Phage loss and the breakdown of a defensive symbiosis in aphids.

Terrestrial arthropods are often infected with heritable bacterial symbionts, which may themselves be infected by bacteriophages. However, what role, if any, bacteriophages play in the regulation and maintenance of insect-bacteria symbioses is largely unknown. Infection of the aphid Acyrthosiphon pisum by the bacterial symbiont Hamiltonella defensa confers protection against parasitoid wasps, but only when H. defensa is itself infected by the phage A. pisum secondary endosymbiont (APSE). Here, we use a controlled genetic background and correlation-based assays to show that loss of APSE is associated with up to sevenfold increases in the intra-aphid abundance of H. defensa. APSE loss is also associated with severe deleterious effects on aphid fitness: aphids infected with H. defensa lacking APSE have a significantly delayed onset of reproduction, lower weight at adulthood and half as many total offspring as aphids infected with phage-harbouring H. defensa, indicating that phage loss can rapidly lead to the breakdown of the defensive symbiosis. Our results overall indicate that bacteriophages play critical roles in both aphid defence and the maintenance of heritable symbiosis.

Phenotypically plastic traits regulate caste formation and soldier function in polyembryonic wasps.

Polyembryonic encyrtid wasps are parasitoids that have evolved a clonal form of embryogenesis and a caste system where some progeny become reproducing wasps whereas others develop into a sterile soldier caste. Theory based on the biology of Copidosoma floridanum predicts that the primary role of soldier larvae is to mediate conflict over sex ratio, which also favours female-biased soldier production. Other data, however, suggest that female-biased soldier production reflects a developmental constraint. Here, we assessed whether female-biased soldier function by polyembryonic wasps reflects sex-specific adaptation or constraint by conducting comparative studies with Copidosoma bakeri, a species that produces clutch sizes similar to C. floridanum yet rarely produces broods associated with sex ratio conflict. Our results indicate that the oviposition behaviour of adults, development of progeny and function of soldier larvae differ greatly between C. bakeri and C. floridanum. These findings indicate that caste formation and soldier function in polyembryonic encyrtid wasps are regulated by phenotypically plastic traits. Our results further suggest that the primary function of the soldier caste in some species is defence of host resources from competitors whereas in others it is the resolution of sex ratio conflict.

Infection by a symbiotic polydnavirus induces wasting and inhibits metamorphosis of the moth Pseudoplusia includens.

Insect pathogens and parasites often affect the growth and development of their hosts, but understanding of these processes is fragmentary. Among the most species-rich and important mortality agents of insects are parasitoid wasps that carry symbiotic polydnaviruses (PDVs). Like many PDV-carrying wasps, Microplitis demolitor inhibits growth and pupation of its lepidopteran host, Pseudoplusia includens, by causing host hemolymph juvenile hormone (JH) titers to remain elevated and preventing ecdysteroid titers from rising. Here we report these alterations only occurred if P. includens was parasitized prior to achieving critical weight, and were fully mimicked by infection with only M. demolitor bracovirus (MdBV). Metabolic assays revealed that MdBV infection of pre-critical weight larvae caused a rapid and persistent state of hyperglycemia and reduced nutrient stores. In vitro ecdysteroid assays further indicated that prothoracic glands from larvae infected prior to achieving critical weight remained in a refractory state of ecdysteroid release, whereas infection of post-critical weight larvae had little or no effect on ecdysteroid release by prothoracic glands. Taken together, our results suggest MdBV causes alterations in metabolic physiology, which prevent the host from achieving critical weight. This in turn inhibits the endocrine events that normally trigger metamorphosis.

Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.

Hemocytes are an essential component of the mosquito immune system but current knowledge of the types of hemocytes mosquitoes produce, their relative abundance, and their functions is limited. Addressing these issues requires improved methods for collecting and maintaining mosquito hemocytes in vitro, and comparative data that address whether important vector species produce similar or different hemocyte types. Toward this end, we conducted a comparative study with Anopheles gambiae and Aedes aegypti. Collection method greatly affected the number of hemocytes and contaminants obtained from adult females of each species. Using a collection method called high injection/recovery, we concluded that hemolymph from An. gambiae and Ae. aegypti adult females contains three hemocyte types (granulocytes, oenocytoids and prohemocytes) that were distinguished from one another by a combination of morphological and functional markers. Significantly more hemocytes were recovered from An. gambiae females than Ae. aegypti. However, granulocytes were the most abundant cell type in both species while oenocytoids and prohemocytes comprised less than 10% of the total hemocyte population. The same hemocyte types were collected from larvae, pupae and adult males albeit the absolute number and proportion of each hemocyte type differed from adult females. The number of hemocytes recovered from sugar fed females declined with age but blood feeding transiently increased hemocyte abundance. Two antibodies tested as potential hemocyte markers (anti-PP06 and anti-Dox-A2) also exhibited alterations in staining patterns following immune challenge with the bacterium Escherichia coli.

