Properties of olfactory conditioning in the housefly, Musca domestica

The housefly, Musca domestica, was conditioned to odours using the proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response and the properties of conditioning were investigated. Among trials including forward pairing of the conditioned stimulus (CS) with the unconditioned stimulus (US), backward pairing and isolated presentations of CS and US, only forward pairing is effective on the acquisition of conditioning. Backward pairing combined with forward pairing does not influence the effectiveness of the forward pairing. CS given overlapping with a US presentation permits only weak conditioning. The acquisition of conditioning decreases with increase of the CS-US interval. In the differential conditioning situation to two odours, discriminative responses are observed. In the flies conditioned with one antenna, the conditioned response is elicited not only by stimulation of the antenna used for conditioning but also by stimulation of the antenna not used for conditioning, although the response using the former is higher than with the latter. The ability to be conditioned is reduced immediately after fastening on a clay bed and increases with time. Ability can also be improved by transection of the ventral nerve cord.     

棉铃虫成虫对两种关键花香气味的联系性学习

为了明确棉铃虫对苯乙醛和乙酸苯甲酯两种关键花香气味的联系性学习行为,在室内分别测定了不同学习训练的棉铃虫雌雄成虫对两种关键花香气味的选择偏好性反应和雄成虫的触角电位反应。偏好性测定结果表明,无花香气味接触经历的棉铃虫对苯乙醛和乙酸苯甲酯的选择频次均无显著差异,且单纯的花香气味接触经历也不能诱导棉铃虫气味偏好性的显著改变,唯有花香气味接触经历伴随蔗糖溶液进行强化训练时,才能诱导气味偏好性的显著性改变。从对两种花香气味的联系性学习看,棉铃虫更容易将苯乙醛与蔗糖溶液进行联系性学习,而不容易将乙酸苯甲酯与蔗糖溶液进行联系性学习。从性别差异看,雌虫比雄虫具有更强的将花香气味与蔗糖溶液进行联系性学习的能力。触角电位测定结果表明,不同学习训练的棉铃虫雄成虫对两种花香气味和绿叶气味顺-3-己烯-1-醇的EAG反应值之间不存在显著性差异。总之,花香气味伴随食物资源的联系性学习经历,能够明显提高棉铃虫成虫对花香气味的选择偏好,可以此为基础进一步优化花香引诱剂的配方设计和应用技术。     

Classical conditioning to visual stimuli in the housefly, Musca domestica

In the housefly, Musca domestica, classical conditioning to monochromatic light was demonstrated by using a proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response. Sequential and temporal relationships between the conditioned stimulus (CS) and unconditioned stimulus (UCS) to form conditioning were studied; conditioning occurred most readily when the CS was presented before the UCS without an interstimulus interval. Spectral response curves, which were obtained by examining the responsiveness of flies conditioned for one colour to other colours, were divided into two groups; one was that of 462 nm-conditioned flies and the other those of 516 nm-, 579 nm-, and 642 nm-conditioned flies.     

Classical reward conditioning in Drosophila melanogaster

Negatively reinforced olfactory conditioning has been widely employed to identify learning and memory genes, signal transduction pathways and neural circuitry in Drosophila. To delineate the molecular and cellular processes underlying reward-mediated learning and memory, we developed a novel assay system for positively reinforced olfactory conditioning. In this assay, flies were involuntarily exposed to the appetitive unconditioned stimulus sucrose along with a conditioned stimulus odour during training and their preference for the odour previously associated with sucrose was measured to assess learning and memory capacities. After one training session, wild-type Canton S flies displayed reliable performance, which was enhanced after two training cycles with 1-min or 15-min inter-training intervals. Higher performance scores were also obtained with increasing sucrose concentration. Memory in Canton S flies decayed slowly when measured at 30 min, 1 h and 3 h after training; whereas, it had declined significantly at 6 h and 12 h post-training. When learning mutant t beta h flies, which are deficient in octopamine, were challenged, they exhibited poor performance, validating the utility of this assay. As the Drosophila model offers vast genetic and transgenic resources, the new appetitive conditioning described here provides a useful tool with which to elucidate the molecular and cellular underpinnings of reward learning and memory. Similar to negatively reinforced conditioning, this reward conditioning represents classical olfactory conditioning. Thus, comparative analyses of learning and memory mutants in two assays may help identify the molecular and cellular components that are specific to the unconditioned stimulus information used in conditioning.     

