the insect preference because it allows capturing the behaviour of the insect continuously. Moreover, the use of cameras with time lapse mode made it possible to process all data since it condenses several days of filming in movies of few minutes. So, by tracking the feeding behaviour, a clear bug preference could be observed for visiting and feeding on P. pinea cones. Furthermore, the individuals spent twice more time feeding, by each feeding meal, on this host species than on the other two species. A higher reward per seed would probably keep insects feeding longer times. It has also to be noted that the seed coats were not yet totally hardened inside cones because we used last year cones collected in the spring. Under natural conditions, the larger cones of P. pinea could be more attractive since visual stimuli are important to this bug in the process of host selection (
In respect to seed trials, no clear preference between mature seeds of P. pinea, P. pinaster and P. halepensis was observed if we compare the number of seeds consumed. However, P. pinea kernels are much larger than the others. Furthermore, it has been shown that the same insect feeds several times on the same seed and that different insects may also feed on that same seed by sharing the feeding hole (from video recording observations, data not showed) (
Other preference study at the seed level showed that L. occidentalis appeared capable of differentiating a viable seed from one infested by chalcid, Megastigmus spermotrophus (Hymenoptera: Torymidae), discarding the latter (
Despite the larger size of P. pinea seeds, which constitute a more significant reward, the seed coat implies a higher cost, being three and twelve times thicker than that of P. pinaster and P. halepensis, respectively. Even so, a benefit/cost analysis pointed to P. pinea seeds as being more advantageous. Feeding behaviour videos show that drilling a hole in P. pinea seeds can take more than 8 hours to complete (unpublished data) but then the benefit is high and, most importantly, it is shared by the remaining insects of the box as other bugs use the same hole to feed. We should note that, although easy to replicate under laboratory conditions, host selection trials resourcing to mature seeds have a limited ecological significance because the seeds, enclosed within the cones, are not subjected to selection in natural conditions.
In spring or early summer, depending on the climatic conditions, this insect becomes active and begins to search for a site with coniferous where to feed and reproduce. No data can be found on the bug behaviour when leaving its winter shelter except for an inconclusive study by
Among the three main pine species in the Mediterranean Basin, P. pinea, P. pinaster and P. halepensis, our results support evidence that the cones and seeds of P. pinea are highly rewarding for L. occidentalis. From a nutritional point of view, we may then expect that P. pinea trees and plantations may favour L. occidentalis population growth. Whereas in seeds and cones there was always a preference trend for P. pinea, no clear preference for host species was detected when we used potted branches.
It must be highlighted that the quality of the host plant rather than the species, and the aggregation behaviour of this bug are important factors to take into account when designing the methodology of future host preference studies. Furthermore, larger scale trials are required. The population dynamics of this insect must be a priority research topic. No management plan will succeed without understanding which factors influence the distribution and abundance of this pest, including the availability of, and its performance on, different hosts.