Closely related parasitoids induce different pupation and foraging responses in Drosophila larvae

Few examples exist where parasites manipulate host behaviour not to increase their transmission rate, but their own survival. Here we investigate fitness effects of parasitism by Asobara species in relation to the pupation behaviour of the host, Drosophila melanogaster . We found that Asobara citri parasitized larvae pupate higher in rearing jars compared to unparasitized controls, while A. tabida pupated on or near the medium. No change in pupation site was found for three other species. A follow-up experiment showed a non-random distribution of parasitized and unparasitized pupae over the different jar parts. To test the adaptiveness of these findings, we performed pupal transfer experiments. Optimum pupation sites were found to be different between host individuals; wall individuals survived better than bottom individuals, but bottom individuals did worse at the wall. Two parasitoid species that alter pupation site significantly showed high rates of diapause at their 'preferred' pupation site. For one of them, A. citri , pupation occurred at the optimal site for highest survival (emergence plus diapause). From literature we know that pupation height and foraging activity are genetically positively linked. Therefore, we implement a short assay for rover/sitter behavioural expression by measuring distance travelled during foraging after parasitism. For one out of three species, foraging activity was reduced, suggesting that this species suppresses gene expression in the for pathway and thereby reduces pupation height. The parasitoid species used here, naturally inhabit widely different environments and our results are partly consistent with a role for ecology in shaping the direction of parasite-induced changes to host pupation behaviour. More parasitoids are found on the wall of the rearing jar when they originate from dry climates, while parasitoids from wet climates pupate on the humid bottom.     

Host regulation and release of parasitism-specific proteins in the system Toxoneuron nigriceps-Heliothis virescens

The braconid wasp Toxoneuron nigriceps induced qualitative and quantitative changes in the protein composition of the moth Heliothis virescens host hemolymph. Total protein concentration was found to be higher in parasitized host 4 days after parasitism as compared to control hosts, mainly due to changes in a particular group of proteins. Host proteins with a molecular mass of 173 and 72 kDa were found in higher levels in the hemolymph of parasitized larvae as control hosts approached pupation, while an 80 kDa peptide was found in reduced concentration in the hemolymph of parasitized hosts. Levels of these three peptides were maintained throughout parasitoid development, while two of them (173 and 72 kDa) were cleared from the host hemolymph close to pupation. Besides the regulation of host proteins, three parasitism-specific proteins (PSPs) were released into the host hemolymph. Two of them (PSP1-MW=116 kDa, pI=6.3; PSP2-MW=114 kDa, pI=6.2) first appeared in the hemolymph of parasitized hosts soon after pupation of control host and increased in concentration as the parasitoid developed. The third PSP (PSP3-MW=56 kDa, pI=5.8) was produced towards the end of parasitoid larval development, close to parasitoid egression. Database searches based on the amino acid composition and amino terminal sequence of PSP1 and PSP2 did not produce any significant matches, while PSP3 was identified as a putative chitinase. Incubation of host derived tissues, parasitoid larvae and teratocytes in 35S conditioned media suggested PSPs were a product of teratocytes. The role of the regulation of host proteins and release of PSPs by teratocytes for the successful development of T. nigriceps are discussed.     

Intra- and interspecies variations in pupation height in Drosophila.

In order to study the pupation site preference which is an important component of larval behaviour, pupation height was scored in Drosophila ananassae, D. bipectinata and D. malerkotliana by using a large number of strains of these species. The mean pupation height of different strains ranged from 1.1 to 8.7 mm in D. ananassae, from 0.41 to 0.75 mm in D. bipectinata and from 1.0 to 1.3 mm in D. malerkotliana. The analysis of variance and t-test were performed to test intre- and interspecies variations in pupation height. These tests revealed significant variation among three species. Significant variations among different strains of the same species were also found in D. ananassae and D. bipectinata. These observations provide evidence for intra- and interspecies variations in pupation height in Drosophila. Variations among different strains of the same species in pupation height are attributable to genetic heterogeneity among the strains.     