Mosquito Peptide Hormones: Diversity, Production, and Function.

Mosquitoes, like other insects, produce a diversity of peptide hormones that are processed from different precursor proteins and have a range of activities. Early studies relied on purification of bioactive peptides for hormone identification, but more recently genomic data have provided the information needed to more comprehensively identify peptide hormone genes and associated receptors. The first part of this chapter summarizes the known or predicted peptide hormones that are produced by mosquitoes. The second part of this chapter discusses the sources of these molecules and their functions.

Intensive linkage mapping in a wasp (Bracon hebetor) and a mosquito (Aedes aegypti) with single-strand conformation polymorphism analysis of random amplified polymorphic DNA markers.

The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito. Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A aygpti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.

Linkage analysis of sex determination in Bracon sp. near hebetor (Hymenoptera: Braconidae).

To test whether sex determination in the parasitic wasp Bracon sp. near hebetor (Hymenoptera: Braconidae) is based upon a single locus or multiple loci, a linkage map was constructed using random amplified polymorphic DNA (RAPD) markers. The map includes 71 RAPD markers and one phenotypic marker, blonde. Sex was scored in a manner consistent with segregation of a single "sex locus" under complementary sex determination (CSD), which is common in haplodiploid Hymenoptera. Under haplodiploidy, males arise from unfertilized haploid eggs and females develop from fertilized diploid eggs. With CSD, females are heterozygous at the sex locus; diploids that are homozygous at the sex locus become diploid males, which are usually inviable or sterile. Ten linkage groups were formed at a minimum LOD of 3.0, with one small linkage group that included the sex locus. To locate other putative quantitative trait loci (QTL) for sex determination, sex was also treated as a binary threshold character. Several QTL were found after conducting permutation tests on the data, including one on linkage group I that corresponds to the major sex locus. One other QTL of smaller effect had a segregation pattern opposite to that expected under CSD, while another putative QTL showed a female-specific pattern consistent with either a sex-differentiating gene or a sex-specific deleterious mutation. Comparisons are made between this study and the in-depth studies on sex determination and sex differentiation in the closely related B. hebetor.

Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includens.

Inducible expression of antimicrobial peptides and other humoral immune factors by the insect fat body is well documented. Hemocytes comprise the second essential arm of the insect immune system but it is unclear whether antimicrobial peptide genes are expressed by all or only some types of hemocytes. Here we report the cloning of cecropin A (Pi-cecA), lebocin (Pi-leb) and lysozyme (Pi-lys) homologs from the moth Pseudoplusia includens. Relative-quantitative real-time PCR (rq-rtPCR) indicated that transcript abundance for each antimicrobial gene increased in fat body and hemocytes following immune challenge with the Gram-negative bacterium Escherichia coli. Relative transcript abundance of Pi-cecA was much higher in fat body than hemocytes. In contrast, transcript levels of Pi-leb were three-fold lower in hemocytes than fat body while transcript levels of Pi-lys were three-fold higher. Estimates for the overall contribution of the fat body and hemocytes to antimicrobial peptide expression suggested that hemocytes contribute significantly to Pi-lys transcript levels in larvae but produce much smaller amounts of Pi-cecA and Pi-leb compared to the fat body. Each antimicrobial peptide was also inducibly expressed in hemocytes following challenge with the Gram-positive bacterium Micrococcus luteus or when hemocytes formed capsules around chromatography beads. Analysis of hemocyte types indicated that granulocytes and plasmatocytes expressed all three antimicrobial peptides, whereas spherule cells and oenocytoids expressed only lysozyme. Transcriptional profiles of these antimicrobial genes were similar in granulocytes and plasmatocytes in vivo but were very different in vitro.

Follow-up of protein release from Pseudoplusia includens hemocytes: a first step toward identification of factors mediating encapsulation in insects.

