The Neuro-Ecology of Drosophila Pupation Behavior

Many species of Drosophila form conspecific pupa aggregations across the breeding sites. These aggregations could result from species-specific larval odor recognition. To test this hypothesis we used larval odors of D. melanogaster and D. pavani, two species that coexist in the nature. When stimulated by those odors, wild type and vestigial (vg) third-instar larvae of D. melanogaster pupated on conspecific larval odors, but individuals deficient in the expression of the odor co-receptor Orco randomly pupated across the substrate, indicating that in this species, olfaction plays a role in pupation site selection. Larvae are unable to learn but can smell, the Syn^97CS and rut strains of D. melanogaster, did not respond to conspecific odors or D. pavani larval cues, and they randomly pupated across the substrate, suggesting that larval odor-based learning could influence the pupation site selection. Thus, Orco, Syn^97CS and rut loci participated in the pupation site selection. When stimulated by conspecific and D. melanogaster larval cues, D. pavani larvae also pupated on conspecific odors. The larvae of D. gaucha, a sibling species of D. pavani, did not respond to D. melanogaster larval cues, pupating randomly across the substrate. In nature, D. gaucha is isolated from D. melanogaster. Interspecific hybrids, which result from crossing pavani female with gaucha males clumped their pupae similarly to D. pavani, but the behavior of gaucha female x pavani male hybrids was similar to D. gaucha parent. The two sibling species show substantial evolutionary divergence in organization and functioning of larval nervous system. D. melanogaster and D. pavani larvae extracted information about odor identities and the spatial location of congener and alien larvae to select pupation sites. We hypothesize that larval recognition contributes to the cohabitation of species with similar ecologies, thus aiding the organization and persistence of Drosophila species guilds in the wild.     

Leaders benefit followers in the collective movement of a social sawfly

The challenges of maintaining cohesion while making collective decisions in social or aggregating insects can result in the emergence of a leader or leaders. Larval aggregations of the steel-blue sawfly Perga affinis forage nocturnally, and some larvae lead the aggregation on foraging trips more often than expected by chance. We investigated the relationship between these leader and follower roles by comparing the weight and growth of individual larvae with different roles. Our observations reveal no significant difference between the growth of leaders and followers, suggesting that the role of leadership may not provide direct foraging benefits. However, by experimentally manipulating the social structure of larval aggregations, we found that individuals within aggregations that comprise a mixture of leaders and followers enjoy higher growth rates than those in aggregations comprising a single behavioural type. These data demonstrate, for the first time, individual benefits to maintaining a balance of leader and follower roles within larval aggregations, and highlight the importance of considering the perspectives of both leaders and followers when investigating the evolutionary significance of this behavioural variation within animal groups.     

Drosophila adult and larval pheromones modulate larval food choice

Insects use chemosensory cues to feed and mate. In Drosophila, the effect of pheromones has been extensively investigated in adults, but rarely in larvae. The colonization of natural food sources by Drosophila buzzatii and Drosophila simulans species may depend on species-specific chemical cues left in the food by larvae and adults. We identified such chemicals in both species and measured their influence on larval food preference and puparation behaviour. We also tested compounds that varied between these species: (i) two larval volatile compounds: hydroxy-3-butanone-2 and phenol (predominant in D. simulans and D. buzzatii, respectively), and (ii) adult cuticular hydrocarbons (CHs). Drosophila buzzatii larvae were rapidly attracted to non-CH adult conspecific cues, whereas D. simulans larvae were strongly repulsed by CHs of the two species and also by phenol. Larval cues from both species generally reduced larval attraction and pupariation on food, which was generally—but not always—low, and rarely reflected larval response. As these larval and adult pheromones specifically influence larval food search and the choice of a pupariation site, they may greatly affect the dispersion and survival of Drosophila species in nature.     