Chemical cues influence pupation behavior of Drosophila simulans and Drosophila buzzatii in nature and in the laboratory

In the wild, larvae of several species of Drosophila develop in heterogeneous and rapidly changing environments sharing resources as food and space. In this scenario, sensory systems contribute to detect, localize and recognize congeners and heterospecifics, and provide information about the availability of food and chemical features of environments where animals live. We investigated the behavior of D. simulans and D. buzzatii larvae to chemicals emitted by conspecific and heterospecific larvae. Our goal was to understand the role of these substances in the selection of pupation sites in the two species that cohabit within decaying prickly pear fruits (Opuntia ficus-indica). In these breeding sites, larvae of D. simulans and D. buzzatii detect larvae of the other species changing their pupation site preferences. Larvae of the two species pupated in the part of the fruit containing no or few heterospecifics, and spent a longer time in/on spots marked by conspecifics rather than heterospecifics. In contrast, larvae of the two species reared in isolation from conspecifics pupated randomly over the substrate and spent a similar amount of time on spots marked by conspecifics and by heterospecifics. Our results indicate that early chemically-based experience with conspecific larvae is critical for the selection of the pupation sites in D. simulans and D. buzzatii, and that pupation site preferences of Drosophila larvae depend on species-specific chemical cues. These preferences can be modulate by the presence of larvae of the same or another species.     

Receptor binding of pancreatic spasmolytic polypeptide (PSP) in rat intestinal mucosal cell membranes inhibits the adenylate cyclase activity

The recently isolated pancreatic spasmolytic polypeptide, PSP, interacted with specific binding sites in the gastrointestinal tract and inhibited the adenylate cyclase activity in rat intestinal mucosal cell membranes. The binding sites appeared to be heterogeneous and Scatchard analysis of the binding data indicated the presence of at least two classes of sites. The high-affinity low-capacity binding sites and the low-affinity high-capacity binding sites had apparent dissociation constants of 1.3 X 10(-7) mol/l and 4.2 X 10(-6) mol/l, respectively. The PSP induced inhibition of the adenylate cyclase activity was independent of the stimulatory state of the enzyme. The basal activity as well as that stimulated by VIP and secretin was half maximally inhibited at approximately 3 X 10(-5) mol/l of PSP. The inhibitory effect of PSP was independent of the agonist concentration employed. PSP did not affect the receptor binding of VIP nor did VIP affect the receptor binding of PSP.     

Differences in larval emergence chronotypes for sympatric Rhagoletis brncici Frías and Rhagoletis conversa (Bréthes) (Diptera,Tephritidae)

In central Chile, Rhagoletis brncici and R. conversa, can be found in sympatry, associated with the fruit of their native host plants: Solanum tomatillo and S. nigrum (Solanaceae), respectively. Third-stage larvae must emerge from its host in search of pupation sites, and during this period larvae must find an appropriate pupation microhabitat while avoiding predation and adverse abiotic factors. In this study, we explored whether these sympatric species differ in terms of the timing of their larval exit from the host fruit in search of pupation sites. Field-collected fruits from host plants were checked daily for larval emergence, within 24 h, under laboratory conditions, in order to determine the time of the event. We found that these species differed significantly in their diel larval emergence. For R. brncici, most larvae left the host fruit between late evening and past midnight, meanwhile larvae from R. conversa concentrated their peak of emergence near midnight and early morning. We discuss these findings in terms of the ecological and evolutionary implications of the temporal separation of larval emergence regarding the use of pupation sites, abiotic stress and risk of predation for these sympatric species.     

Dispersal and prepupation behavior of Chilean sympatric Drosophila species that breed in the same site in nature

We investigated dispersal patterns of Drosophila larvae searchingfor pupation sites over three substrates to determine the roleof spatial heterogeneity and presence of other species on prepupationbehavior. We used D. melanogaster, D. hydei, and D. pavani whoseparents emerged from apples collected in one orchard. Each speciesshowed different preferences for substrates on which to pupate,particularly in the presence of another Drosophila species.Larval locomotion rate and turning behavior in D. melanogaster,D. hydei, and D. pavani were modified depending this upon thetype of substrate (agar and sand) on which the larvae crawled.These two behaviors are involved in dispersal and aggregationof pupae. Distance between pupae of the same species decreaseswhen larvae of another species pupate on the same substrate.Aggregated distributions over the substrates lead to patcheswith few or no individuals. These could serve as pupation sitesfor other Drosophila species that, in nature, also emerge fromsmall breeding sites.     

Environmental control of pupal colour in swallowtail butterflies (Lepidoptera: Papilioninae): Battus philenor (L.) and Papilio polyxenes Fabr.

Abstract. 1. Some swallowtail butterflies produce both green and brown pupae. The phenotypes result from the joint action of genotype and environment and usually make the pupae cryptic in their habitats.
2. The major environmental cues influencing pupal colour in two swallowtail species were determined to be textural and optical.
3. Differences in the usage of these kinds of cues in the two species are thought to have evolved because of major differences in the pupation habitats. P.polyxenes , which usually pupates on slender stems amidst vegetation, responds more strongly to optical cues. B.philenor , which usually pupates on exposed surfaces of tree trunks and cliffs, responds more strongly to textural cues.
4. Differences in the overall tendency to produce brown pupae ('sensitivity': Hazel, 1977) are thought to be related to the frequency of brown pupation sites utilized by these two species: high average sensitivity in philenor , which often uses brown sites, and lower average sensitivity in polyxenes , which often uses green sites.     

