Mother Colony Research Articles

Influence of Queen and Diet on Nestmate Recognition and Cuticular Hydrocarbon Differentiation in a Fission-Dispersing Ant,Aphaenogaster senilis

In social insects, colony fission is a particular mode of dispersal by which an adult colony splits into two or more independent nests. In the monogynous ant Aphaenogaster senilis, field data suggest that new queens may be produced in queenless daughter nests after nest relocation. Because workers do not fly, colony fission limits dispersal distance, leading young sister colonies to compete together and with the mother queen. In the present study we analysed the effects of queen loss and diet change on nestmate recognition. Queenright colonies were separated into two queenless and one queenright fragments. One queenless group received the same food as the queenright group, while the other queenless group received a different diet for 150 days. Recognition bioassays revealed that aggression between queenright and queenless former nestmates increased progressively until day 20, when they could no longer be reunited. Different diets also induced aggression between orphaned groups. Chemical analyses indicated that cuticular hydrocarbon profiles were already different between groups after 5 days. Overall, our results are in accordance with the graded model of nestmate recognition and suggest that the loss of the mother queen progressively leads to the independence of the related nests after fission. This may also allow queenless ants to merge again with their mother colony during a short time window after fission.

Algal Symbionts in the Larval Tunic Lamellae of the Colonial Ascidian Lissoclinum timorense (Ascidiacea, Didemnidae)

Lissoclinum timorense is a colonial ascidian that harbors the prokaryotic alga Prochloron . The algal photosymbionts adhere to the lamellae of the tunic on the posterior half of the trunk of larvae, which aggregate in the common cloacal cavity of the mother colony. Bead-adhesion tests demonstrated that the lamellae are adhesive, whereas the anterior half of the larval trunk is not. The anterior half is covered with a thin layer of outer tunic, which probably prevents Prochloron cells from attaching and interfering with sensory receptors and adhesive organs. The larval structures and the mode of algal transmission between generations are very similar to those of the Prochloron -harboring ascidian Didemnum molle . Molecular phylogenetic analyses have suggested that photosymbiosis was independently established in each genus, and thus the apparent similarity in the larvae probably resulted from convergence. The distribution pattern of photosymbionts is probably more determinative of algal transmission than phylogenetic constraints.

In situ localisation and quantification of surfactins in a Bacillus subtilis swarming community by imaging mass spectrometry

Surfactins are a family of heptacyclopeptides in which the C-terminal carbonyl is linked with the beta-hydroxy group of a fatty acid acylating the N-terminal function of a glutamic acid residue. The fatty acyl chain is 12-16 carbon atoms long. These compounds, which are secreted by the Gram-positive bacterium Bacillus subtilis in stationary phase in liquid cultures, play an important role in swarming communities on the surface of agar media in the formation of dendritic patterns. TOF secondary ion MS (TOF-SIMS) imaging was used to map surfactins within 16-17 h swarming patterns, with a 2 mum spatial resolution. Surfactins were mainly located in the central mother colony (the site of initial inoculation), in a 'ring' surrounding the pattern and along the edges of the dendrites. In the mother colony and the interior of the dendrites, surfactins with shorter chain lengths are present, whereas in the ring surrounding the swarm community and between dendrites, surfactins with longer fatty acyl chain lengths were found. A quantitative analysis by MALDI-TOF MS showed a concentration gradient of surfactin from the mother colony to the periphery. The concentration of surfactin was approximately 400 pmol/mL in the mother colony and approximately 10 pmol/mL at the base of the dendrites, decreasing to 2 pmol/mL at their tips.

THE SUCCESSFUL TRANSPLANTATION OF ACROPORA MICROPHTHALMA AT BARRANG LOMPO REEF EDGE< SOUTH SULAWESI

Research on transplantation of Acropora microphthalma was conducted at Barrang Lompo Island reef edge of South Sulawesi from September to December 2002. The aim of this research was to know the successful rate of several coral transplantation methods for rehabilitation of coral reefs, covering the survival and growth rates. The methods used were framework earthenware (FE), iron stake (IS), dead coral tying (DCT), where the mother colony (MC) was used as control. These transplantation techniques were applied at five meter depth in two stations, north and south sites of the island. The survival rate for each method FE, IS, DCT and MC at the northern station was 85.71; 42.86; 85.71; 57.14%, while for southern station was 85.71; 85.71; 71.43 and 85.71%, respectively. The growth rates observed for each method at the first station were 2.18; 2.02; 1.40; 1.05 cm, and for the second station were 2.56; 2.21; 1.61; 1.04 cm, respectively.

