Research on burrowing arthropods as indicators of change - behavioural bioassays

Authors: Felicita Scapini, Mariella Nardi, Claudia Rossano, Lucia Fanini, Simone Gambineri, Serena Benedetti, Giovanni M. Marchetti, Elfed Morgan, Derek D. Green (Partner 1)


Locomotor activity rhythms were analysed in three species of talitrid amphipods colonizing different habitats in the Maremma Regional Park (Italy): Orchestia montagui from the Posidonia banquette, Talitrus saltator from the beach and a channel near river mouth, Orchestia gammarella from the river bank upstream and near river mouth and from a cave. Water conductivity was measured at all stations in summer and autumn; seasonal changes were observed near the river mouth (higher conductivity in summer). Endogenous rhythm were recorded automatically in dark conditions and constant temperature for 21 days in the laboratory in, and individuals were tested singly, 36 at the same time, two populations each time. Experiments lasted from May 2002 to October 2004. Individual features of the locomotory activity rhythms were analysed; populations features were analysed and intra-specific comparisons were made; differences between populations were analysed. The descriptors analysed were: survival, percentage of active individuals, periodicity, period, signal to noise ratio. The results showed that biological clocks expressed circadian rhythmicity in all the three studied species with an evident individual variability; The characteristics of the rhythm mainly depended on species: Orchestia gammarella had weak rhythms and high variability in circadian period, Talitrus saltator had intermediate rhythm features between the two other species, Orchestia montagui had well defined rhythms with respect to both the other species. A certain plasticity existed inside each species and emerged from intra-specific differences related to the colonized habitat. It is concluded that rhythms can be differently expressed depending on how much they are adaptive in the locality where the organisms live.



The seasonal variation of the circadian activity rhythm of the Talitrus saltator population living in the Italian study site of the MEDCORE project was analysed. The rationale of the study was an in-depth analysis of the rhythmic behaviour throughout a year of the population chosen as a case study, allowing successive comparisons with a wide range of different ecological and geographical conditions. Different profiles of rhythmic patterns of circadian activity were highlighted and the possible causes of the found variability were analysed; qualitative comparisons were made with other researches carried out in the framework of the MEDCORE Project (Karima Nasri and Dhouha Bholi, Partner 7). The common experimental protocol was: animals collected by hand on the beach and immediately transferred by car (in boxes containing wet sand) to the laboratory in Florence; animals put individually in the 39 recording chambers (constructed at the Bioscience workshop, Birmingham) (sand from the beach of origin); experiments performed in controlled laboratory conditions: constant temperature = 18 ± 1°C; continuous darkness (free-running conditions); recording sessions lasting 10 days; 1 recording session in each season from summer 2004 to spring 2005; at the end of the experiments each individual was put in alcohol 75% to check of the morphometric parameters: sex, number of the second antennae tagma, cephalic length. Rhythms features analysed were: survival, periodicity, mean circadian period, definition of the period (signal to noise ratio); in each recording session 39 individuals were tested. Seasonal differences were analysed. Results were:  NO significant difference in the survival (CHIsquare = 2.120, df = 3, p > 0.05); NO significant difference in the frequencies of the rhythmic animals (CHIsquare = 3.755, df = 3, p > 0.05); NO significant difference in the mean circadian period (Kruskall-Wallis CHIsquare = 0.7498, df = 3, p > 0.05); but at last there was a SIGNIFICANT difference in the signal to noise ratio (Kruskall-Wallis CHIsquare = 22.5948, df = 3, p < 0.001), that expresses the clearness (i.e. the definition) of the rhythm with respect to the random noise. Animals showed a more defined circadian period in summer. These data confirmed previous results obtained with other Italian populations. Then the focus was made on sample composition (males, female and juveniles) to test whether season was the only source of variation. As juveniles were found in February only, differences in period between males and females were analysed only . Females showed a more defined circadian period than males. 


These data confirm  the results obtained in the orientation choices of the two sexes. A study has been started to analyse eventual differences of the rhythmic behaviour in animals coming from different points along the coastline, as had already been done with orientation (see below). Up to the moment, two points only have been tested in summer (point 4000m and 5000m from the mouth of the river Ombrone), and no significant difference has been found. It can be concluded that the population of T. saltator living at Collelungo in the Maremma Regional Park is a healthy population, being abundantly present on the beach all year long and showing a good circadian rhythm of activity. These results confirmed the seasonal trend of the circadian rhythm in this population, as already demonstrated in other Italian populations. The clearness of the circadian rhythm expressed by the signal to noise ratio was the parameter of the rhythm significantly different in the four seasons (while the period was not). These results showed for the first time the differences between males and females in the definition of the circadian rhythm, as already demonstrated in the precision of orientation in the same species: females (which carry juveniles in early stages of development in the pouch), show a “seasonally changing” precision in the activity rhythm in order to better adapt to the environmental ecological conditions.



