Michael R. Strand

Isolation and identification of a plasmatocyte-spreading peptide from the hemolymph of the lepidopteran insect Pseudoplusia includens

Insect blood cells (hemocytes) play an essential role in defense against parasites and other pathogenic organisms that infect insects. A key class of hemocytes involved in insect cellular immunity is plasmatocytes. Here we describe the isolation and identification of a peptide from the moth Pseudoplusia includens that mediates the spreading of plasmatocytes to foreign surfaces. This peptide, designated plasmatocyte-spreading peptide (PSP1), contains 23 amino acid residues in the following sequence: H-ENFNGGCLAGYMRTADGRCKPTF-OH. In vitro assays using the synthetic peptide at concentrations ≥2 nm induced plasmatocytes from P. includens to spread on the surface of culture dishes. Injection of this peptide into P. includenslarvae caused a transient depletion of plasmatocytes from circulation. Labeling studies indicated that this peptide induced 75% of plasmatocytes that were double-labeled by the monoclonal antibodies 49G3A3 and 43E9A8 to spread, whereas plasma induced significantly more plasmatocytes to spread. This suggests that only a certain subpopulation of plasmatocytes responds to the peptide and that other peptidyl factors mediate plasmatocyte adhesion responses.

THE DEVELOPMENTAL STRATEGIES OF ENDOPARASITOID WASPS VARY WITH HOST FEEDING ECOLOGY

Among the most important trade-offs in life history evolution is whether to grow larger at the cost of longer development time, or to develop more rapidly at the cost of reduced size. For insect herbivores, resolution of this trade-off is thought to be strongly influenced by feeding ecology and mortality risks. In contrast, how these factors might affect the developmental strategies of third trophic level organisms, like parasitoid wasps, is less understood. To address this question, we compared the development of larval en- doparasitoids in the families Ichneumonidae and Braconidae that parasitize larval stage herbivores in the order Lepidoptera. The campoplegine ichneumonid Venturia canescens parasitized concealed hosts and exhibited a developmental strategy that favored progeny size over development time. In contrast, the closely related ichneumonid Campoletis son- orensis parasitized exposed hosts and exhibited the opposite strategy of favoring rapid development time over size. The microgastrine braconid Microplitis croceipes attacks par- tially concealed hosts and showed evidence of a trade-off between maximizing body size and minimizing development times. These results suggested that parasitoids attacking ap- parent, foliar-feeding hosts may favor rapid development time over size while parasitoids that attack concealed hosts favor size over development time. A broader survey of the literature supported the trends found in our experimental studies. The braconids and ich- neumonids examined in this study also exhibit distinct differences in larval feeding and pupation behavior. These developmental traits did not appear to affect the size-development time continuum. However, these traits may affect the size range of hosts that larval en- doparasitoids can successfully exploit.

Alterations in the haemocytes of Pseudoplusia includens after parasitism by Microplitis demolitor

Abstract Parasitism by the braconid Microplitis demolitor suppressed the encapsulation response of its host, Pseudoplusia includens. Total haemocyte counts were higher in parasitized larvae than unparasitized larvae. Differential haemocyte counts did not differ between parasitized and unparasitized hosts, but the in vitro spreading behaviour of haemocytes was altered significantly by parasitism. Greater than 80% of plasmatocytes and granular cells from P. includens were unable to spread in vitro 2 h after oviposition by M. demolitor, and spreading of plasmatocytes remained suppressed through the course of parasitoid development. In addition, phenoloxidase activity in haemolymph was greatly reduced in parasitized larvae when compared to unparasitized larvae. Similar alterations in haemocyte spreading behaviour, phenoloxidase activity and encapsulation response were observed after injection of calyx fluid or purified polydnavirus from M. demolitor. The activity of polydnavirus was abolished by pretreatment with psoralen and ultraviolet irradiation which suggests the virus must be transcriptionally active to alter the host encapsulation response. Injection of venom did not affect haemocyte spreading or phenoloxidase activity, but dose-response studies indicated venom synergized the effects of calyx fluid. At equal doses, injection of calyx fluid plus venom had a greater and more persistent effect on plasmatocyte and granular cell spreading than injection of calyx fluid alone.

