Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator.

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.

Non-spatial information on the presence of food elevates search intensity in ant workers, leading to faster maze solving in a process parallel to spatial learning.

Experience can lead to faster exploitation of food patches through spatial learning or other parallel processes. Past studies have indicated that hungry animals either search more intensively for food or learn better how to detect it. However, fewer studies have examined the contribution of non-spatial information on the presence of food nearby to maze solving, as a parallel process to spatial learning. We exposed Cataglyphis niger ant workers to a food reward and then let them search for food in a maze. The information that food existed nearby, even without spatial information, led to faster maze solving compared to a control group that was not exposed to the food prior to the experiment. Faster solving is probably achieved by a higher number of workers entering the maze, following the information that food is present nearby. In a second experiment, we allowed the ants to make successive searches in the maze, followed by removing them after they had returned to the nest and interacted with their naïve nestmates. This procedure led to a maze-solving time in-between that displayed when removing the workers immediately after they had reached the food and preventing their return to the colony, and that of no removal. The workers that interacted upon returning to the nest might have transferred to naïve workers information, unrelated to spatial learning, that food existed nearby, and driven them to commence searching. Spatial learning, or an increase in the correct movements leading to the food reward relative to those leading to dead-ends, was only evident when the same workers were allowed to search again in the same maze. However, both non-spatial information on the presence of food that elevated search intensity and spatial learning led to faster maze solving.

The effect of maze complexity on maze-solving time in a desert ant.

One neglected aspect of research on foraging behavior is that of the effect of obstacles that increase habitat complexity on foraging efficiency. Here, we explored how long it takes individually foraging desert ant workers (Cataglyphis niger) to reach a food reward in a maze, and examined whether maze complexity affects maze-solving time (the time elapsed till the first worker reached the food reward). The test mazes differed in their complexity level, or the relative number of correct paths leading to the food reward, vs. wrong paths leading to dead-ends. Maze-solving time steeply increased with maze complexity, but was unaffected by colony size, despite the positive correlation between colony size and the number of workers that searched for food. The number of workers observed feeding on the food reward 10 min after its discovery decreased with complexity level but not colony size. We compared our experimental results to three simulation models, applying different search methods, ranked them according to their fit to the data and found the self-avoiding random search to fit the best. We suggest possible reasons for the model deviations from the observational findings. Our data emphasize the necessity to refer to habitat complexity when studying foraging behavior.

Consistent differences in foraging behavior in 2 sympatric harvester ant species may facilitate coexistence.

The co-occurrence of 2 similar species depends on their ability to occupy different ecological niches. Here, we compared the consistency of different aspects of foraging behavior in 2 co-occurring harvester ant species (Messor ebeninus and Messor arenarius), under field conditions. The 2 species are active concomitantly and display a similar diet, but M. arenarius features smaller colonies, larger workers on average, and a broader range of foraging strategies than M. ebeninus. We characterized the flora in the 2 species' natural habitat, and detected a nesting preference by M. arenarius for more open, vegetation-free microhabitats than those preferred by M. ebeninus. Next, we tested the food preference of foraging colonies by presenting 3 non-native seed types. Messor arenarius was more selective in its food choice. Colonies were then offered 1 type of seeds over 3 days in different spatial arrangements from the nest entrance (e.g., a seed plate close to the nest entrance, a seed plate blocked by an obstacle, or 3 plates placed at increasing distances from the nest entrance). While both species were consistent in their foraging behavior, expressed as seed collection, under different treatments over time, M. ebeninus was more consistent than M. arenarius. These differences between the species may be explained by their different colony size, worker size, and range of foraging strategies, among other factors. We suggest that the differences in foraging, such as in food preference and behavioral consistency while foraging, could contribute to the co-occurrence of these 2 species in a similar habitat.

Within-colony genetic diversity differentially affects foraging, nest maintenance, and aggression in two species of harvester ants.

There is accumulating evidence that genetic diversity improves the behavioral performance and consequently the fitness in groups of social animals. We examined the behavioral performance of colonies of two co-occurring, congeneric harvester ant species (Messor arenarius and a non-described Messor sp.) in fitness-related behaviors, pertaining to foraging performance, nest maintenance, and aggression. We linked these behaviors to the colonial genetic diversity, by genotyping workers, using six and five microsatellite markers for M. arenarius and M. sp., respectively. Correlations of genetic diversity with colony performance and aggression level contrasted between the two species. In M. arenarius, genetic diversity was correlated with foraging performance and nest maintenance but not with the overall aggression level, while in M. sp., genetic diversity was correlated with the overall aggression level, but not with foraging performance or nest maintenance. The two species exhibited similar specific aggression levels, with higher aggression shown towards heterospecifics and lower towards non-nestmate conspecifics and nestmates. However, M. sp. workers displayed a tendency to interact for longer with heterospecifics than did M. arenarius. We speculate that the different foraging strategies, group vs. individual foraging, and possibly also the different mating systems, contribute to the differences found in behavior between the two species.

The interplay between maze complexity, colony size, learning and memory in ants while solving a maze: A test at the colony level.

Central-place foragers need to explore their immediate habitat in order to reach food. We let colonies of the individually foraging desert ant Cataglyphis niger search for a food reward in a maze. We did so for three tests per day over two successive days and an additional test after a time interval of 4-20 days (seven tests in total). We examined whether the colonies reached the food reward faster, consumed more food and changed the number of workers searching over time, within and between days. Colonies' food-discovery time shortened within and between days, indicating that some workers learnt and became more efficient in moving through the maze. Such workers, however, also forgot and deteriorated in their food-discovery time, leveling off back to initial performance after about two weeks. We used mazes of increasing complexity levels, differing in the potential number of wrong turns. The number of workers searching increased with colony size. Food-discovery time also increased with colony size in complex mazes but not in simple ones, perhaps due to the more frequent interactions among workers in large colonies having to move through narrow routes. Finally, the motivation to solve the maze was probably not only the food reward, because food consumption did not change over time.