S.B. Vinson

Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus

Abstract Campoletis sonorensis is a habitual parasitoid of 3rd-instar larvae of Heliothis virescens. C. sonorensis eggs and small glass rods were encapsulated in 5th-instar host larvae implanted in the absence of wasp calyx fluid; prior injection of calyx fluid into larvae suppressed the encapsulation response. Within 8 h of calyx fluid injection there was a removal of approx. 75% of the circulating capsule-forming haemocytes (plasmatocytes). The remaining subpopulation of plasmatocytes, in addition to being incapable of encapsulating targets in vivo , spread at a significantly reduced rate in vitro . Identical changes in plasmatocyte count and behaviour were observed after injection of virus purified from calyx fluid. Additionally, the activity of calyx fluid was abolished after ultraviolet irradiation. The onset of haemocytic abnormalities occurred more rapidly after natural parasitism of 3rd-instar host larvae. The cell-free haemolymph of calyx fluid-injected 5th-instar larvae also retarded the spreading of plasmatocytes from non-injected control larvae in vitro . We conclude that the abnormalities induced in H. virescens plasmatocytes by C. sonorensis virus contribute to the suppression of encapsulation.

Ecdysteroid-titre reduction and developmental arrest of last-instar Heliothis virescens larvae by calyx fluid from the parasitoid Campoletis sonorensis

Abstract Fifth-instar Heliothis virescens larvae did not pupate after injections of Campoletis sonorensis calyx fluid in or before the burrow-digging stage of development. Arrested development occurred in 40% of larvae injected at the cell-formation stage. Further experiments showed that the particles in calyx fluid were responsible for developmental arrest. Arrested development due to calyx fluid could be reversed by injecting 10 μg of either ecdysone or 20-hydroxyecdysone, although a second injection of 20-hydroxyecdysone was needed for some larvae 3 days after the first treatment. Ecdysteroid production ceased for up to 10 days in 5th-instar H. virescens after calyx-fluid injection. After 10 days, some experimental larvae began to produce ecdysteroids again but remained developmentally arrested. The head, thorax, or abdomen of larvae were isolated by ligations and calyx fluid injected into the isolated body region. After 24 h, ligatures were released and the larvae observed for developmental arrest. Only injections into the isolated thorax stopped development. This, along with ecdysteroid data, indicated that C. sonorensis calyx fluid may directly affect the prothoracic glands of 5th-instar H. virescens.

Passive evasion by eggs of braconid parasitoid Cardiochiles nigriceps of encapsulation in vitro by haemocytes of host Heliothis virescens. Possible role for fibrous layer in immunity

Abstract We have utilised a novel in vitro encapsulation system using haemolymph of the host, Heliothis virescens and eggs of the parasitoid, Cardiochiles nigriceps (as targets for encapsulation) to examine the properties of the egg surface with respect to recognition by the host haemocytes. Many biotic and abiotic targets were readily encapsulated in vitro, with the exception of negatively charged cation exchange beads and allogenic tissue. However, mature eggs, which had a 0.5 – 1.0 μm thick fibrous layer on their outer surface, were not encapsulated in vitro regardless of whether they were developing, dormant or had been killed by ultra-violet irradiation. Conversely, immature eggs, which had a sheath of follicle cells over the fibrous layer, were readily encapsulated; similarly, experimental removal of the fibrous layer with driselase or H. zea haemolymph also caused the eggs to elicit an in vitro encapsulation response. Identical encapsulation responses occur if eggs are injected manually into H. virescens larvae. The data suggest that eggs of the parasitoid, C. nigriceps can evade encapsulation by the host haemocytes independently of other “immunosuppressants” such as calyx fluid/virus, venom or teratocytes. It is proposed that the fibrous layer may, among other possible functions, delay encapsulation until a more permanent means of suppressing encapsulation of the egg is established in the host.