Kin recognition in the ant Rhytidoponera confusa I. Environmental odour

The relative contribution of genetic and environmental components to the colony-specific recognition odour was assessed for the ant Rhytidoponera confusa, using an aggression assay. Colonies maintained in the laboratory for 6 months were significantly less aggressive to each other than they were to colonies that had been collected from the field within the previous 48 h. Colonies tested 12–24 h after collection from the field showed significantly more aggression against laboratory ants than did the same colonies after they had been kept in the laboratory for 4–5 weeks. Both results indicate that environmental odours can contribute to the colony odour. Nestmates supplied with different diets and nest materials for 4–5 weeks were attacked more than the controls. Though slight, the increase in aggression was significant. This suggests that environmental odours, though significant when odour differences are extreme enough, may nevertheless be less important than genetic odours in nature. This and other studies suggest this pattern may be common in social insects generally.     

No sex in fungus-farming ants or their crops

Asexual reproduction imposes evolutionary handicaps on asexual species, rendering them prone to extinction, because asexual reproduction generates novel genotypes and purges deleterious mutations at lower rates than sexual reproduction. Here, we report the first case of complete asexuality in ants, the fungus-growing ant Mycocepurus smithii, where queens reproduce asexually but workers are sterile, which is doubly enigmatic because the clonal colonies of M. smithii also depend on clonal fungi for food. Degenerate female mating anatomy, extensive field and laboratory surveys, and DNA fingerprinting implicate complete asexuality in this widespread ant species. Maternally inherited bacteria (e.g. Wolbachia, Cardinium) and the fungal cultivars can be ruled out as agents inducing asexuality. M. smithii societies of clonal females provide a unique system to test theories of parent–offspring conflict and reproductive policing in social insects. Asexuality of both ant farmer and fungal crop challenges traditional views proposing that sexual farmer ants outpace coevolving sexual crop pathogens, and thus compensate for vulnerabilities of their asexual crops. Either the double asexuality of both farmer and crop may permit the host to fully exploit advantages of asexuality for unknown reasons or frequent switching between crops (symbiont reassociation) generates novel ant–fungus combinations, which may compensate for any evolutionary handicaps of asexuality in M. smithii.     

Sensu stricto individual conditioning,and imitation,in the ant Myrmica sabuleti (Hymenoptera: Formicidae)

Summary

Conditioning of isolated ants was attempted in the ant Myrmica sabuleti. Isolated ants did not die and could be visually as well as olfactorily conditioned. They acquired and kept ? or partly lost ? their visual ? or their olfactory ? conditioned responses as do ants living in a colony. Each individual of an ant colony is thus able to learn and memorize by itself. Any collective conditioning, learning and memory actually reflect the individuals’ performance. Naive workers paired with isolated previously conditioned ants apparently acquired olfactory conditioning in a shorter time than isolated ants, reaching a conditioning score identical to that of isolated ants. But they lost their apparent conditioning as soon as the olfactory cue was removed. Thus, imitation of nestmates exists in ants but does not lead, by itself, to learning sensu stricto since nothing of what has been imitated is retained.     

Predator-prey interaction between hatchery-reared Japanese flounder juvenile, Paralichthysolivaceus, and sandy shore crab, Matutalunaris: daily rhythms, anti-predator conditioning and starvation

Predator-prey interaction between sandy shore crab, Matuta lunaris (Forskål, 1775), and juvenile Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel), was investigated under controlled laboratory conditions. Possibility of training and conditioning hatchery-reared flounder to avoid predators was also examined. Crabs took over 75% of their daily ration at night when they were given access to prey 24 h a day. Large (64.8±5.4 g)- and medium (30.68±3.33 g)-sized crabs ate ca. 5.5±1.45 and 3.9±1.99 individuals of flounder (TL=4.96±0.23 cm) a day, respectively. When flounder juveniles that have experienced predation pressure by crabs encountered predators again, they exhibited better survival compared to the naive fish. Flounder juveniles were also conditioned either using small and, thus, benign predators, or large crabs over fence. The conditioned fish with either method were better able to avoid capture by crabs than naive fish, revealing that learning process should play an important role in their predator avoidance. Anti-predator performance was also compared between starved and fed flounder juveniles. Fed fish were rarely eaten by predators after 3 h of exposure, whereas starved fish continued to be eaten. Our results suggest that stock-enhancement program of Japanese flounder can be improved by applying proper feeding protocol and conditioning to avoid predators prior to release. Present research supports the idea that behavioural and ecological consideration for the target species is indispensable for the success of stock enhancement.     

Aspects of compound-conditioning to gustatory stimuli in the housefly Musca domestica L.