A Theoretical Approach to Study the Evolution of Aggregation Behavior by Larval Codling Moth, <Emphasis Type="BoldItalic">Cydia pomonella</Emphasis> (Lepidoptera: Tortricidae)

Pupation site-seeking larvae of the codling moth, Cydia pomonella, aggregate in response to aggregation pheromone produced by cocoon-spinning conspecific larvae. Larvae that pupate in an aggregation rather than in solitude may experience a lower rate of parasitism by the parasitoid Mastrus ridibundus. Additionally, adults eclosing from a larval aggregation may encounter mates more rapidly at the site of eclosion (on-site) than away from that site (off-site). We employed an evolutionary simulation to determine the effect of several ecological parameters on the evolution of larval aggregation behavior. These parameters included (i) the probability of mate encounter off-site; (ii) the time available for finding a mate; and (iii) the population density of parasitoids and their rate of larval parasitism. The model predicts that larval aggregation behavior is selected for when the probability of off-site mate encounter is low, the time to locate mates is short, and egg-limited parasitoids are at high population levels. We also show that aggregations reduce the risk of parasitism through dilution effects. The parameters found to favour the evolution of larval aggregation behavior are consistent with life history traits exhibited by C. pomonella.     

Natural selection in the laboratory for a change in resistance byDrosophila melanogaster to the parasitoid waspAsobara tabida

The selection response of the polymorphic hostD. melanogaster (Meigen) to the braconid waspA. tabida (Nees) is addressed. Cages of flies with and without wasps were initiated with a population ofD. melanogaster that exhibited variation both in larval foraging behavior and in encapsulation ability. Encapsulation ability was measured as the proportion of parasitized larvae that produce a hardened capsule which encapsulates the wasp egg and ultimately kills the wasp larva. We determined whether the host population changed its encapsulation ability and/or its foraging behavior in response to the wasp. Both species were collected from a local orchard whereA. tabida is the only wasp known to parasitizeD. melanogaster larvae. The naturally occurring genetic polymorphism for rover and sitter larval foraging behavior inD. melanogaster is also found in this field population.A. tabida's vibrotactic search behavior enables it to detect rover more frequently than sitter larvae. Rover larvae move significantly more while feeding than do sitter larvae. In this field population, rover larvae also show higher encapsulation abilities than do sitter larvae. Six cage populations, three without wasps and three with wasps, each containing an equal mixture of rover and sitter flies, were established in the laboratory and maintained for 19 fly generations. Selection pressure in the laboratory was similar to that found in the field population from which the flies and wasps were derived. We found that larvae from cages with wasps developed a significantly higher frequency of encapsulation than those reared without wasps. We were, however, unable to detect a change in larval movement (rover or sitter behavior) in larvae from cages subject to selection from wasps compared to larvae from cages containing no wasps. This may have resulted from a balance between two selective forces, selection against rovers by the wasps' use of vibrotaxis, and selection for rovers resulting from their increased encapsulation abilities     

The foraging locus: behavioral tests for normal muscle movement in rover and sitter Drosophila melanogaster larvae

We used Drosophila melanogaster larvae with different alleles at the foraging (for) locus in a variety of behavioral tests to evaluate normal muscle usage of rover and sitter phenotypes. The results show that sitter and lethal sitter alleles of for do not affect larval behavior through a mutation which affects larval muscle usage. In general the behavior of rovers and sitters differed on food but not on non-nutritive substrates. Rovers and sitters moved equally well on non-nutritive substrates, and measures such as the time to roll over and length of forward stride showed no significant strain differences. Larvae with different alleles at for did not differ in body length. Rovers took more strides, not longer ones, than sitters while on foraging substrates. We conclude that differences in larval locomotion during foraging found in larvae with different alleles at for can not be explained on the basis of muscle usage alone. It is more likely that for affects larval ability to perceive or respond to the foraging environment.     