Nest volatiles as modulators of nestmate recognition in the ant Camponotus fellah

When ants from alien colonies encounter each other the stereotypic reaction is usually one of aggressive behavior. It has been shown that factors such as queen-derived cues or nest-odors modulate this reaction. Here we examined whether nest volatiles affect nestmate recognition by observing the reaction of nestmates towards individual workers under one of four regimes: completely isolated; isolated but receiving a constant airflow from the mother colony; as previous but with the passage of nest volatiles towards the isolated ants blocked by adsorption on a SuperQ column; or reversed airflow direction-from the isolated ants to the nest interior.Ants that had been completely isolated for three weeks were subjected to aggressive behavior, but not those that had continued to receive airflow from the mother colony. Adsorbing the nest volatiles from the airflow by SuperQ abolished this difference, with these ants now also being subjected to aggression, indicating that nest volatiles can modulate nestmate recognition. Reverse airflow also reduced the level of aggression but to a lesser extent than airflow directed from the mother colony. In queenless colonies the overall aggression was reduced under all regimes, and there was no effect of flow, suggesting that the volatiles involved are queen-borne. The SuperQ adsorbed volatiles originated from Dufour's gland secretions of both workers and queen, implicating them in the process. Cuticular hydrocarbon profiles were not affected by exposure to nest volatiles, suggesting that the latter either constitute part of the recognition cues or affect worker behavior via a different, as yet elusive mechanism.

Production of sexuals in a fission-performing ant: dual effects of queen pheromones and colony size

Models based on the kin selection theory predict that in social hymenopterans, queens may favor a lower investment in the production of sexuals than workers. However, in perennial colonies, this conflict may be tuned down by colony-level selection because of the trade off between colony survival and reproductive allocation. In this study, we present a survey of sexual production in colonies of Aphaenogaster senilis, a common species of ant in the Iberian Peninsula. Similar to most species that reproduce by fission, males were found in large excess compared to gynes (172:1). Sexuals were more likely to be found in queenless than in queenright (QR) field colonies. However, we also found a few gynes and numerous males in very large QR colonies. We compared these data with those available in the literature for A. rudis, a congeneric species from North America that has independent colony founding. The sex ratio in this species was only five males for each female, and sexuals were mostly found in QR nests, irrespective of colony size. We confirmed queen inhibition of sexual production in A. senilis in laboratory experiments and provide evidence that this inhibition is mediated by a nonvolatile pheromone. To seek the potential source of such a queen pheromone, we analyzed the secretions of two conspicuous exocrine glands, the Dufour’s and postpharyngeal glands (DG and PPG, respectively) in both queens and workers. Both secretions were composed of hydrocarbons, but that of DG also contained small quantities of tetradecanal and hexadecanal. The hydrocarbon profile of the DG and PPG showed notable caste specificity suggesting a role in caste-related behavior. The PPG secretions also differed between colonies suggesting its role in colony-level recognition. We suggest that in A. senilis, there are two modes of colony fission: First, in very large colonies, gynes are produced, probably because of the dilution of the queen pheromone, and consequently one or more gynes leave the mother colony with workers and brood to found a new nest. This is beneficial at the colony level because it avoids the production of costly sexuals in small colonies. However, because the queen and workers have different optima for sexual production, we hypothesize that queens tend to overproduce the pheromone to delay their production. This in turn may drive workers to leave the mother colony during nest relocation and to produce sexuals once they are away from the queen’s influence, creating a second mode of colony fission.