Larval pupation site preference on fruit in different species of Drosophila

Larval pupation site preference (PSP) of different species of Drosophila was analyzed on fruit in the laboratory. The larvae of D. melanogaster, D. ananassae, D. virilis, D. novamexicana and D. hydei pupated on the surface of glass vials; D. simulans, D. yakuba, D. mauritiana and D. malerkotliana pupated in/on fruit; and D. rajasekari pupated on cotton plugs in all experiments. D. bipectinata larvae changed their preference from fruit in the control to glass surface for all of the fruits tested. The statistical analysis of PSP (glass and fruit) found a significant result in that compared to other species, D. mauritiana and D. ananassae preferred to pupate on cotton compared to the control.     

Variability of intra- and interspecific competitions of bamboo stump mosquito larvae over small and large spatial scales

We carried out field experiments to examine the variability of interspecific competition of mosquito larvae among microcosms in a bamboo grove (small spatial scale) and between bamboo groves at two sites, with single and multiple mosquito species (large spatial scale). Four types of microcosms that differed in capacity and litter input were set. In the hillside bamboo grove, where multiple species occurred, succession of the predominant species from Aedes albopictus to Tripteroides bambusa was observed in control microcosms from which no mosquito larvae were removed. Weekly removal of competitive species resulted in increased pupation of A. albopictus and adult body weight under both rich and poor resource conditions. In the late period of the experiments, the effect of competitor removal on pupation of A. albopictus was greater in deep containers that never dried than in shallow containers that were dried in the laboratory. The number of eggs showed a slight difference between competitor‐excluded and deep control microcosms. These results indicate that interspecific competition limits pupation of A. albopictus more strongly in deep containers than in shallow and drought‐prone containers.
Compared with the hillside site, the larval density of A. albopictus attained a higher density in the bamboo grove in the plain where no competitive species occurred, due to a higher oviposition rate. Lower rate of pupation and lower adult weight at the plain site than at the hillside site indicated that resource limitation was more severe at the plain site. Populations of A. albopictus at hillside and plain sites appeared to suffer from strong inter‐ and intraspecific competition, respectively. At the hillside site, the intensity of interspecific competition appeared to increase later in the breeding season, with a high larval density of T. bambusa. In contrast, at the plain site, intensity of intraspecific competition appeared to be reduced later in the breeding season with decreasing larval density of A. albopictus.     

Receptor binding of pancreatic spasmolytic polypeptide in intestinal mucosal cells and membranes

Rat intestinal mucosal cells contain receptors for pancreatic spasmolytic polypeptide (PSP). The binding of 125I-PSP was rapid, saturable, reversible and specific. PSP competed with 125I-PSP for binding to the receptors and 10(-7) M of PSP half-maximally inhibited 125I-PSP binding. The normalized PSP dose-response graphs in intact cells and crude membranes were superimposable. Scatchard plots of PSP binding to membranes were curvilinear, indicating multiple classes of binding sites, negative cooperative interaction between sites or a combination of both. PSP increased the rate of dissociation of the 125I-PSP-receptor complex compared to the rate observed by dilution only, thus giving evidence that negative cooperative interaction may occur between PSP binding sites. The half-life of the fast dissociating complex was about 1.5 min and that of the slow dissociating complex 38 min. These values were independent of the receptor occupancy. The increased rate of dissociation at high receptor occupancy stemmed from a shift in the ratio of the pool sizes of fast and slow dissociating receptor complexes.     

Fine-scale abundance and distribution of wild silk moth pupae

Although several factors influence herbivore insect distributions at any particular scale, the most important determinants are likely to differ between species with different life histories. Identifying what these factors are and how they relate to life history forms an important component of understanding the population dynamics of species, and the habitat requirements necessary for their conservation. The pupal stage of two wild silk moth species, Gonometa postica Walker and G. rufobrunnea Aurivillius (Lepidoptera: Lasiocampidae), is the target of harvesting practices that are totally dependent on the availability of pupae from natural populations. Consequently, and partly due to poor knowledge of the species' biology, there is substantial interest in the distribution of pupae among and within trees for both these species. It was investigated whether between- and within-tree pupal distributions in these two species are non-random, and if so, whether there are relationships between pupation site use and tree characteristics such as tree size, available pupation space and branch position. Between-tree patterns in pupal abundance were random in terms of absolute spatial position, but markedly non-random with respect to tree characteristics. The apparent G. postica pupae were aggregated on large larval host plants, whereas the cryptic G. rufobrunnea pupae were aggregated on non-host plants. These patterns reflect the life history differences of the two species. In contrast, at the within-tree scale, branch position, aspect and tree shape influenced pupation site choice similarly for both species. These patterns might be related to microclimate. Documenting between-tree and within-tree patterns in Gonometa pupal distributions is the first step towards explaining pupation site selection, as well as identifying possible evolutionarily selective factors in the species, and generating testable hypotheses from these.     