Predators,Parasites and Heterospecific Aggregations in Chrysomeline Larvae

Gregariousness is a common feature in aposematic insect prey and offers the additional benefit of enhancing the effectiveness of their toxic defences. Aggregations of the aposematic larvae of two species of leaf beetles, Paropsis atomaria and Paropsisterna variicollis, occur together on the same Eucalyptus trees over spring and summer. Conventionally, the colouration of these larvae is thought to provide aposematic protection against vertebrate predators, but supporting evidence is limited. We determine whether environmental preferences contribute to the heterospecific aggregations, and the potential costs for larvae of living in heterospecific aggregations. We surveyed natural aggregations of the larvae of both species in the field and recorded environmental variables of these aggregations. This revealed that heterospecific aggregations occurred more commonly at higher leaf temperatures, and in less visually conspicuous locations. Paropsis atomaria larvae were twice as likely to be found in heterospecific aggregations than Pa. variicollis. Next, we manipulated larval aggregations in the field to investigate the survival and gregarious behaviour of larvae in heterospecific aggregations. The gregarious behaviour of both species of larvae did not differ between heterospecific and monospecific aggregations. Further, the survival of larvae did not significantly differ between heterospecific and monospecific aggregations. We suggest that the preference for P. atomaria larvae to aggregate with Pa. variicollis at higher leaf temperature results in the observed heterospecific aggregations, with a potential benefit of lowerer parasitoidism rates for P. atomaria.     

Drosophila Food-Associated Pheromones: Effect of Experience,Genotype and Antibiotics on Larval Behavior

Animals ubiquitously use chemical signals to communicate many aspects of their social life. These chemical signals often consist of environmental cues mixed with species-specific signals—pheromones—emitted by conspecifics. During their life, insects can use pheromones to aggregate, disperse, choose a mate, or find the most suitable food source on which to lay eggs. Before pupariation, larvae of several Drosophila species migrate to food sources depending on their composition and the presence of pheromones. Some pheromones derive from microbiota gut activity and these food-associated cues can enhance larval attraction or repulsion. To explore the mechanisms underlying the preference (attraction/repulsion) to these cues and clarify their effect, we manipulated factors potentially involved in larval response. In particular, we found that the (i) early exposure to conspecifics, (ii) genotype, and (iii) antibiotic treatment changed D. melanogaster larval behavior. Generally, larvae—tested either individually or in groups—strongly avoided food processed by other larvae. Compared to previous reports on larval attractive pheromones, our data suggest that such attractive effects are largely masked by food-associated compounds eliciting larval aversion. The antagonistic effect of attractive vs. aversive compounds could modulate larval choice of a pupariation site and impact the dispersion of individuals in nature.     

The impact of food type, temperature and starvation on larval development of Balanus amphitrite Darwin (Cirripedia: Thoracica)

The impact of diatom food species (Chaetoceros calcitrans and Skeletonema costatum), temperature and starvation on the larval development of Balanus amphitrite was evaluated. Starvation threshold levels for different ages of larvae (0- to 5-day-old) fed with C. calcitrans and S. costatum and then starved at 5, 15 and 25 °C temperature were estimated as ultimate recovery hour (URH; denoting the starvation point in hours at the end of which larvae can recover and continue development). Effect of temperature on starvation threshold varied significantly with larval age and food species. The URH declined with larval age at 5 °C, but not at 15 and 25 °C. The URH and grazing rates were high for early instars fed on C. calcitrans, and for advanced instars fed on S. costatum. Carbon gain through feeding was maximum for 2-day-old larvae when fed with C. calcitrans and decreased with larval age. However, when fed with S. costatum carbon gain increased with larval age. This confirms that with development the utility of food types changes. The differences in the carbon gain can be attributed to differences in grazing rate due to variations in the size of the diatom cells, larval intersetular distance, diatom sinking rate and the photo-taxic behavior of larvae. Molting was observed at times when larvae were undergoing starvation and this could be viewed as stress-induced molting, and it differed with the larval age and food organisms.     

Does aggregation behavior of codling moth larvae,Cydia pomonella,increase the risk of parasitism by Mastrus ridibundus?