Polydomy in ants: what we know, what we think we know, and what remains to be done

The correct identification of colony boundaries is an essential prerequisite for empirical studies of ant behaviour and evolution. Ant colonies function at various organizational levels, and these boundaries may be difficult to assess. Moreover, new complexity can be generated through the presence of spatially discrete subgroups within a more or less genetically homogeneous colony, a situation called polydomy. A colony is polydomous only if individuals (workers and brood) of its constituent nests function as a social and cooperative unit and are regularly interchanged among nests. This condition was previously called polycalic, and the term polydomy was used in a broader sense for a group of daughter nests of the same mother colony (implying limited female dispersal), without regard to whether these different nests continued to exchange individuals. We think that this distinction between ‘polycaly’ and ‘polydomy’ concerns two disparate concepts. We thus prefer the narrower definition of polydomy, which groups individuals that interact socially. Does this new level of organization affect the way in which natural selection acts on social traits? Here, after examining the history of terms, we review all ant species that have been described as expressing polydomous structures. We show that there is no particular syndrome of traits predictably associated with polydomy. We detail the existing theoretical predictions and empirical results on the ecology of polydomy, and the impact of polydomy on social evolution and investment strategies, while carefully distinguishing monogynous from polygynous species. Finally, we propose a methodology for future studies and offer ideas about what remains to be done.

Gene Diversity and Mode of Reproduction in the Brooded Larvae of the Coral Heteroxenia fuscescens

The soft coral Heteroxenia fuscescens is a common shallow-reef brooding species in the Red Sea. By means of amplified fragment length polymorphism markers, we studied modes of reproduction of planulae (sexual vs. vegetative) and levels of gene diversity of a population residing in the northern Gulf of Eilat, Red Sea. Eighty-nine larvae were collected from 9 mother colonies at 3 locations over a distance of 5 km. Amplifications revealed 221 putative loci, of which 81.4% were polymorphic; gene diversity was 0.29, allowing good identification of individual genotypes. No 2 identical DNA samples were present, so no asexual reproduction of planulae was indicated. The sampled planulae did not exhibit any genetic structure characteristic to a specific location, indicating one large gene pool and extensive gene flow among H. fuscescens specimens inhabiting the northern Gulf of Eilat.

Vertical Transmission of Photosymbionts in the Colonial Ascidian Didemnum molle: The Larval Tunic Prevents Symbionts from Attaching to the Anterior Part of Larvae

Morphological processes in the vertical transmission of photosymbionts were investigated in the Prochloron-bearing ascidian Didemnum molle. Prochloron cells were found exclusively in the common cloacal cavity of the colony, attached mainly to the tunic lining of the cavity wall. Oocytes were found in the abdominal region of each zooid, but no Prochloron cells were associated with this stage. During embryogenesis, embryos moved into the tunic core of the colony and were always separated from Prochloron cells in the cloacal cavity by the tunic matrix, until they hatched out from the tunic core. In swimming larvae, Prochloron cells covered the surface of the posterior half of the larval trunk, whereas a thin larval tunic layer covered the anterior half, where no Prochloron cells were found. The tunic of the posterior half of the larval trunk had many folds that enfolded the Prochloron cells and may be adhesive in order to acquire Prochloron cells from the mother colony. The thin larval tunic layer is probably not adhesive and protects the anterior half of the trunk from interference by Prochloron cells with sensory receptors and adhesive organs.

Morphological process of vertical transmission of photosymbionts in the colonial ascidian Trididemnum miniatum Kott, 1977

In obligate symbioses between didemnid ascidians and prokaryotic algae (cyanobacteria), the larvae of the host ascidian usually possess Prochloron cells that are acquired from their mother colony. The process of vertical transmission of the photosymbionts has been histologically described in several species harboring Prochloron in the common cloacal cavity, where Prochloron cells are attached to the hairy surface of the tunic of the larvae during or just before larval spawning. Since the process has never been described in species harboring the photosymbionts in the tunic of the colony, here we describe the histological and ultrastructural process of vertical transmission in a photosymbiotic didemnid, Trididemnum miniatum. No photosymbionts were associated with gametes of the ascidian. Prochloron cells appeared in the epithelial pouch enclosing the embryo and were then incorporated in the tunic of embryos in late embryonic stages. Although Prochloron cells in the tunic of the colony were rarely associated with the host cells, some amoeboid-shaped tunic cells containing Prochloron cells were occasionally found around the epithelial pouch. Therefore, the host cell is thought to be a vehicle transporting the photosymbionts in the tunic of the colony to the tunic of the embryos. The photosymbiont cells were directly embedded in the tunic of the larval trunk. They were not contained in the host cells, as are those in the tunic of the colony. These observations revealed that the mechanism of vertical transmission in T. miniatum is very different from that in didemnids harboring Prochloron in the common cloacal cavity of the colonies, such as Trididemnum cyclops. The mechanisms of symbiont transmission are diverse even within the genus Trididemnum.