Correlating pathological symptoms in Heliothis virescens eggs with development of the parasitoid Telenomus heliothidis

Abstract The parasitoid Telenomus heliothidis was able to develop successfully in Heliothis virescens eggs throughout nearly all phases of host embryogenesis. Development of H. virescens eggs ceased subsequent to parasitism because of an arrestment factor injected by the parasitoid female at oviposition. This factor appeared to be produced by exocrine cells of the T. heliothidis common oviduct. Microscopical studies indicated host cells were pycnotic prior to T. heliothidis first-instar eclosion; however, very rapid decomposition of host tissues occurred after first instar eclosion. The adult produced arrestment factor was found to be partially responsible for the host symptoms prior to T. heliothidis first-instar eclosion, but teratocytes associated with the parasitoid larva were primarily responsible for host decomposition. The T. heliothidis larva did not appear to have an active role in host pathogenesis.

A possible mechanism for the physiological suppression of conspecific eggs and larvae following superparasitism by solitary endoparasitoids

Competition for possession of a host by internal solitary parasitoids has been attributed to physical combat and physiological suppression, but the mechanisms that result in what has been referred to as physiological suppression is poorly understood. Some insights are provided by the studies reported here using the solitary endoparasitoid, Campoletis sonorensis (Cameron). Embryos of C. sonorensis less than ten hours old rarely hatch in various artificial media, while embryos twenty hours or older generally hatch. These results suggest that young embryos in which the embryonic membranes have not yet formed are only able to develop in a narrow range of environments represented by the nonparasited hemolymph. In contrast, embryos in which the embryonic membranes are formed are able to develop in a wide range of environments represented by parasitized hemolymph which has been shown by a number of studies to change. These ideas were given support by studies reported here, where young and older eggs were incubated singly or paired. We suggest the general changes in the hemolymph of a parasitized host become unfavorable for the development of newly oviposited eggs.

Degeneration of last instar Heliothis virescens prothoracic glands by Campoletis sonorensis polydnavirus

Abstract The development of last instar Heliothis virescens larvae was experimentally arrested by ligation between the head and thorax or by injection with calyx fluid from the endoparasitoid Campoletis sonorensis . Prothoracic glands were removed from these larvae at various times after treatment and the gland cells were either measured or prepared for electron microscopy. At 4 days postligation, gland cell ultrastructure was similar to that of cells from unligated larvae and cell size was not significantly reduced until 7 days after ligation. In contrast to ligated controls, cells from calyx fluid-injected larvae were significantly smaller 24 hr after injection. Significant degeneration in gland cell ultrastructure was observed 2 days after injection and by 7 days postinjection, prothoracic glands showed gross degeneration. Similarly, injection of purified polydnavirus caused gross gland degeneration by 7 days after injection. Irradiating calyx fluid abolished its ability to cause gland degeneration. Our results indicate that C. sonorensis polydnavirus stimulates prothoracic gland degeneration. This virally induced degeneration of the prothoracic glands may account for our previously reported observation that injection of calyx fluid causes a drastic reduction in hemolymph ecdysteroid titer and consequent developmental arrest.

Campoletis sonorensis virus: Expression in Heliothis virescens and identification of expressed sequences

Abstract Northern blot analysis of polyadenylated mRNA from Heliothis virescens larvae parasitized by Campoletis sonorensis wasps revealed at least 10 viral mRNAs that hybridized with 32P-labelled C. sonorensis virus DNA. Cloned C. sonorensis virus DNA fragments which contained expressed sequences were identified by screening virus genomic libraries with cDNAs synthesized from mRNAs isolated from parasitized larvae. Viral transcripts were mapped on several of the cloned cDNA positive C. sonorensis virus DNAs. Three additional, previously undescribed superhelical DNAs were detected in the virus genome.

Interference with function of plasmatocytes of Heliothis virescens in vivo by calyx fluid of the parasitoid Campoletis sonorensis.