Mastrus ridibundus is a specialist hymenopteran parasitoid that parasitizes last-instar larvae or prepupae of the codling moth, Cydia pomonella. Foraging females eavesdrop on an aggregation pheromone produced by cocooning larvae. We investigated whether larvae that cocoon in aggregation experience a greater rate of parasitism than larvae that cocoon in isolation. In wind tunnel experiments, 10 larvae in aggregations were more readily located by female M. ridibundus than 10 larvae well separated from each other. Similarly, aggregations of 30 larvae were more attractive to female M. ridibundus than those of 3 larvae. In cage experiments, larval cocooning in aggregation or isolation had no effect on the mean rate of parasitism and the mean number of eggs deposited per parasitized host. In Petri-dish experiments, the location of larvae within an aggregation significantly affected their rate of parasitism, with those in the center of an aggregation completely shielded from parasitism. Our data suggest that aggregation behavior by C. pomonella larvae does not appear to increase the rate of parasitism. The increased risk of aggregated larvae to be detected by M. ridibundus is likely offset by diluted parasitism risk and structural refugia effects that larvae in aggregation experience. As an egg-limited parasitoid, female M. ridibundus can parasitize on average only one larva in an aggregation, with the likelihood of parasitism for each larva being inversely proportional to the number of larvae in that aggregation.     

Studies on the characteristics of parasitism ofPlutella xylostella (Lep.: Plutellidae) by a larval parasiteDiadegma semiclausum (Hym.: Ichneumonidae)

Laboratory experiments were conducted to study the characteristics of parasitism of diamondback moth,Plutella xylostella (L.), a worldwide pest of crucifers, by a larval parasite,Diadegma semiclausum Hellén. Results showed that the greater the host larval density, the lesser was the percentage of larvae parasitized. The area of discovery decreased with increasing parasite density, but k-value remained practically unchanged. Parasitism reduced food consumption in parasitised larvae. No difference was observed in duration of larval instars between the parasitized and healthy larvae. As the frequency of superparasitism increased, the proportion of production of female progeny also increased. Presence of parasite larva within the host larva deterred superparasitism greater than the presence of parasite egg. Presence of larva of eitherD. semiclausum or another larval parasite,Cotesia plutellae Kurdjumov, within host larva deterred multiparasitism by either species greater than the presence of egg of either parasite species.Diadegma semiclausum was much more aggressive thanC. plutellae in parasitisingP. xylostella larvae.     

Microbiology of feces of the larval honey bee,Apis mellifera

Methods for collection and microbiological examination of feces of larval honey bees, Apis mellifera, are described. Feces collected on sterile agar were inoculated onto selective media, some of which were acidified to approximate more closely the pH of larval food and the larval gut. A total of 104 microbial isolates were obtained from fecal collections of 20 larvae, although the feces of 4 of these larvae contained no detectable microbes. Microorganisms isolated in order of frequency were Bacillus spp., Gram-variable pleomorphic bacteria (Achromobacter eurydice?), molds (primarily Penicillia), actinomycetes, Gram-negative bacterial rods, and yeasts. It appears that larvae can become inoculated with microorganisms which are found in adult bees and pollen from ingestion of contaminated food. However, evidence for a constant symbiotic microflora which could contribute significant amounts of biochemicals to larvae is lacking.     

A larval hunger signal in the bumblebee Bombus terrestris

Larvae of Bombus terrestris, a pollen-storing bumblebee, are dependent on progressive provisioning by workers. We test the hypothesis that larval cuticular chemicals can act as a hunger signal. We first show with a new classical conditioning experiment, using a Y-shaped tube, that workers can be trained to prefer the extracts of normally fed larvae over those of starved larvae. This proves the ability of workers to discriminate between larval extracts. Second, we show in a bioassay that workers also use these perceived differences to feed larvae according to their nutritional status. Larval broods sprayed with the extracts of the starved larvae were fed significantly more than larval broods sprayed with the extracts of normally fed larvae or with the solvent (n-pentane) only. We therefore conclude that B. terrestris larvae signal their need for food via their cuticular chemicals, and discuss the extent to which this form of communication could give larvae some control over their development. Received 2 September 2005; revised 11 April; accepted 24 April 2006.     