Vertical transmission of American foulbrood ( Paenibacillus larvae) in honey bees ( Apis mellifera)

The mode of transmission between hosts (horizontal versus vertical) of disease agents is important for determination of the evolution of virulence in pathogens. For disease management, it is imperative that the epidemiology of the disease is understood and pathogen transmission rates between hosts is a key factor for this understanding. Surprisingly little is known about transmission rates in honey bee pathology. We have studied the rate of vertical transmission of Paenibacillus larvae, the causative agent of American foulbrood (AFB) in honey bee colonies, as colonies reproduce by colony fission (swarming), by culturing for the spores from repetitive samples of adult bees. The results demonstrate vertical pathogen transmission to daughter swarms. The spore density declines over time in both mother colonies and daughter swarms if mother colonies do not exhibit clinical disease symptoms. Occasional positive samples more than a year post swarming, also in daughter swarms, indicate production of infectious spores from diseased larvae, without clinical disease observable by beekeepers, and/or maintenance of infective spores in the hive environment, allowing both horizontal and vertical transmission to be maintained. The results suggest that the virulence of AFB, being lethal at colony level in contrast to other bee diseases shaped by evolution, could be dependent on apicultural practices and that the pathogen probably would be maintained without causing frequent colony mortality in a natural system.

Colony growth and evidence for colony multiplication in Phaeocystis pouchetii (Prymnesiophyceae) isolated from mesocosm blooms

Using well plates of Phaeocystis pouchetii colonies isolated from experimental mesocosms in western Norway, increases in colony size and division were documented. Median, longest linear dimensions increased 0-7 μm h -1 ; literature Phamcystis globosa values are 0.9-4.7 μm h -1 . Ten to twelve percent of colonies divided at rates of 0.21-0.28 divisions day -1 . Daughter colonies were 100 μm smaller than mother colonies. Colonies delayed 3.5-4.9 days to first division, compared with literature values of 4-5 days for P. globosa. This study provides the first experimental evidence for colony division of wild P. pouchetii.

A scientific note on the lack of nepotism in queen larval feeding during emergency queen rearing in a naturally mated honey bee colony

Honey bee (Apis mellifera L.) colonies consist of many patrilines (Estoup et al., 1994) and workers can potentially increase their inclusive fitness during queen replacement if they can favour a full-sister offspring queen (Visscher, 1998). There is evidence of preferential rearing of some patrilines in emergency queen rearing (Tilley and Oldroyd, 1997; Osborne and Oldroyd, 1999; Châline et al., 2003), although the reason for this is unclear. Some patrilines could act nepotistically or a “royalty allele” could make some larvae more attractive to workers (Osborne and Oldroyd, 1999). Early studies on behavioural interactions and larval preferences during queen rearing used colonies with limited numbers of patrilines or cross-fostering of larvae between unrelated colonies with naturally mated queens. They showed weak or no evidence of nepotism (Breed et al., 1984; Noonan, 1986; Page et al., 1989). To avoid the problem of unnaturally low number of patrilines and the use of artificial insemination, we used microsatellites to analyse the kinship of feeding workers and queen larvae during emergency queen rearing, for the first time in a colony with a naturally mated queen. The experiment was carried out in double blind in a queenless observation hive containing individually marked bees. This was done by marking bees emerging from two brood combs of the test colony over 6 consecutive days. Before the experimental colony set-up, the marked bees were returned to their mother colony to ensure a normal ontogeny. Twentyfour hours before the set-up, 50 queen cells were initiated in a queen rearing colony by grafting larvae from the test colony. Thirteen of these were then attached to a brood comb from the test colony and transferred in an observation hive with 2000 of the marked workers. During the experiment, three additional queen cells were built naturally. Each queen cell was observed individually for 1/2 h every day until capping. The total observation time was 2130 min. The bees feeding each queen larvae were recorded, as this seemed the best behaviour for the

Sexual reproduction of the photosymbiotic ascidian Diplosoma virens in the Ryukyu Archipelago, Japan: vertical transmission, seasonal change, and possible impact of parasitic copepods