Hemocyte types involved in encapsulation were characterized using monoclonal antibodies (mAbs). This approach revealed that four hemocyte types in Pseudoplusia includens could be classified into two antigenically distinct cell lines. The first line comprised granulocytes (GR) and spherulocytes (SP) and the second line comprised plasmatocytes (PL) and oenocytoids (OE). One of the mAbs labeled a subpopulation of plasmatocytes that spread on culture plate surfaces. This subclass represented approximately 70% of all plasmatocytes. The cytoplasmic punctate staining of granulocytes clearly decreased upon short term culture, suggesting the associated antigens were released into the culture medium during cell spreading. A follow-up of protein secretion into culture medium by Western blotting confirmed this hypothesis for both granulocytes and plasmatocytes. In a few cases, discharged proteins exhibited a short half-life suggesting they may behave as regulatory molecules. Among them, plasmatocyte proteins of +/-25 kDa might be mobilized at an early stage of encapsulation. The same proteins appeared to accumulate at the periphery of the median plasmatocyte multilayer in late capsules. This location coincides with where an outer monolayer of granulocytes attaches and causes termination of capsule growth. These preliminary results raise the possibility that released proteins regulate hemocyte recruitment during encapsulation.

Insect hemocytes and their role in immunity.

The innate immune system of insects is divided into humoral and cellular defense responses. Humoral defenses include antimicrobial peptides, the cascades that regulate coagulation and melanization of hemolymph, and the production of reactive intermediates of oxygen and nitrogen. Cellular defenses refer to hemocyte-mediated responses like phagocytosis and encapsulation. In this review, we discuss the cellular immune responses of insects with emphasis on studies in Lepidoptera and Diptera. Insect hemocytes originate from mesodermally derived stem cells that differentiate into specific lineages identified by morphology, function, and molecular markers. In Lepidoptera, most cellular defense responses involve granular cells and plasmatocytes, whereas in Drosophila they involve primarily plasmatocytes and lamellocytes. Insect hemocytes recognize a variety of foreign targets as well as alterations to self. Both humoral and cell surface receptors are involved in these recognition events. Once a target is recognized as foreign, hemocyte-mediated defense responses are regulated by signaling factors and effector molecules that control cell adhesion and cytotoxicity. Several lines of evidence indicate that humoral and cellular defense responses are well-coordinated with one another. Cross-talk between the immune and nervous system may also play a role in regulating inflammation-like responses in insects during infection.

Variable sex ratio strategy of Telenomus heliothidis (Hymenoptera: Scelionidae): adaptation to host and conspecific density.

The sex allocation behavior of the solitary egg parasitoid Telenomus heliothidis Ashmead was investigated by examining the response of females reared in isolation and under crowded conditions. Females reared in isolation adjusted their sex ratio with foundress and host number per patch in accordance with the predictions of local mate competition (LMC) theory. However, females did not shift their sex ratio in response to conspecifics foraging on the same host patch or to contact with previously parasitized hosts. Instead, shifts were associated with encounter rate and a sequence of oviposition. Females maintained under crowded conditions responded to host patches much differently. One-day-old females which had lived under crowded conditions for 24 h produced sex ratios similar to those of continuously isolated females. However, females reared under crowded conditions for 7 days consistently produced unbiased sex ratios, and exhibited a different sequence of oviposition. This shift appeared to be due directly to crowding rather than age, oviposition experience or sperm depletion since the effect could be reversed by subsequent isolation.

Cross-protection experiments with parasitoids in the genus Microplitis (Hymenoptera: Braconidae) suggest a high level of specificity in their associated bracoviruses.

The immunological and developmental effects of bracoviruses (BVs) from three parasitoids in the genus Microplitis (Braconidae: Microgastrinae) were compared in the hosts Pseudoplusia includens and Heliothis virescens (Lepidoptera: Noctuidae). Southern blotting experiments indicated that viral DNAs from Microplitis demolitor bracovirus (MdBV) cross-hybridized with viral DNAs from Microplitis croceipes bracovirus (McBV) and Microplitis mediator bracovirus (MmBV) under conditions of high stringency. Injection of calyx fluid plus venom from each parasitoid species dose-dependently delayed development of P. includens and H. virescens. Each virus also inhibited pupation of P. includens but not H. virescens. In situ hybridization experiments indicated that MdBV and McBV persistently infect hemocytes in both hosts while MmBV persistently infects hemocytes in P. includens but not H. virescens. While MdBV infection induced a loss of adhesion by most plasmatocytes, McBV and MmBV infection induced a loss of adhesion in less than 50% of cells. Cross-protection experiments indicated that calyx fluid plus venom from one species usually protected progeny of another species from encapsulation but did not always promote successful development.