Nosema ceranae Can Infect Honey Bee Larvae and Reduces Subsequent Adult Longevity

Nosema ceranae causes a widespread disease that reduces honey bee health but is only thought to infect adult honey bees, not larvae, a critical life stage. We reared honey bee (Apis mellifera) larvae in vitro and provide the first demonstration that N. ceranae can infect larvae and decrease subsequent adult longevity. We exposed three-day-old larvae to a single dose of 40,000 (40K), 10,000 (10K), zero (control), or 40K autoclaved (control) N. ceranae spores in larval food. Spores developed intracellularly in midgut cells at the pre-pupal stage (8 days after egg hatching) of 41% of bees exposed as larvae. We counted the number of N. ceranae spores in dissected bee midguts of pre-pupae and, in a separate group, upon adult death. Pre-pupae exposed to the 10K or 40K spore treatments as larvae had significantly elevated spore counts as compared to controls. Adults exposed as larvae had significantly elevated spore counts as compared to controls. Larval spore exposure decreased longevity: a 40K treatment decreased the age by which 75% of adult bees died by 28%. Unexpectedly, the low dose (10K) led to significantly greater infection (1.3 fold more spores and 1.5 fold more infected bees) than the high dose (40K) upon adult death. Differential immune activation may be involved if the higher dose triggered a stronger larval immune response that resulted in fewer adult spores but imposed a cost, reducing lifespan. The impact of N. ceranae on honey bee larval development and the larvae of naturally infected colonies therefore deserve further study.     

Influence of parasitism byEucelatoria bryani [Dip.: Tachinidae] on the consumption and utilisation of chickpea flour diet byHeliothis armigera [Lep.: Noctuidae]

Fourth and 5th instar larvae ofHeliothis armigera (Hübner) parasitised byEucelatoria bryani Sabrosky consumed significantly less chickpea flour diet than unparasitised larvae of the same age in the laboratory. Significant reduction in diet consumption, larval weight gain and frass produced at 24 and 48 h following parasitisation was observed in 5th instar host larvae. Parasitised larvae retained a greater percentage of food ingested (AD) than did the unparasitised ones. Unparasitised 5th instar larvae ofH. armigera were more efficient in converting the ingested food (ECI) and digested food (ECD) into body substance than larvae of similar age parasitised byE. bryani.     

DENSITY-DEPENDENT NATURAL SELECTION IN DROSOPHILA: EVOLUTION OF GROWTH RATE AND BODY SIZE

Drosophila melanogaster populations subjected to extreme larval crowding (CU lines) in our laboratory have evolved higher larval feeding rates than their corresponding controls (UU lines). It has been suggested that this genetically based behavior may involve an energetic cost, which precludes natural selection in a density-regulated population to simultaneously maximize food acquisition and food conversion into biomass. If true, this stands against some basic predictions of the general theory of density-dependent natural selection. Here we investigate the evolutionary consequences of density-dependent natural selection on growth rate and body size in D. melanogaster. The CU populations showed a higher growth rate during the postcritical period of larval life than UU populations, but the sustained differences in weight did not translate into the adult stage. The simplest explanation for these findings (that natural selection in a crowded larval environment favors a faster food acquisition for the individual to attain the same final body size in a shorter period of time) was tested and rejected by looking at the larva-to-adult development times. Larvae of CU populations starved for different periods of time develop into comparatively smaller adults, suggesting that food seeking behavior in a food depleted environment carries a higher cost to these larvae than to their UU counterparts. The results have important implications for understanding the evolution of body size in natural populations of Drosophila, and stand against some widespread beliefs that body size may represent a compromise between the conflicting effects of genetic variation in larval and adult performance.     

Early social context does not influence behavioral variation at adulthood in ants

Early experience can prepare offspring to adapt their behaviors to the environment they are likely to encounter later in life. In several species of ants, colonies show ontogenic changes in the brood-to-worker ratio that are known to have an impact on worker morphology. However, little information is available on the influence of fluctuations in the early social context on the expression of behavior in adulthood. Using the ant Lasius niger, we tested whether the brood-to-worker ratio during larval stages influenced the level of behavioral variability at adult stages. We raised batches of 20 or 180 larvae in the presence of 60 workers until adulthood. We then quantified the activity level and wall-following tendency of callow workers on 10 successive trials to test the prediction that larvae reared under a high brood-to-worker ratio should show greater behavioral variations. We found that manipulation of the brood-to-worker ratio influenced the duration of development and the size of individuals at emergence. We detected no influence of early social context on the level of between- or within-individual variation measured for individual activity level or on wall-following behavior. Our study suggests that behavioral traits may be more canalized than morphological traits.     