Houseflies (Musca domestica L.) were trained in a compound-conditioning paradigm where the conditioning stimuli were water and 1% sodium chloride solution. The unconditioned stimulus was 16% sucrose solution. A high degree of conditioning was produced. Control experiments for pseudoconditioning and sensitization revealed that the response of the flies to the procedure were due to an associative process. Experiments with double-water conditioned stimuli and with an interval between stimulus presentations indicate that the marked response to the first conditioned stimulus may be due to stimulus generalization in which the water, rather than the salt component of both stimuli served as the learning cue.     

Kin recognition in the ant Rhytidoponera confusa II. Gestalt odour

Two models have been proposed for the transfer of genetically determined colony odour pheromones in social insects. The ‘Gestalt’ model suggests a complete transfer of pheromones amongst all nestmates whereas the ‘Individualistic’ model suggests no significant odour transfer with nestmates bearing individually distinct odours. In experiments with the ant Rhytidoponera confusa, colonies showed significantly more aggression (12% aggression) towards nestmates that had been kept in cages with non-nestmates than they did to nestmate controls (0% aggression). Colonies also showed significantly less aggression against non-nestmates that had been kept with nestmates in a separate cage (84% aggression) than they did to non-nestmate controls (100% aggression). A second experiment indicated that workers can absorb colony-specific odours from the nest materials of other colonies. However, whilst both experiments indicated a partial Gestalt component to the colony odour (ca. 28%), the Individualistic component seemed to be more important (ca. 72%).     

Olfactory conditioning of proboscis activity in Drosophila melanogaster

Olfactory learning and memory processes in Drosophila have been well investigated with aversive conditioning, but appetitive conditioning has rarely been documented. Here, we report for the first time individual olfactory conditioning of proboscis activity in restrained Drosophila melanogaster. The protocol was adapted from those developed for proboscis extension conditioning in the honeybee Apis mellifera. After establishing a scale of small proboscis movements necessary to characterize responses to olfactory stimulation, we applied Pavlovian conditioning, with five trials consisting of paired presentation of a banana odour and a sucrose reward. Drosophila showed conditioned proboscis activity to the odour, with a twofold increase of percentage of responses after the first trial. No change occurred in flies experiencing unpaired presentations of the stimuli, confirming an associative basis for this form of olfactory learning. The adenylyl cyclase mutant rutabaga did not exhibit learning in this paradigm. This protocol generated at least a short-term memory of 15 min, but no significant associative memory was detected at 1 h. We also showed that learning performance was dependent on food motivation, by comparing flies subjected to different starvation regimes.     

Innate and enhanced predator recognition in hatchery-reared chinook salmon

We used a laboratory behaviour assay to investigate how innate predator recognition, handling stress, retention time, and number of conditioning events might affect chemically mediated anti-predator conditioning for hatchery-reared chinook salmon, Oncorhynchus tshawytscha. Juvenile chinook salmon with no prior exposure to predatory stimuli exhibited innate fright responses to northern pikeminnow, Ptychocheilis oregonensis, odour, regardless of whether the salmon came from a population that exists in sympatry or allopatry with northern pikeminnows. Juvenile chinook salmon exhibited enhanced predator recognition following a single conditioning event with conspecific extract and northern pikeminnow odour. Handling similar to what hatchery salmon might experience prior to release did not substantially reduce the conditioned response. When we conditioned juvenile chinook salmon in hatchery rearing vessels, fish from tanks treated once exhibited a conditioned response to northern pikeminnow odour in aquaria, but only for one behaviour (feeding response), and fish treated twice did not respond. The results suggest that enhanced recognition of predator stimuli occurs quickly, but may be to some extent context-specific, which may limit conditioned fright responses after release into the natural environment.     

Dynamics of odour learning in Leptopilina boulardi, a hymenopterous parasitoid

We investigated the dynamics of odour learning involved in host location by a parasitoid insect,Leptopilina boulardi (Hymenoptera: Figitidae). Females of this species find their Drosophila host larvae by probing fruits with their ovipositor. They can be conditioned to respond to an odour when the odour exposure is associated with oviposition. We investigated the effect of the number of conditioning trials, sensitization tests and extinction tests on the retention of the conditioned response. Results showed that: (1) a single odour-oviposition association produced a strong short-term memory (1-2 h), which rapidly decayed over 24 h; (2) multiple odour-oviposition associations produced a memory trace that was strong in both the short term and the longer term (24 h); (3) sensitization to the odour through mere oviposition experience (without odour) was low after a single trial and high after multiple trials, but was only observed for a short period; (4) all memory traces were erased by three successive extinction tests, regardless of the intertest interval. We conclude that the probing behaviour of a Drosophila parasitoid is characterized by great plasticity shaped in the short term by sensitization, and in the longer term by associative learning. We compare olfactory plasticity in this parasitoid foraging for hosts and that of the honeybee foraging for food, suggesting common underlying processes in the central nervous system. Finally our results may relate to the dynamics of the foraging activity of L. boulardi. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.      