Diplosoma virens is a colonial ascidian hosting prokaryotic algae Prochloron sp. in the common cloacal cavity of the colonies and is sometimes parasitized by notodelphyid copepods. In ascidian–Prochloron symbiosis, it is generally known that the host larvae acquire the algal symbionts from their mother colonies to maintain the symbiosis. A histological study of the sexually mature colonies of D. virens showed that the algal symbionts attach to pre-hatching larvae on the rastrum (plant rake) projected from the postero-dorsal part of the larval trunk, and then the rastrum is packed in the posterior half of the larval trunk that will become a cloacal cavity after metamorphosis. This process is the same as that of D. simile. Monthly sampling of D. virens colonies showed that they have embryos in summer in Ryukyus, situated near the northern-most limit of the coral reefs in the West Pacific. While the frequencies of copepod parasitism were variable among the populations, the colonies from a highly parasitized population had a significantly smaller number of eggs/embryos per zooid than the colonies from the less parasitized populations. The parasites probably have an inhibitory impact on the sexual reproduction of the host colonies.

Worker policing persists in a hopelessly queenless honey bee colony ( Apis mellifera )

In queenright colonies of Apis mellifera, worker policing normally eliminates worker-laid eggs thereby preventing worker reproduction. However, in queenless colonies that have failed to rear a replacement queen, worker reproduction is normal. Worker policing is switched off, many workers have active ovaries and lay eggs, and the colony rears a last batch of male brood before dying out. Here we report a colony which, when hopelessly queenless, did not stop policing although a high proportion of workers had active ovaries (12.6%) and many eggs were laid. However, all these eggs and also worker-laid eggs transferred from another colony were policed. This unusual pattern was repeated eight weeks later by a second queenless colony made using worker bees from the same mother colony, which strongly suggests genetic determination.

Food influence on colonial recognition and chemical signature between nestmates in the fungus-growing ant Acromyrmex subterraneus subterraneus

Neotropical “Fungus-growing” leaf-cutting ants (tribe Attini) live in obligatory symbiosis with a fungus, which they grow on fresh leaves harvested by workers. Colonial recognition is likely based on chemical cues provided by cuticular hydrocarbons that have been found to be partly influenced by environmental odor sources. The diet breadth of Acromyrmex subterraneus subterraneus enabled us to test the impact of different plant diets on colonial recognition. The intermediary of the fungus in the ants’ feeding habit adds a special angle to the question. From a queenright (QR) mother colony of A. s. subterraneus we formed several groups of queenless (QL) workers with fungus (approx. 700 ants). The QR colony and two of the QL-groups were fed with the same diet of fresh bramble leaves. Two other QL-groups were fed with privet leaves and two with rose flowers. After 4 months, QR workers were significantly more aggressive towards the QL-group fed with rose flowers or privet leaves than towards workers of the QL-groups fed with fresh bramble leaves. Rose-fed QL workers were aggressive towards privet-fed QL workers and vice versa, but never towards workers of their counterpart group that fed on the same diet. These results suggest that the absence of the queen or the separation time between groups played a minor role in shaping nestmate recognition cues as compared to the diet. The behavioral studies were supplemented by chemical analyses of cuticles, postpharyngeal glands (PPG) and plant-food extracts revealing profiles variations that were correlated with the dietary changes. However, although the plant extract contained several hydrocarbons there was no congruency between the plant profile and the respective diet-group ants. These results support the hypothesis that the diet influences indirectly the chemical profiles and consequently the recognition cues in A. s. subterraneus.

Blending in with the crowd: social parasites integrate into their host colonies using a flexible chemical signature.

Social parasites are able to exploit their host's communication code and achieve social integration. For colony foundation, a newly mated slave-making ant queen must usurp a host colony. The parasite's brood is cared for by the hosts and newly eclosed slave-making workers integrate to form a mixed ant colony. To elucidate the social integration strategy of the slave-making workers, Polyergus rufescens, behavioural and chemical analyses were carried out. Cocoons of P. rufescens were introduced into subcolonies of four potential host species: Formica subgenus Serviformica (Formica cunicularia and F. rufibarbis, usual host species; F. gagates, rare host; F. selysi, non-natural host). Slave-making broods were cared for and newly emerged workers showed several social interactions with adult Formica. We recorded the occurrence of abdominal trophallaxis, in which P. rufescens, the parasite, was the donor. Social integration of P. rufescens workers into host colonies appears to rely on the ability of the parasite to modify its cuticular hydrocarbon profile to match that of the rearing species. To study the specific P. rufescens chemical profile, newly emerged callows were reared in isolation from the mother colony (without any contact with adult ants). The isolated P. rufescens workers exhibited a chemical profile closely matching that of the primary host species, indicating the occurrence of local host adaptation in the slave-maker population. However, the high flexibility in the ontogeny of the parasite's chemical signature could allow for host switching.