Changes in digestive rate of a predatory beetle over its larval stage: Implications for dietary breadth

Prey and non-prey foods differ substantially in their suitability for zoophytophagous omnivores, but the relative quality of these foods depends on the stage-specific digestive capabilities of the organism in question. Quantitative (or real-time) PCR was used to amplify food-specific DNA and measure consumption rates and digestion efficiencies of four foods - two prey (Aphis glycines and Leptinotarsa decemlineata eggs) and two non-prey (Zea mays pollen and the yeast Saccharomyces cerevisiae) species - over different larval stages of Coleomegilla maculata. The amount of Z. mays pollen consumed increased as larvae aged, but not proportionately with larval size, such that consumption rates decreased uniformly with insect age. While aging larvae fed A. glycines had a similar pattern in their diminishing consumption rates, they consumed similar amounts of A. glycines regardless of age, suggesting a negative feedback mechanism for consumption of this species of aphids. Older larvae digested three of the four foods significantly more efficiently than younger larvae, the exception being larvae fed A. glycines which was digested at a similar rate throughout the larval stage. There was a significant effect of time on food quantity detected for all four species of food. We conclude that C. maculata expands its physiological capacity for digesting prey and non-prey foods as they age in order to better accommodate the increased nutritional needs of the older larvae. This strategy has important implications for the life history strategies of zoophytophagous insects and how they function within foods webs.     

Effect of group size on parasitism in a natural population of the Baltimore checkerspot Euphydryas phaeton

Summary The effect of group size of early instars on parasitism of Euphydryas phaeton (Nymphalidae) was examined. Different numbers of larvae were stocked per web to determine the effect of group size on parasitism. Larval aggregations of moderate size (the size occurring naturally) had the least parasitism. Larger larval groups had a disproportionately high rate of parasitism. The major larval parasitoids located vulnerable larvae within webs, instead of attacking larvae available on the outside of webs. Parasitism rates were similar for larvae of damaged and undamaged webs, a consequence of the behavior and location of larvae in the webs. Lower limit to group size was a function of facilitation of larval numbers in reaching the first feeding site, the top of the host plant. Feeding facilitation by larval aggregations was not a factor in larval survival or growth.     

Genetics and ecology of Drosophila melanogaster larval foraging and pupation behaviour

In this paper we show that, (1) Drosophila melanogaster larvae utilize a variety of pupal microhabitats in an orchard, (2) variation in larval foraging path length, pupation distance from the food and pupal microhabitat preference (on or off the fruit) is genetically based and, (3) variation in these behaviours can be maintained in a spatially heterogenous environment since there is a reversal in pupation site suitability in wet and dry pupal microhabitats. Differences in path length in both laboratory and natural populations can be attributed to genes on the second pair of chromosomes and is under simple genetic control, whereas differences in pupal height are polygenically inherited (the second pair of chromosomes influences pupal height three times more than the third pair). Pupae collected from on-fruit sites had shorter foraging path lengths and lower pupal heights than off-fruit populations. Populations from the orchard maintained their field pupal microhabitat preferences even after 1 year of rearing them in the laboratory. Larvae with the sitter larval phenotype (short path lengths and low pupal heights tended to pupate more on-fruit than those with the rover phenotype (long path lengths and high pupal heights). To determined if these genetically based differences in microhabitat preference contributed to fitness, larval pupation behaviour was studied in a “field assay” (dish with fruit on soil) with soil water content varied. At low soil water contents, pupal survivorship was significantly better on the fruit whereas, at high soil water contents, survivorship was better in the soil. There was a reversal in which microhabitat (dry or wet) was a better site for pupation. In the field environment where soil water content fluctuates in space and time, such a reversal would explain the maintenance of genetic variation for these larval behaviours. Another selective agent acting on D. melanogaster larvae in our orchard is parasitization by Asobara tabida. This parasitoid parasitizes larvae with high locomotory scores (e.g. rovers) significantly more than those with low scores (sitters). This study relates laboratory phenotypes to field phenotypes thereby linking the ecological, behavioural and genetic components of larval habitat selection in D. melanogaster.