Infection characteristics of Solenopsis invicta virus 2 in the red imported fire ant, Solenopsis invicta

Solenopsis invicta virus 2 (SINV-2) is the second virus identified from the fire ant, S. invicta Buren. SINV-2 is unique among positive-strand RNA viruses from insects by possessing four cistrons in a monopartite genome. Fire ant colonies testing positive for SINV-2 by RT-PCR did not exhibit any discernable symptoms. RT-PCR-based surveys for SINV-2 among 688 fire ant mounds in Alachua County, Florida, sampled during the period January, 2006 through December, 2007 showed that the prevalence of SINV-2 among nests ranged from 1.6% to 16.4%. Unlike S. invicta virus 1, no seasonal-associated prevalence was observed with regard to SINV-2 infection among fire ant colonies. No social form specificity was evident; SINV-2 was found in both monogyne and polygyne S. invicta ants. Real-time quantitative PCR experiments showed that SINV-2 genome equivalents per individual ant ranged from 1.9 × 10⁷ in. pupae to 4.3 × 10¹¹ in. inseminated queens. The SINV-2 infection was detected in all ant stages examined (eggs, larvae, pupae, workers, and queens). Tissue tropism studies indicated that the alimentary canal (specifically the midgut) is most likely the susceptible tissue. SINV-2 was successfully transmitted to uninfected S. invicta ants by feeding a partially purified homogenate of SINV-2-infected ants. The SINV-2 transmission rate ranged from 30% to 80%, and both positive (genomic) and negative (replicative) SINV-2 RNA strands accumulated in recipient ants over the course of the experiment. These results indicated that SINV-2 replicates within S. invicta.     

Time-course of memory formation differs in honey bee lines selected for good and poor learning

Six lines of the honey bee Apis mellifera capensis were selected for good and poor learning, and compared with respect to the time-course of their memory after a single learning trial to an odour stimulus (geraniol). The good learners showed the well known bi-phasic time-course with a high rate of conditioned responses immediately after learning, followed by a reduction 1–3 min later and a consolidation into a long-term memory after an interval longer than 5 min. Poor learners differed significantly with respect to their consolidation period and the initial high response rate. The initial period (of less than 1 min) is controlled not only by the associative memory of the learning trial but also by a sensitizing effect of the sucrose stimulus which was used as an unconditioned stimulus. It is concluded that the lines selected for learning performance differ with respect to the establishment of a long-term memory trace. As in Drosophila learning mutants, differences between good and poor learners are also affected by non-associative processes involved in sensitization.     

Macroevolutionary assembly of ant/plant symbioses: Pseudomyrmex ants and their ant-housing plants in the Neotropics

Symbioses include some of the clearest cases of coevolution, but their origin, loss or reassembly with different partners can rarely be inferred. Here we use ant/plant symbioses involving three plant clades to investigate the evolution of symbioses. We generated phylogenies for the big-eyed arboreal ants (Pseudomyrmecinae), including 72% of their 286 species, as well as for five of their plant host groups, in each case sampling more than 61% of the species. We show that the ant-housing Vachellia (Mimosoideae) clade and its ants co-diversified for the past 5 Ma, with some species additionally colonized by younger plant-nesting ant species, some parasitic. An apparent co-radiation of ants and Tachigali (Caesalpinioideae) was followed by waves of colonization by the same ant clade, and subsequent occupation by a younger ant group. Wide crown and stem age differences between the ant-housing genus Triplaris (Polygonaceae) and its obligate ant inhabitants, and stochastic trait mapping, indicate that its domatium evolved earlier than the ants now occupying it, suggesting previous symbioses that dissolved. Parasitic ant species evolved from generalists, not from mutualists, and are younger than the mutualistic systems they parasitize. Our study illuminates the macroevolutionary assembly of ant/plant symbioses, which has been highly dynamic, even in very specialized systems.     

Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila

Juvenile hormone (JH) signaling underpins both regulatory and developmental pathways in insects. However, the JH receptor is poorly understood. Methoprene tolerant (Met) and germ cell expressed (gce) have been implicated in JH signaling in Drosophila. We investigated the evolution of Met and gce across 12 Drosophila species and found that these paralogs are conserved across at least 63 million years of dipteran evolution. Distinct patterns of selection found using estimates of dN/dS ratios across Drosophila Met and gce coding sequences, along with their incongruent temporal expression profiles in embryonic Drosophila melanogaster, illustrate avenues through which these genes have diverged within the Diptera. Additionally, we demonstrate that the annotated gene CG15032 is the 5′ terminus of gce.In mosquitoes and beetles, a single Met-like homolog displays structural similarity to both Met and gce, and the intron locations are conserved with those of gce. We found that Tribolium and mosquito Met orthologs are assembled from Met- and gce-specific domains in a modular fashion. Our results suggest that Drosophila Met and gce experienced divergent evolutionary pressures following the duplication of an ancestral gce-like gene found in less derived holometabolous insects.     

Direct observation of swimming behaviour in a shallow-water scavenging amphipod Scopelocheirus onagawae in relation to chemoreceptive foraging

Direct observations on foraging behaviour of scavenging lysianassid amphipods have been limited, and no previous study has examined the effect of food odour quantitatively on the behaviour. The present study recorded the swimming behaviour of the amphipod Scopelocheirus onagawae using videographic techniques before and after the introduction of food odour (amino acid solution). S. onagawae showed consistent nocturnal activity swimming at a high speed (16.8 cm s^− 1) with an approximately straight trajectory in various directions before and after the introduction of odour in which the amino acid concentration was below the behavioural threshold concentration for this species (1.0 × 10^− 7 mol l^− 1). High speed multidirectional linear swimming is thought to be advantageous for these amphipods, enabling them to survey across a broad area. After the first encounter with the odour plume above the behavioural threshold concentration, the amphipods slowed down their swimming speed (ca. 9.7 cm s^− 1) with a short time-lag (ca. 0.42 s), and thereafter they frequently turned so that they remained within the odour plume. Once moved out of the odour plume, the amphipods quickly returned to the plume with a shorter response time (ca. 0.1 s) than that in the first detection of the odour plume, suggesting that the sensory adaptation is involved with the tracking of the odour. Our study demonstrated that chemoreception is a major factor causing behavioural change in scavenging amphipods at the edge of the odour plume.     

Alkaloids in the venom of Messor ants

Tobacco alkaloids of the anabasine type have been found or confirmed in the venom of five species of arid-dwelling Messor ants. They are frequently accompanied by alkylpyrazines. Messor mediorubra contains four alkaloids, with anabasine the major component and also minor pyrazines. Anabasine was found alone in the venom of Messor semirufus and confirmed in Messor ebeninus. Messor rugosus from Tel Aviv contained a mixture of alkaloids and pyrazines, but those from Ein Yahav contained 3-ethyl-2,5-dimethylpyrazine, some 2-phenylethylamine, and N-ethylidene 2-phenylethylamine. Messor arenarius is confirmed as having a complex but variable mixture of alkaloids and pyrazines.     

Distribution of phenotypes among Bacillus thuringiensis strains

An extensive collection of Bacillus thuringiensis isolates from around the world were phenotypically profiled using standard biochemical tests. Six phenotypic traits occurred in 20–86% of the isolates and were useful in distinguishing isolates: production of urease (U; 20.5% of isolates), hydrolysis of esculin (E; 32.3% of isolates), acid production from salicin (A; 37.4% of isolates), acid production from sucrose (S; 34.0% of isolates), production of phospholipase C or lecithinase (L; 79.7% of isolates), and hydrolysis of starch (T; 85.8% of isolates). With the exception of acid production from salicin and hydrolysis of esculin, which were associated, the traits assorted independently. Of the 64 possible combinations of these six phenotypic characteristics, 15 combinations accounted for ca. 80% of all isolates, with the most common phenotype being TL (23.6% of isolates). Surprisingly, while the biochemical traits generally assorted independently, certain phenotypic traits associated with the parasporal crystal were correlated with certain combinations of biochemical traits. Crystals that remained attached to spores (which tended to be non-toxic to insects) were highly correlated with the phenotypes that included both L and S. Among the 15 most abundant phenotypes characterizing B. thuringiensis strains, amorphous crystals were associated with TLE, TL, T, and Ø (the absence of positive tested biochemical traits). Amorphous crystal types displayed a distinct bias toward toxicity to dipteran insects. Although all common phenotypes included B. thuringiensis isolates producing bipyramidal crystals toxic to lepidopteran insects, those with the highest abundance of these toxic crystals displayed phenotypes TLU, TLUA, TLUAE, and TLAE.