Life cycle and mode of infection of Leptolegnia chapmanii (Oomycetes) parasitizing Aedes aegypti

The life cycle and mode of infection of mosquito larvae by Leptolegnia chapmanii (Oomycetes: Saprolegniales) were determined. The life cycle is typical of saprolegniaceous fungi, as the species is dimorphic producing diplanetic biflagellate zoospores. Sexual reproduction is by means of gametangial contact and results in the production of a characteristic papillate oogonium containing a subcentric or eccentric oospore. L. chapmanii is capable of infecting Aedes aegypti larvae both by germination of encysted secondary zoospores on the exterior cuticle and by germination of ingested zoospore cysts in the larval midgut. Once the fungus is established in the host, the disease, a coelomomycosis, is fatal. The encystment pattern of secondary zoospores on the larval cuticle appcars preferential. Scanning electron microscopy indicates that mechanical pressure is not the sole force utilized by the fungus for cuticle penetration.     

Histological and ultrastructural studies of Beauveria bassiana infection in Leptinotarsa decemlineta larvae during ecdysis

Studies of Leptinotarsa decemlineata larvae infected by Beauveria bassiana during ecdysis have enabled us to define the modes of fungal penetration employed to enter the ecdysial cuticle. We have observed the mechanically active passage of the penetrant hyphae and have followed the growth of the filaments and blastospore formation in the molting fluid. The attack of the new integument and its consequent alteration and the entry into the body cavity have also been studied. The infection develops rapidly in some of the larvae which die in premolt, while others are able to molt. Conditions rendered abnormal due to the presence of the fungus cause integumentary injuries which serve as an important factor in pathogenesis since they enhance the entry of fungal elements and bacteria thereby inducing septicemia. Contaminated larvae are able to molt, showing no signs of injury or disease, and survive for a long time, until the fungus finally invades the organism and causes death. This postponement of mortality shows that molting and hemocytic reactions are, to a certain extent, an effective defense mechanism. These last observations can be useful in the understanding of pathological processes associated with a hidden phase of fungal infection.     

Regulation of Haemonchus contortus populations in sheep exposed to continuous infection

Groups of sheep were infected three times weekly for 15 weeks with infective larvae of Haemonchus contortus at four rates ranging from 600 to 4800 larvae per week. At 3-weekly intervals, sheep from each group were killed for total worm counts after receiving a dose of radiolabelled larvae which enabled a direct measurement of establishment of incoming larvae during the continuing infection. Peak H. contortus burdens were reached between 6 and 9 weeks of infection, and were related to infection rate. In the groups receiving 2400 and 4800 larvae per week, worm numbers then declined rapidly and by the end of the experiment were lower than those in the groups given 600 or 1200 larvae per week. Establishment of incoming larvae was not influenced by infection rate, and declined from 45% in the first 4 weeks of infection to insignificant levels during the final 6 weeks. The proportion of incoming larvae arrested in their development increased as the infection progressed. It was concluded that H. contortus numbers were regulated by development of resistance to infection and by a loss of established worms which was related to the current rate of larval intake, and to the host's previous experience of infection.     

Pathogenesis of infection by the hyphomycetous fungus,Tolypocladium cylindrosporum inAedes sierrensis andCulex tarsalis [Dip.: Culicidae]

The mode of infection and cycle of development ofTolypocladium cylindrosporum Gams was examined inAedes sierrensis andCulex tarsalis. Larvae were found to be infected through the external cuticle, the pharynx and the midgut. Blastospores and conidia were both infective although for equal numerical concentrations blastospores proved more virulent causing high mortality within the first 48 h after inoculation (80 % for L2 larvae exposed to 5×10⁵ spores/ml), while conidia generally took 7–10 days to produce the same results. Sporulation did not occur on submerged cadavers. Conidia were produced only on floating cadavers in contact with air. Conidial production on floating 4th instar larvae was found to average 1.8×10⁷ conidia/larva. Invasion of the haemocoele and fairly extensive growth of the fungus almost invariably occurred before larvae were killed. This was particularly true forAedes sierrensis larvae. Details are presented of growth within the host and post-mortem penetration of the fungus out of the cadaver. AdultA. sierrensis sprayed with a conidial suspension proved susceptible to infection with 100 % mortality being recorded at 10 days. Infections originated in the thorax, suggesting, the integument or possibly the thoracic spiracles to be the most probable site of infection.     