Presence of soldier larvae determines the outcome of competition in a polyembryonic wasp.

Soldier-producing polyembryonic waSPS are the only social animals that develop as parasites inside the bodies of other insects. Characterizing the kin composition of broods is central to understanding the evolution of the soldier caste in these unique social insects. Here we studied the role of soldiers in mediating the outcome of competition among clones of the polyembryonic wasp Copidosoma floridanum. Soldier-producing female clones usually monopolized host resources, whereas soldierless male clones usually coexisted in hosts. Behavioural experiments further indicated that early-emerging soldiers are specialized to combat intraspecific competitors and later-emerging soldiers are specialized for defence against interspecific competitors. Taken together, our results point to intraspecific competition as a major selective force in the evolution of the soldier caste. Our data also present an evolutionary conundrum: given the benefit of soldiers, why are male clones functionally soldierless?

Microplitis demolitor polydnavirus infects and expresses in specific morphotypes of Pseudoplusia includens haemocytes.

Microplitis demolitor is a polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens. M. demolitor eggs are never encapsulated by host haemocytes when coinfected with its associated polyndnavirus (MdPDV) whereas eggs are encapsulated within 36 h when injected into hosts without virus. In this study, infection of specific classes of P. includens haemocytes by MdPDV was examined. Electron microscopic studies indicated that MdPDV entered all haemocyte morphotypes. Northern blot analysis revealed that similar size classes of viral mRNAs were produced in granular cells, plasmatocytes and spherule cells. Expression of a 1.6 kb MdPDV mRNA in haemocytes from parasitized hosts was detectable by in situ hybridization at 2 h post-parasitism (p.p.) and continued through until day 6 p.p. By 12 h p.p., viral expression was detected in greater than 80% of the haemocytes in circulation but thereafter the percentage of haemocytes exhibiting a hybridization signal declined. Similar patterns were observed in haemocytes from larvae injected with calyx fluid or MdPDV plus venom. Granular cells and plasmatocytes from unparasitized larvae were purified on Percoll cushions and maintained in vitro. Both morphotypes were successfully infected with MdPDV and exhibited changes in morphology and adhesiveness very similar to cells from parasitized hosts. Cell-free plasma from parasitized larvae had a variable effect on haemocyte adhesion. Haemocytes cultured in plasma from 1 or 4 day p.p. larvae rapidly spread whereas cells cultured in 7 day p.p. plasma did not. Reciprocally, adhesion of haemocytes from parasitized larvae could not be rescued by cell-free plasma from unparasitized larvae. Together, these data suggest that disruption of the host encapsulation response is medicated primarily by direct infection of granular cells and plasmatocytes by MdPDV.

Developmental disruption of Pseudoplusia includens and Heliothis virescens larvae by the calyx fluid and venom of Microplitis demolitor.

Calyx fluid and venom from the braconid parasitoid Microplitis demolitor differentially affected the development of Pseudoplusia includens and Heliothis virescens. P. includens exhibited delays in larval development, supernumerary instars, and formed larval-pupal intermediates when injected with 0.01-0.10 wasp equivalents of calyx fluid. In contrast, H. virescens was relatively unaffected by calyx fluid regardless of dose. Venom did not affect the development of either host species, but appeared to synergize the activity of calyx fluid. This was particularly evident in H. virescens, where injection of 0.10-0.20 wasp equivalents of calyx fluid and venom induced the formation of a large number of intermediates while the same amount of calyx fluid did not. The particulate portion of M. demolitor calyx fluid was the only component that caused developmental delays and the formation of intermediates in both host species. Purified virus caused developmental alterations in P. includens, while trioxsalen treated calyx fluid did not affect development of P. includens or H. virescens. These data suggest the requirement for venom in parasitism may differ between host species, and that dosage plays an important role in interpreting the interaction between calyx and venom components.

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.

Granular cells are required for encapsulation of foreign targets by insect haemocytes.