Monoclonal antibodies bind distinct classes of hemocytes in the moth Pseudoplusia includens

Insect hemocytes have historically been identified on the basis of morphology, ultrastructure and hypothesized function. Among insects in the order Lepidoptera, five hemocyte classes are usually recognized: granular cells, plasmatocytes, spherule cells, oenocytoids and prohemocytes. We have generated a panel of monoclonal antibodies (mAbs) against hemocytes of the moth Pseudoplusia includens. In this study, hemocyte identification using 16 different mAbs was compared to identification methods using morphological characters. Three main categories of mAb binding activity were identified: (1) mAbs that specifically labeled only one morphological class of hemocytes, (2) mAbs that labeled granular cells and spherule cells, and (3) mAbs that labeled plasmatocytes and oenocytoids. With one exception, none of the antibodies bound to other tissues in P. includens. However, certain mAbs that specifically labeled granular cells and/or spherule cells in separated hemocyte populations also labeled plasmatocytes co-cultured with granular cells or cultured in granular cell conditioned medium. Overall, our results suggest that granular cells are antigenically related to spherule cells, and that plasmatocytes are antigenically related to oenocytoids. The use of mAbs as hemocyte markers are discussed.

Mating system of Bracon hebetor(Hymenoptera:Braconidae)

Abstract. 1 We report on the mating system of a field population of the parasitic wasp, Bracon hebetor, on a corn pile infested by the Indian meal moth, Plodia interpunctella. We demonstrate that the mating system is based upon male scramble competition polygyny with male aggregations on high places on the corn. 2 The sex ratio among adults was greater than 80% males on the surface of the corn, whereas below the surface the sex ratio was less than 45%. Males actively courted females on the surface, but there were no aggressive interactions among males during courtship or mating. 3 Approximately 20% of the females found on the surface of the corn had no sperm in their spermathecae, regardless of age, but the numbers of unmated females decreased later during the day. 4 In laboratory studies we showed that females from this population oviposit a female biassed sex ratio, and that only 14% of females were mated before dispersing from their place of emergence. 5 Thus sib‐mating is unlikely in this gregarious parasitoid. This outcrossing mating system probably arose because of severe inbreeding depression that B.hebetor suffers via a sex locus: diploids that are heterozygous at the sex locus develop into females, but homozygous diploids are male and are generally inviable. The female biassed sex ratio may have evolved in B. hebetor in response to males being the more expensive sex, females dispersing more frequently from the population than males, or a fraction of females remaining unmated in the population.

Brood-mate avoidance in the parasitic waspBracon hebetorSay

The parasitic waspBracon hebetorsuffers severe inbreeding depression. This study examined two behavioural mechanisms that minimize mating between close relatives. First, the majority of males and females were unwilling to mate immediately upon emergence. Receptivity to mating slowly increased with age of the adult. By the time most individuals were willing to mate, the majority of wasps had dispersed from the natal site. Second, females tended to avoid mating with brood-mates when given a choice between a male that developed on the same host and one that developed on a different host. Experiments using eye-colour mutants and broods composed of relatives and non-relatives indicated that females discriminated against male brood-mates on the basis of environmental cues. Females consistently mated with brothers and non-brothers if they developed on another host, but tended to reject brothers and non-brothers from the same brood as themselves. Females maintained the ability to recognize brood-mates for at least 5 days after eclosion.

Hematopoiesis in larval Pseudoplusia includens and Spodoptera frugiperda

Maintenance of circulating hemocytes in larval Lepidoptera has been attributed to both mitosis of hemocytes already in circulation and the release of hemocytes from hematopoietic organs. In this study, we compared hematopoiesis in the noctuids Pseudoplusia includens and Spodoptera frugiperda. For both species, hemocyte densities per microl of blood increased with instar. Differential hemocyte counts indicated that plasmatocytes were the most abundant hemocyte type during early instars but granular cells were the most abundant hemocyte type in the last instar. Hematopoietic organs were located in the meso- and metathorax of S. Frugiperda and P. Includens. These organs contained large numbers of hemocytes in S. Frugiperda, but contained few hemocytes in P. Includens. The majority of the hemocytes recovered from hematopoietic organs were identified as plasmatocytes. Using hemocyte type-specific markers and bromodeoxyuridine (BrdU) incorporation experiments, we determined that all hemocyte types with the exception of oenocytoids synthesize DNA. BrdU labeling indices for both species also fluctuated with the molting cycle. Ligation experiments suggested that hematopoietic organs are an important source of circulating plasmatocytes in S. Frugiperda but not in P. Includens. Injection of heat killed bacteria into larvae induced higher levels of BrdU labeling than injection of sterile saline, suggesting that infection and wounding induce different levels of hemocyte proliferation. Arch.