The differential mortality at various life stages of the greenhouse whitefly,Trialeurodes vaporariorum (Homoptera: Aleyrodidae), by infection with the fungus Aschersonia aleyrodis (Deuteromycotina: Coelomycetes)

The parasitoid Encarsia formosa is commonly applied to control the greenhouse whitefly Trialeurodes vaporariorum in glasshouse tomatoes and cucumbers. Nevertheless, in some cases the control capacity of this natural enemy is insufficient and an additional selective pest-suppressing agent is desirable. The entomopathogenic fungus Aschersonia aleyrodis was applied to cucumber plants carrying whiteflies in different developmental stages. After spraying each leaf with 2 ml of spore suspension (4 × 10⁶ spores/ml) the plants were kept at 100% RH for 24 hr; thereafter the humidity was lowered to 70% RH at 20°C and the photoperiod was 16 hr. Treated eggs did not become infected, but larvae that hatched from these eggs and settled on the treated abaxial leaf surface were infected at the same rate and to the same degree as treated first instar larvae. This suggests that the spores persist for at least 7 days. The final percentages of infection over all instars when treated as young eggs, old eggs, and first larval instars were 94, 93, and 90%, respectively. The final percentages of infection when treated as third and fourth larval instars and prepupae were 76, 28, and 12%, respectively. The older instars were less susceptible and adults were seldom infected by the fungus. Several applications of A. aleyrodis as a microbial insecticide are needed to achieve sufficient control of whitefly populations in glasshouses.     

Influence of primary infection on the population dynamics of Nematospiroides dubius after challenge infections in mice

Dobson C., Sitepu P. and Brindley P. J. 1985. Influence of primary infection on the population dynamics of Nematospiroides dubius after challenge infections in mice. International Journal for Parasitology15: 353–359. Similar proportions of the inoculum of Nematospiroides dubius larvae reached sexual maturity by 14 days after administration of 50–400 larvae but more adult worms had been expelled by day 63 after infection from those mice infected with 50 vs 400 larvae. There was a significant correlation between time and worm expulsion for all inoculum size groups except for mice given 400 larvae.In mice reinfected with 100 larvae, after termination of primary infections derived from 10 through 400 larvae, more worms from the challenging dose were recovered from mice given greater compared with those given smaller numbers of larvae at primary infection. The N. dubius population size after challenge infection was correlated positively both with number of larvae administered as the primary infection and with the resultant population size during that infection. The serum anti-N. dubius antibody titres after reinfection were higher in mice given 400 compared with those given fewer larvae at primary infection, and the fecundity and female to male sex ratio of the N. dubius populations decreased in proportion to these antibody titres.Protective immunity against challenge N. dubius infection, in mice which had been drenched free of adult worms established from 400 larvae for 5 down to 1 weeks before reinfection, increased from 45% (1 week) to 80% (5 weeks). There was a negative correlation between the population size of N. dubius during challenge infection and the duration between anthelmintic treatment and challenge infection.     

Percutaneous infection of Hylobius pales by Metarrhizium anisopliae

The manner in which Metarrhizium anisopliae infects larval and adult Hylobius pales was investigated histologically and by scanning electron microscopy. In the presence of fungal and bacterial contaminants on beetles, none or few conidia of M. anisopliae germinated. Antibiosis is suggested, since the inhibition could be eliminated by surface sterilization. On larvae, the contaminants were scarce, and spore germination was greater. With germination, the germ tubes typically grew extensively over the procuticle or sclerites before producing appressoria of various shapes and sizes. These appressoria consistently produced a light-transmissible mucoid substance. Conidia, germ tubes, and appressoria were frequently fused into infection cushions of random arrangement and size. Sclerites of larvae and adults were not penetrated by the fungus, whereas procuticle and metawings were.     