Mouse PSP94 expression is prostate tissue-specific as demonstrated by a comparison of multiple antibodies against recombinant proteins

Prostate tissue-specific gene expression is crucial for driving potentially therapeutic genes to target specifically to the prostate. Prostate secretory protein of 94 amino acids (PSP94), also known as beta-MSP (microseminoprotein), is one of the three most abundant secretory proteins of the prostate gland, and is generally considered to be prostate tissue-specific. We have previously demonstrated that the expression of the rat PSP94 gene is strictly prostate tissue-specific by an antibody against a recombinant rat PSP94. In order to study prostate targeting utilizing the PSP94 gene in a mouse pre-clinical experimental model, we need to establish antibodies against mouse PSP94 to confirm if it is prostate tissue-specific as well. In this study, firstly we raised a polyclonal antibody against a recombinant glutathione-S-transferase- (GST-) mouse mature form of PSP94. However, it showed very poor immunoreactivity against prostate tissue PSP94 as tested in Western blotting experiments. Neither antibodies against rat PSP94 nor mouse PSP94 showed significant cross-reactivity. Thus a second antibody was established against a recombinant mouse mature PSP94 containing N-terminal polyhistidines, and stronger immunoreactivity against mouse prostate tissue PSP94 was identified in Western blotting experiments. Both of these antibodies showed immunohistochemical reactivity, while the latter showed stronger reactivity in IHC when tested with different fixatives. By studying tissue distribution, we demonstrated that, as with rat PSP94, mouse PSP94 is strictly prostate tissue-specific in experiments of both Western blotting and immunohistochemistry (IHC). This conclusion was also derived from a comparison among antibodies against human, rat, and mouse PSP94, showing very different immunoreactivities in Western blotting and IHC. Finally, a competitive assay between different species was performed. We demonstrated that antibodies against PSP94 from different species (human, primate, rodents) have poor cross-reactivities. These observations also indicate that the PSP94 gene is a rapidly evolving gene in all species. Results from this study have led to the possibility of utilizing PSP94 as a targeting agent specifically to the prostate in a mouse experimental model.     

Natural pupation sites of swallowtail butterflies (Lepidoptera: Papilioninae): Papilio polyxenes Fabr., P.glaucus L. and Battus philenor (L.)

Abstract. 1. Natural pupation sites have been found in Papilio polyxenes and P.glaucus by releasing prepupal larvae marked with UV-fluorescent paint and locating them at night with a UV lamp, and in Battus philenor by searching a forest habitat where the larval foodplant is abundant.
2. P.polyxenes , a species of weedy habitats, pupates off the ground on a variety of substrates including grasses, weed stalks, posts, etc. The pupae may be green or brown, resembling the substrate.
3. P.glaucus , a species of forest habitats, pupates very close to the ground in the litter and has monomorphic brown pupae.
4. B.philenor , also a forest species, pupates on exposed surfaces (chiefly tree-trunks or cliffs) well off the ground. Its pupae may be brown or green, but the latter were found only on the slenderest twigs.
5. The results for polyxenes and glaucus support the generalization of Clarke & Sheppard (1972) that species of stable habitats are likely to have monomorphic pupae, while those of habitats in which available sites may not be so similar from one generation to the next will be dimorphic.
6. B.philenor is more problematical, but its tendency towards pupal monomorphism (brown) is logical in relation to its common pupation sites.     