The growth and role of Microplitis demolitor teratocytes in parasitism of Pseudoplusia includens

Abstract The growth and function of Microplitis demolitor teratocytes in Pseudoplusia includens larvae was investigated. An average of 542 teratocytes was produced per M. demolitor egg. The number of teratocytes present per host declined during the course of parasitoid development, but the size of individual cells increased. The ploidy levels of teratocytes ranged from 2 to 8C at hatching, but had increased to 128–256C at the completion of parasitoid development. Injection of in vitro cultured teratocytes into fourth stadium P. includens larvae had an inconsistent effect on development. Most larvae pupated without delay when injected with a physiological dose of 600 teratocytes, but 54% of larvae failed to pupate when injected with a superphysiological dose of 2400 teratocytes. Examination of teratocytes indicated most cells were encapsulated by P. includens haemocytes within 24 h of injection. However, prior injection of larvae with M. demolitor calyx fluid or polydnavirus plus venom suppressed the encapsulation response. Further, 87% of larvae injected with polydnavirus plus venom and 600 teratocytes exhibited alterations in growth that were very similar to parasitism by M. demolitor .

Encapsulation of foreign targets by hemocytes of the moth Pseudoplusia includens (Lepidoptera: Noctuidae) involves an RGD-dependent cell adhesion mechanism

Abstract We tested the hypothesis that an insect immune response, encapsulation, involves an RGD-dependent cell adhesion mechanism by examining the effects of the tetrapeptide RGDS on hemocyte spreading and encapsulation. Soluble RGDS at concentrations of 0.5–2 mM inhibited the spreading of the primary encapsulating hemocyte, the plasmatocyte, on the surface of plastic tissue culture plates. At concentrations of 5–10 mM, the spreading of granular cells was also inhibited. RGES did not inhibit plasmatocyte or granular cell spreading, indicating that the effect was specific for RGDS. RGDS-Sepharose beads were encapsulated by hemocytes in vitro, whereas RGES-Sepharose beads were not. Furthermore, soluble RGDS, but not RGES, inhibited in vitro encapsulation of RGDS-Sepharose. When injected into the hemocoel of P. includens larvae, RGDS-Sepharose was encapsulated in 3 h, whereas RGES-Sepharose was not encapsulated until 24 h. The only hemocyte morphotype to encapsulate RGDS-Sepharose was the plasmatocyte. Lastly, RGDS. but not RGES, inhibited plasmatocyte spreading in response to cell-free plasma. These results indicate that the molecular basis of cell adhesion mediating hemocyte spreading and encapsulation in insects involves cell adhesion molecules containing the RGD recognition sequence.

Surface characteristics of foreign targets that elicit an encapsulation response by the moth Pseudoplusia includens

Hemocytes from the moth Pseudoplusia includens encapsulate a variety of biotic and abiotic targets. Prior studies indicated that granular cells are usually the first hemocyte type to attach to foreign targets. Thereafter, large numbers of plasmatocytes attach to the target and form a capsule. To identify surface features that induce an encapsulation response, chromatography beads that differed in matrix composition, charge, and functional groups were tested using in vitro and in vivo bioassays. We first conducted in vitro assays using hemocytes with no plasma components present. These experiments indicated that bead types having sulfonic, diethylaminoethyl, and quaternary amine functional groups were encapsulated significantly more often than beads with other functional groups. Charge also significantly affected encapsulation with positively charged beads being encapsulated more often than negatively charged or neutral beads. In vitro assays using purified populations of hemocytes confirmed that these targets were recognized as foreign by granular cells, and that plasmatocytes only formed capsules after granular cells attached to the target. Bead types that were encapsulated under these in vitro conditions were always rapidly encapsulated when injected into P. includens larvae. However, some bead types, like CM-Sephadex, not encapsulated in vitro were encapsulated in vivo if left in the insect hemocoel for a longer period of time (ca. 24 h). Purified plasmatocytes encapsulated these beads in vitro if they were preincubated in plasma. Basic characterization studies suggest these humoral recognition molecules are proteins or small peptides. Comparative studies with other species of noctuid moths also indicated that encapsulation of some bead types differed significantly among species. Collectively, these results reveal that P. includens recognizes some targets as foreign by pattern recognition receptors on granular cells, whereas others are recognized by pattern recognition molecules in plasma. The binding affinities of these recognition molecules also appear to differ among closely related species of Lepidoptera.