Histopathology of a nuclear polyhedrosis infection in Aedes epactius with observations in four additional mosquito species

Aedes epactius larvae were utilized to study the infection sequence of the nuclear polyhedrosis virus (NPV) from Aedes sollicitans. From 30 min to 6 hr postinoculation, polyhedra and many free virions were observed in the larval midgut lumen. Penetration of the midgut cells by virions was not observed. The first infected nuclei were observed 12 hr postinoculation. Nucleocapsids initially exhibited electron translucent cores which became electron dense before the nucleocapsids acquired an envelope. Envelope acquisition occurred through a process of de novo membrane morphogenesis. Occlusion of the singly embedded virions began by 18 hr postinoculation with the mature rough-surfaced polyhedra averaging approximately 1 by 2 μm. Unusually long nucleocapsids (approximately two or three times the length of other nucleocapsids) were only observed in late infection period nuclei. There was no evidence that long nucleocapsids represented an early developmental stage for nucleocapsids of standard length. Infection was restricted to midgut nuclei and gastric caecae cells. Infected early instar A. epactius larvae became moribund 36 to 40 hr postinoculation and infected midgut nuclei were observed to undergo lysis. The late stages of NPV infection were observed in larvae of A. annandalei, Wyeomyia smithii, Toxorhynchites brevipalpus, and Eretmapodites quinquevittatus. Virion development and occlusion in these species was basically identical to the sequence observed in A. epactius larvae.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

Interactions between a braconid,Microplitis croceipes,and a fungus,Nomuraea rileyi,in laboratory-reared bollworm larvae

The parasite Microplitis croceipes required 1.1 days longer at 26°C to complete development in Heliothis zea larvae than was required for the fungus Nomuraea rileyi to kill the host larvae and sporulate. Host larvae parasitized by M. croceipes or infected with N. rileyi failed to complete a fifth larval molt or pupate. Of the remaining healthy larvae, one-half completed six larval stadia before popation. Larvae parasitized by M. croceipes were predisposed to infection by N. rileyi, but the fungus inhibited development of M. croceipes if host larvae were infected with N. rileyi within 1 day after parasitization.     

Use of quantitative PCR to understand within-host competition between two entomopathogenic fungi

Interactions between the entomopathogenic fungi Zoophthora radicans and Pandora blunckii infecting larvae of Plutella xylostella were investigated. This is the first report to quantify within-host growth of one fungus in the presence of another competing for the same host resource using quantitative PCR (qPCR) at regular time points during the infection process. In larvae inoculated only with Z. radicans, there was a cumulative increase in the quantity of Z. radicans DNA throughout the time course of infection. However, in dual-inoculated larvae, there was an initial accelerated rate of growth of Z. radicans compared to when it was inoculated alone, but by the time of host death it had been effectively excluded by P. blunckii. The implications of these results for co-existence of these fungal pathogens in the field are discussed.     

In vivo events associated with entomopathology of Beauveria bassiana for the corn earworm (Heliothis zea)

When conidia of Beauveria bassiana are injected into the hemocoel of corn earworm larvae, it appears that a positive correlation exists between exocellular proteolytic activity of the fungus and entomopathological manifestations. Once inside the hemolymph, defense mechanisms (including phagocytosis) are incapable of overcoming the fungus and one important event in a terminal mycocidal cascade involves preferential invasion of the gut wall. Such invasion helps explain the observed inhibition of feeding by infected larvae. Although histopathological changes seen in gut tissues suggest that a gut toxin is produced, evidence for such a toxin could not be obtained in preliminary tests. Biochemical changes are seen in hemolymph components; however, these are viewed as being due to general starvation rather than to specific activities of the fungus, at least up to the time that a general mycosis is established. With the host larva under physiological stress (starvation, nutrient depletion, and, possibly, toxin production in gut tissues) and failure of defense mechanisms, the infection spreads quickly and a terminal mycosis results.     

Experimental laboratory infection of mosquito larvae with fungi of the genus Coelomomyces. II. Experiments with Coelomomyces punctatus in Anopheles quadrimaculatus

Experiments on the infection of Anopheles quadrimaculatus with Coelomomyces punctatus indicate that planonts released from sporangia are not infective for mosquito larvae but most likely infect the copepod Cyclops vernalis. Exposure of early-instar larvae to up to 5 × 10³ planonts per larva for as long as 48 hr resulted in no larval infections. Motile planonts were no longer detectable after 48 hr. However, incubation of early-instar larvae in media to which planonts, algae, and copepods had been added several days previously resulted in larval infection. Infection did not occur during the first 6 days after planont introduction. On day 7 and for several days thereafter, copepods were detected in the media which had an extensive mycelium developing in the hemocoel. This mycelium cleaved into thousands of posteriorly uniflagellate planonts. The presence of planonts at the time of mosquito infection, in conjunction with the above results, suggests that Cyclops vernalis is an alternate host for Coelomomyces punctatus and that the latter has a life cycle similar to that proposed for C. psorophorae involving a mosquito and a copepod as obligate alternate hosts. In established infection containers, dilution of the media with water significantly increased levels of infection 6 days later. All larval instars were susceptible to C. punctatus.     