SummaryImmature stages of the ichneumonid parasitoid, Campoletis sonorensis, develop within the haemocoel of its noctuid host, Heliothis virescens. The host cannot encapsulate the parasitoid egg owing to the suppressive effect of the polydnavirus-laden calyx fluid injected by the female parasitoid during oviposition. We have examined the effects of injection of calyx fluid on the following haemocytic manifestations of the immune system of 5th-instar larvae of H. virescens: encapsulation, nodulation, phagocytosis, erythrocyte rosetting and coagulation. Of these phenomena, only those requiring the formation of a multicellular sheath of plasmatocytes were affected. In general, encapsulation was fully suppressed; all of the C. sonorensis eggs and most of the glass rods implanted as targets were devoid of attached haemocytes 3 days after implantation although a few of the latter were coated by a sparsely distributed layer of granulocytes. Plasmatocytes also appeared to be present in thicker depositions of haemocytes. In nodulation, only the second, encapsulation-like phase was inhibited. The resistant first stage, involving the entrapment of particles by haemocytes, only resulted in the formation of amorphous, disorganized nodules. Granulocyte-dependent aspects of the immune system (phagocytosis, rosetting and possibly coagulation and the first stage of encapsulation and nodulation) occurred normally. The data suggest that in 5th-instar hosts injection of calyx fluid acts specifically on plasmatocyte function.

The rôle of the calyx and poison gland of Cardiochiles nigriceps in the host-parasitoid relationship☆

Abstract Parasitism of Heliothis virescens by Cardiochiles nigriceps reduced the growth of the host. Both the poison gland and the calyx of the female parasitoid were important in reducing the growth of the parasitized host. Injections of poison gland contents (0·04 gl/larva) or calyx fluid (0·04 gl/larva) into H. virescens larvae did not affect their growth. However, a mixture of the two glands (1:1) at this low dosage significantly reduced the weight gained by Heliothis larvae . Heliothis larvae parasitized by parasitoids from which the poison gland was removed (poi gl − females) grew significantly larger than those larvae parasitized by either a normal female or by a female from which the alkaline gland was removed (alk gl − female). The injection of poison gland material (0·04 gl/larva) into larvae which were parasitized by poi gl − females reduced the weight of these larvae to within the limits of larvae parasitized by normal females. The development of larval parasitoids which hatched from eggs of poi gl − females is abnormal. The time of larval development for the parasitoids from poi gl − females is significantly longer than that for the parasitoids from normal females or from alk gl − females. In addition 34 per cent of parasitoids from poi gl − females did not successfully complete development whereas only 10 per cent of the parasitoids from normal and alk gl − females failed to complete development. Injection of poison gland contents into a host parasitized by poi gl − females allowed normal larval development of the parasitoid. These results suggest that the poison gland material and the calyx fluid act synergistically to regulate the growth of parasitized H. virescens larvae. It is speculated that the reason for abnormal development of the larval parasitoids from poi gl − females is because of the lack of control of the host development in the absence of the poison gland material.

Infection of the red imported fire ant by Beauveria bassiana through various routes of exposure

Abstract The entomophagous fungus, Beauveria bassiana, is a potential biological control agent of the red imported fire ant, Solenopsis invicta, but the best method of exposing the colony to the pathogen is unknown. Different application methods revealed that immersion of adult ants in a conidial suspension was more effective and practical than spraying. Although B. bassiana is capable of penetrating insect cuticle, there was little evidence that infection occurred by the penetration of the cuticle of most areas of the ants body, with the exception of the tarsi and by oral routes. Scanning electron microscopy revealed the presence of conidia, many of which were germinating on the tarsi suggesting this as a possible site of penetration. Another route of infection was found through the use of conidia formulated in baits. Liquid baits containing conidia were more effective in causing mortality among workers, whereas solid baits containing conidia were more effective in causing mortality among brood. Through dissections, microscopic examinations, and the use of a culture method to reveal the presence of viable fungus, adult workers were found capable of filtering the conidia from liquid. While germinating conidia were found in the crops of large major workers that had ingested conidia-containing liquid baits, most workers filter out the conidia and place them in their buccal cavity, which appears to a source of fungal invasion. The contamination of soil with conidia did not cause the ants to abandon the soil as a nest media nor was increased ant mortality observed. Infected ants were removed by nest mates from the colony and placed on the refuse pile prior to possible fungal sporulation and where dry conditions further reduced sporulation. The removal of infected colony members prior to fungal sporulation and subsequent reduced sporulation may reduce colony reinfection.