When do honey bee guards reject their former nestmates after swarming?

Honey bees (Apis mellifera L.) reproduce by swarming wherein the mother queen leaves the nest with approximately two thirds of her workers (the prime swarm). Several daughter queens are raised in the original nest, and these start to emerge shortly after the first swarm departs. One or several of these daughter queens may then leave the original colony sequentially with smaller afterswarms. Here we study the change in acceptance of former nestmates after colony reproduction using free-flying honey bee colonies. We used a total of four colonies each of which we divided to make four new colonies: two artificial swarms (the offspring colonies) and two colonies that mimic established ('old') colonies after swarming. The way the original (mother) colonies were divided allowed us to determine the relative importance of wax comb on cue divergence. Half of the divisions contained the original wax combs from the mother colony whereas the other divisions were not provided with comb requiring them to construct their own. We then tested the acceptance of former nestmates by introducing foragers at the hive entrances and observing the behaviour of guard bees. Our results did not show a consistent change in acceptance of former nestmates after swarming. In two out of four replicates, workers originating from the colonies that contained the original wax combs were rejected by guards from the initially comb-less colonies. This suggests that comb wax plays an important role in nestmate recognition. However, the remaining two replicates did not show a response; all former nesmates were still accepted two weeks after artificial swarming.

Intranidal Mated Offspring Hibernation (IMOH) in the Ant species Leptothorax gredosi (Hymenoptera: Formicidae: Formicoxenini)

In a number of ant species belonging to the Myrmicinae, tribus Formicoxenini, the young females (♀♀) mate within, or in the vicinity of, the maternal nests. They shed wings and spend the winter there. In the monogynous Spanish species Leptothorax (Myrafant) gredosi Espadaler & Collingwood 1982, dealate and mated young ♀♀ were found in the nests in fall. Daughter colonies with one such ♀ each and a few workers could easily be established. Several years of laboratory observations revealed that usually in spring the young mated ♀♀ fight and mutilate each other, and are eventually evicted from the colonies. In the field, the ♀♀ probably leave the nests during this period, perhaps together with a few workers, in order to establish own, monogynous colonies. However, young ♀♀ that have been reared and have mated in orphaned colonies begin to fight already in fall. Presumably they develop their ovaries earlier than under the influence of a queen. Survival chances for single ♀♀ during the winter are probably lower than within the mother colonies. Such temporary Intranidal Mated Offspring Hibernation' (IMOH) should not be confused with long-term functional monogyny, but it may constitute an evolutionary route towards the development of functional monogyny as well as of (facultative) polygyny, and for the evolution of social parasitism.

Establishment of single-genotype axenic cultures from the haploid stage of the pine blister rust Cronartium flaccidum

The haplophase of the pine blister rust Cronartium flaccidum was used to grow mycelial clones axenically from single genotypes. Single telia were suspended over the media in order to obtain a direct cast of uninucleate basidiospores, providing nurse cultures from a mass basidiospore inoculum. Nurse culture and medium composition were crucial factors in successful germination and establishment of single-spore colonies. Significant differences in percentage germination between treatments and controls suggested that an extracellular matrix secreted by high density seeded basidiospores could be involved in the germination of single propagules. Proteins present in this matrix could exert a surface contact stimulus triggering germination of newly seeded basidiospores. Pre-production of extracellular enzymes in the medium could also initiate early substrate catabolism and thus create a favourable environment to support subsequent colony establishment. Growth was good on various media, and excellent on HG1Y + BSA medium. Primary mother colonies were divided into several smaller ones, re-grown axenically, then macerated. Aliquots of the resulting mycelial fragments were dispensed with a micropipette into fresh agar media. Clones derived from each pure culture were in general morphologically similar in texture, margin, compactness, and surface topography. These genetically pure lines grew at a faster rate and rapidly produced a considerable amount of mycelium. The significance of culturing rust fungi from single genotypes is discussed in relation to potential applications and new directions of research.