Atkinsiella dubia infection in the larvae of Japanese mitten crab,Eriocheir japonicus

Heavy mortalities reaching 100% among larvae of the Japanese mitten crab,Eriocheir japonicus, occurred in Yamaguchi Prefectural Naikai Fisheries Experimental Station, Yamaguchi, Japan. Under the microscope, infected zoeal larvae were seen to be filled with numerous aseptate hyphae. An investigation was carried out to identify the pathogenic fungus and determine its pathogenicity under laboratory conditions. The pathogenic fungus was identified asAtkinsiella dubia. Its optimum growth temperature was 25°C, and it grew only at 2.5% NaCl. Under laboratory conditions, it showed pathogenicity to the larvae of the swimming crab,Portunus trituberculatus. This is the first report of mass mortality in crustaceans due toA. dubia infection in Japan.     

Regulation of infection by a nematode (Muellerius capillaris) in the snail Theba pisana

The regulation of infection by Muellerias capillaris was studied in three groups of Theba pisana. The regulation prior to penetration was very important for all the groups as assessed by the use of snail-conditioned water: a lytic activity was observed and the surviving larvae were less active than the control. The mortality rate of larvae after penetration was low in preinfected local group of snails, medium in the naive nonlocal group, and high in the naive local group.     

The mode of transmission of Tipula iridescent virus: I. Source of infection

A virus was isolated from a diseased tipulid larva and identified as Tipula iridescent virus (TIV) on the basis of the size and morphology of the virion, the production of iridescence in vitro and in infected tipulid larvae, and a serological reaction between antiserum against the virus and an isolate of TIV.A stock of Tipula oleracea was bred in the laboratory. Subjection of larvae to several stress factors did not result in any evidence for activation of a latent virus. Healthy T. oleracea larvae did not develop iridescence when confined in petri dishes with either live TIV-infected larvae or with large amounts of their feces, although these feces were found to contain infective virus by injecting extracts into healthy larvae. It appears that the concentration of virus in the feces of infected larvae is not high enough for them to serve as a source of infection. It was shown that the cadavers of TIV-infected larvae can serve as a source of infection for healthy first- and fourth-instar larvae.     

A mosquito parasite from a mosquito predator

The fungus Coelomomyces macleayae has been reported from treehole mosquito larvae of three Aedes subgenera in Australia, Fiji, and the United States. This fungus is now recorded for the first time from a mosquito of the genus Toxorhynchites, the large predatory larvae of which are of some importance in the naturalistic control of associated mosquito pests and vectors. A single parasitized larva of T. rutilus septentrionalis was collected from a magnolia treehole (previously used for A. triseriatus infection experiments) near West Lake, Louisiana, in September, 1971. Larval A. triseriatus and Orthopodomyia signifera were also present but were uninfected.     

Reduced parasitism of a leaf-mining moth on trees with high infection frequencies of an endophytic fungus

We investigated relationships between endophytic fungi and a leaf-mining moth, Phyllonorycter sp., along an elevational gradient from 2255 to 2895 m. The fungi and moth larvae inhabit leaves of Quercus gambelii. Fungal frequencies and larval densities varied with elevation. However, larval densities were not associated with the frequencies of infection by endophytic fungi. Survival of larvae was positively associated with the most dominant fungus, Gnomonia cerastis, owing to reduced parasitism of moth larvae on trees with high frequencies of Gnomonia.     

Influence of a baculovirus infection on molting and food consumption by Spodoptera litura

The effect of a baculovirus (nuclear polyhedrosis virus) infection on molting, neurosecretory activity, and food consumption of the tobacco leaf eating caterpillar. Spodoptera litura, is studied. The virus was administered by allowing the larvae to feed on castor leaves contaminated with purified polyhedral suspension (5.57 × 10⁸/ml). Host larvae failed to molt more than once after infection. Infected last-instar larvae failed to pupate and had prolonged larval stage. Activity of median neurosecretory cells (MNC) of brain increased during disease development. However, the larval—pupal molt was inhibited, possibly due to lack of release of neurosecretion from the MNC. The surface area of corpus allatum of last-instar larvae increased during infection indicating high activity. Infected larvae continued to feed almost until death. The indices of digestion and utilization were significantly lowered due to disease development.