Haemocytes play an essential role in defending invertebrates against pathogens and parasites that enter their haemocoel. A primary defense response is encapsulation; a process in which haemocytes attach to the foreign organism and kill it. Whether encapsulation requires cooperation between specific subpopulations of haemocytes is unknown. Using purified subpopulations of haemocytes and an in vitro encapsulation assay, we investigated the process of capsule formation in the insect Pseudoplusia includens. Immunocytochemical staining revealed that capsule formation involves a three step process. Encapsulation began when granular cells attached to the foreign target. This was followed by attachment of multiple layers of plasmatocytes. Termination of capsule formation occurred when a subpopulation of granular cells formed a monolayer around the periphery of the capsule. Neither granular cells nor plasmatocytes were capable of forming a capsule independently. However, plasmatocytes encapsulated targets if granular cells were present or if targets were preincubated in medium conditioned by granular cells. The effect of granular cell-conditioned medium could be blocked by the addition of the cell adhesion recognition sequence, RGDS, but not by RGES. These results demonstrate experimentally that granular cells are required for encapsulation of foreign targets by plasmatocytes in vitro, and that the role of granular cells in this process involves an RGD-dependent cell adhesion mechanism.

Haemocytes from Pseudoplusia includens express multiple alpha and beta integrin subunits.

Cellular immune responses such as encapsulation involve the adhesion of one or more classes of haemocytes. How insect haemocytes recognize encapsulation targets as foreign or the identity of the molecules regulating haemocyte adhesion is unknown. One of the most important classes of adhesion receptors in mammalian immune cells is the integrins, which form functional heterodimers through different combinations of alpha and beta subunits. Prior studies with the moth Pseudoplusia includens indicated that encapsulation depends on two classes of haemocytes called granulocytes and plasmatocytes. Here we report the cloning and identification of three alpha integrin subunits (alphaPi1-3) and one beta subunit (betaPi1) from P. includens. Northern blot analysis indicated that all four subunits are expressed in granulocytes and that three of the four subunits are expressed in plasmatocytes. Quantification of transcription patterns using real-time PCR revealed that expression of alphaPi2 and betaPi1 increased in granulocytes and plasmatocytes when binding to a foreign surface or forming a capsule. alphaPi2 transcription in plasmatocytes was further increased by granulocyte conditioned medium, plasmatocyte spreading peptide, and the integrin recognition peptide RGD. Collectively, these results suggest that one or more integrins play an important role in regulating haemocyte adhesion during encapsulation.

Microplitis demolitor polydnavirus induces apoptosis of a specific haemocyte morphotype in Pseudoplusia includens.

Microplitis demolitor polydnavirus (MdPDV) is associated with Microplitis demolitor, a parasitic wasp that attacks the larval stage of the lepidopteran Pseudoplusia includens. Previously, we observed that MdPDV induced several alterations in the granular cells and plasmatocytes of P. includens, the primary haemocytes involved in regulating the cellular immune response toward M. demolitor and other parasites. In examining the mechanisms underlying immunosuppression of this host, we found that MdPDV induced apoptosis of granular cells. Granular cells underwent apoptosis both when virus was injected into the haemocoel of P. includens larvae and after infection with MdPDV in vitro. Characteristics of MdPDV-induced apoptosis included condensation of chromatin, cell surface blebbing and fragmentation of DNA into a 200 bp ladder. Although MdPDV induced changes in the ability of plasmatocytes to adhere to foreign surfaces, apoptosis of this morphotype was not observed. Examples from the literature suggest that some viruses promote their own survival by suppressing apoptosis of host cells. However, since polydnaviruses are likely to be transmitted vertically, we suggest that MdPDV promotes its own survival by inducing apoptosis of host immune cells which would otherwise kill the developing M. demolitor egg.

Immunological basis for compatibility in parasitoid-host relationships.

The insect immune system serves as a key defense against attack by parasitoids. Incompatible hosts often eliminate parasitoids by encapsulation, a process in which hemocytes form a multilayered envelope around the invading organism. Capsule formation involves cooperation between one or more classes of hemocytes and is likely mediated by cytokines and adhesion molecules. Reciprocally, parasitoids have evolved a variety of strategies for overcoming host immune responses. Some parasitoids passively avoid elimination by developing in locations inaccessible to host hemocytes or by possessing surface features that fail to elicit an immune response. Other species actively disrupt the host immune system by injecting specific factors into the host at oviposition. In particular, polydnaviruses associated with several taxa of parasitoids disrupt capsule formation by killing hemocytes or altering their ability to adhere to foreign surfaces. These symbionts have likely played a critical role in evolution of host range and in defining parasitoid-host compatibility.