The second bio-assay was based on orientation and its adaptation to the shoreline. During the day, the “safe place” for Talitrus saltator is represented by the wet sand zone, where it can burrow, avoiding both to get dry and to be swashed away by the sea. Therefore, orientation capability is needed to recover the safe place, in case of displacement. Cues for orientation may be visual (sun and landscape), slope (beach substrate) and the magnetic field. The same cue could be used for multiple mechanisms, e.g. the sun vision could be used for a sun compass or for a simple phototaxis. In the framework of MEDCORE, we compared performances of talitrids at three different study sites: Maremma (Italy), Oued Laou (Morocco) and Berkoukesh (Tunisia). On field a circular arena was used placed directly on the beach to test the orientation of animals collected in loco. Traps at the arena’s rim, subtending 5° each, were used to calculate the direction chosen by the animals. The landscape vision was avoided, when needed, by placing a white cardboard 10cm height all around arena’s periphery. Air temperature, air humidity, sun visibility and sky cover were recorded at every release of the animals. In the laboratory, individual characteristics of the sample were measured: size (cephalon length), age (number of 2nd antennae tagma) and sex. The orientation data were analysed with the statistics of angular distribution (software developed ad hoc by G.M. Marchetti, and made available to the partners and other interested researchers and students). The effects of multiple variables and factors on orientation distributions were analysed with SPLM (spherical projected linear models) and different models were compared with the Akaike criterion to choose the “best model”, with the minimum number of factors and the best likelihood. 


At Berkoukesh (Tunsia), one site only was analysed on the left bank of Oued Berkoukech; experiments were conducted in April and June 2005, with and without landscape. The “best model” included a significant interaction with the factor “month”, which could include abiotic factors such as air humidity and temperature, and biotic, as the sample structure or the place were the sandhoppers were found, the dune or the shoreline. In April the orientation was towards the dunes, and the best model included time of day, age and sun azimuth as significant factors; in June, the sandhoppers oriented seaward, and the best model included landscape vision, age, air temperature and sky cover as significant factors. 


At Oued Laou (Morocco) two sites were tested, on the left and right bank of the Laou river (2km far each other), experiments were made with and without landscape vision (screen on/screen off). The SPLM analysis showed a significant interaction with the “oued bank”. The abiotic factors were the same as the tests had been made at the same time, slight differences were found between the sample structures. But the impacts of human frequentation on the two banks were clearly different and were worth of consideration. On the left bank (Oued Laou village) the talitrids appeared very scattered  and the SPLM analysis showed that variation in orientation was dependent on time of day, age of the sample, sex, air temperature, air humidity, sky cover and sun visibility. The orientation mechanism used was here a simple phototaxis, changing with the sun azimuth change. On the other hand, sandhoppers from the right river bank (Ka’a Asrass village) were well concentrated seawards and orientation depended on the day of the experiment, solar time, age and sun visibility. The mechanism used was here the sun compass, as in well adapted populations.


At the Maremma site (Italy) four sites were analysed (the same as in the biodiversity study), subjected to different shoreline dynamics; seasonal replicates were made; experiments without landscape only were carried out. The best model did not show any interactions; it included season, sun visibility, time of day, trampling by visitors, distance from river mouth, air humidity and sex as significant factors. 


On the whole, it can be concluded that:

  1. The use of sun vision could be different, depending of the stability of environmental conditions; 

  2. The sun compass mechanism is used in cases of environmental stability, when the population can fix (likely genetically) in its behaviour the direction towards safety, being sure that they will not change through time;

  3. Sun compass use can also be combined with environmental experience, to face sudden but temporary disturbances; 

  4. The phototaxis is mainly used in cases of environmental instability, co-occurring with less precision, and it is often related with fringe populations or explorative behaviour;

  5. Landscape vision is also a cue for orientation, but with a minor weight in the choice of the escape direction; 

  6. Differences in behaviour regarding sex and age of the individuals were also observed; 

  7. Such differences are related with the precision of the direction, not with the direction chosen. 

The results from the orientation experiments have been crossed with those from other analyses conducted by partners on the same sites within different tasks (geomorphology, sedimentology, ecology and also socioeconomics). In collaboration with Prof. Khattabi (Partner 10), Lucia Fanini analysed the human impacts to the beaches of Oued Laou and Ka’Asrass (on the left and right sides of the river respectively) and correlated the differences with those observed in the behaviour of beach arthropods. The study at the Maremma site also included an estimate of trampling on the beach, that was possible because the site is within a natural park with controlled visits. Interesting perspectives are also the correlation of these data with the genetic analyses by the Roma team, and with the trends of change of the shorelines, as estimated by G.Ciampi (geographic research in the Maremma Regional Park) and A. Oueslati (Tunisia and Morocco).


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