Gregarious pupation act as a defensive mechanism against cannibalism and intraguild predation

Coccinellid pupae use an array of defensive strategies against their natural enemies. This study aims to assess the efficiency of gregarious pupation as a defensive mechanism against intraguild predators and cannibals in coccinellid. The study was designed specifically (i) to determine the natural occurrence of gregarious pupation in the field for different coccinellid species, and (ii) to evaluate the adaptive value of gregarious pupation as a defensive mechanism against 2 types of predators (i.e., cannibals and intraguild predators). In the field, gregarious pupation consisted of a group of 2–5 pupae. The proportion of gregarious pupation observed varied according to species, the highest rate being observed with Harmonia axyridis Pallas (Coccinellidae; 14.17%). Gregarious pupation had no impact on the probability that intraguild predators and cannibals locate pupae. Intraguild predation occurred more often in site with gregarious pupation, while cannibalism occurred as often in site with gregarious pupation as in site with isolated pupa. However, for a specific pupa, the mortality rate was higher for isolated pupae than for pupae located in a gregarious pupation site both in the presence of intraguild predators and in the presence of cannibals. The spatial location of pupae within the group had no impact on mortality rate. Since it reduces the risk of predation, it is proposed that gregarious pupation act as a defensive mechanism for H. axyridis pupae.     

Convergent evolution of neuroendocrine control of phenotypic plasticity in pupal colour in butterflies

Phenotypic plasticity in pupal colour occurs in three families of butterflies (the Nymphalidae, Papilionidae and Pieridae), typically in species whose pupation sites vary unpredictably in colour. In all species studied to date, larvae ready for pupation respond to environmental cues associated with the colour of their pupation sites and moult into cryptic light (yellow–green) or dark (brown–black) pupae. In nymphalids and pierids, pupal colour is controlled by a neuroendocrine factor, pupal melanization-reducing factor (PMRF), the release of which inhibits the melanization of the pupal cuticle resulting in light pupae. In contrast, the neuroendocrine factor controlling pupal colour in papilionid butterflies results in the production of brown pupae. PMRF was extracted from the ventral nerve chains of the peacock butterfly Inachis io (Nymphalidae) and black swallowtail butterfly Papilio polyxenes (Papilionidae). When injected into pre-pupae, the extracts resulted in yellow pupae in I. io but brown pupae in P. polyxenes. These results suggest that the same neuroendocrine factor controls the plasticity in pupal colour, but that plasticity in pupal colour in these species has evolved independently (convergently).     

Evolution of pupation site and interspecific competitive ability in Drosophila hydei

A competition experiment conducted over a period of 50 weeks resulted in stable coexistence of Drosophila hydei and D. melanogaster. In a repeat of this experiment the results for the first 25 weeks were similar to the results of the earlier experiment, but after this time the state of coexistence collapsed and a fairly rapid trend towards competitive exclusion was observed. Extinctions of D. melanogaster occurred in 11 of the 12 experimental cages before termination of the experiment at 50 weeks. At about the same time as the competitive ability of D. hydei increased, an alteration in the range of pupation sites utilized by this species was noted. The change in pupation site was demonstrated to be at least partially heritable, and it appears to be responsible for D. hydei's increased competitive ability. This experiment provides an example of natural selection within a competing population acting against the stability of the overall two-species system.     

Selection for pupation height in Drosophila melanogaster

Divergent directional selection for high and low pupation height was practiced in D. melanogaster. A quick response was observed in the two directions of selection. This is the first time selection for low pupation sites was successful. Realized heritabilities were 18% and 13% for the high and low lines. Reciprocal crosses between divergent lines showed little or no dominance for low pupation sites. The need for a strict control of environmental factors when measuring pupation height is emphasized.     

Identification of CRISP2 from human sperm as PSP94‐binding protein and generation of CRISP2‐specific anti‐peptide antibodies

Cysteine‐rich secretory proteins (CRISPs) are mainly found in the mammalian male reproductive tract and reported to be involved at different stages of fertilization. CRISPs have been shown to interact with prostate secretory protein of 94 amino acids (PSP94) from diverse sources, and the binding of these evolutionarily conserved proteins across species is proposed to be of functional significance. Of the three mammalian CRISPs, PSP94–CRISP3 interaction is well characterized, and specific binding sites have been identified; whereas, CRISP2 has been shown to interact with PSP94 in vitro. Interestingly, human CRISP3 and CRISP2 proteins are closely related showing 71.4% identity. In this study, we identified CRISP2 as a potential binding protein of PSP94 from human sperm. Further, we generated antisera capable of specifically detecting CRISP2 and not CRISP3. In this direction, specific peptides corresponding to the least conserved ion channel regulatory region were synthesized, and polyclonal antibodies were generated against the peptide in rabbits. The binding characteristics of the anti‐CRISP2 peptide antibody were evaluated using competitive ELISA. Immunoblotting experiments also confirmed that the peptide was able to generate antibodies capable of detecting the mature CRISP2 protein present in human sperm lysate. Furthermore, this anti‐CRISP2 peptide antibody also detected the presence of native CRISP2 on sperm.Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.