Mucosal delivery of native and recombinant protein vaccines against Trichostrongylus colubriformis

This paper describes a series of five pilot trials to test the feasibility of inducing a protective mucosal immune response against a non-blood-feeding intestinal nematode by delivery of antigens across the mucosal epithelium. A number of antigen preparations from Trichostrongylus colubriformis (viable larvae, larval homogenate and recombinant 17 kDa excretory-secretory protein) were delivered to the luminal surface of the mucosal epithelium overlying jejunal or rectal lymphoid tissue in cellulose or chitosan formulations. Significant protection was induced following delivery of viable larvae, larval homogenate or recombinant protein to the epithelium overlying rectal Peyer’s patches, and recombinant protein to the epithelium overlying jejunal Peyer’s patches. Viable larvae were associated with a jejunal IgE/IgG1 response, while the 17 kDa antigen was associated with a jejunal IgA response. The results demonstrate that delivery of Trichostrongylus native and recombinant antigens across the epithelium overlying rectal lymphoid patches can result in significant protective immunity even in the absence of adjuvant. They warrant the further investigation of appropriate mucosal delivery methods and adjuvants for induction of protective mucosal responses to stages and species of gastrointestinal helminths which do not ingest serum antibodies.     

Detection and semi-quantification of Strongylus vulgaris DNA in equine faeces by real-time quantitative PCR

Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available to differentiate among strongyle species in a faecal sample is by identifying individual L3 larvae following a two week coproculture procedure. The aim of the present study is to overcome this diagnostic obstacle by developing a fluorescence-based quantitative PCR assay capable of identifying S. vulgaris eggs in faecal samples from horses. Species-specific primers and a TaqMan probe were designed by alignment of published ribosomal DNA sequences of the second internal transcribed spacer of cyathostomin and Strongylus spp. nematodes. The assay was tested for specificity and optimized using genomic DNA extracted from identified male worms of Strongylus and cyathostomin species. In addition, eggs were collected from adult female worms and used to evaluate the quantitative potential of the assay. Statistically significant linear relationships were found between egg numbers and cycle of threshold (Ct) values. PCR results were unaffected by the presence of cyathostomin DNA in the sample and there was no indication of PCR inhibition by faecal sources. A field evaluation on faecal samples obtained from four Danish horse farms revealed a good agreement with the traditional larval culture (kappa-value=0.78), but with a significantly higher performance of the PCR assay. An association between Ct values and S. vulgaris larval counts was statistically significant. The present assay can reliably and semi-quantitatively detect minute quantities of S. vulgaris eggs in faecal samples.     

Therapeutic Potential of Myrrh and Ivermectin against Experimental Trichinella spiralis Infection in Mice

Trichinosis is a parasitic zoonosis caused by the nematode Trichinella spiralis. Anthelmintics are used to eliminate intestinal adults as well as tissue-migrating and encysted larvae. This study aimed to investigate the effects of ivermectin and myrrh obtained from the aloe-gum resin of Commiphora molmol on experimental trichinosis. Ninety albino mice were orally infected with 300 T. spiralis larvae. Drugs were tested against adult worms at day 0 and day 5 and against encysted larvae on day 15 and day 35 post-infection (PI). Mature worms and encysted larvae were counted in addition to histopathological examination of muscle specimens. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, albumin, globulin, urea, and creatinine values were estimated. Significant reductions in mean worm numbers were detected in ivermectin treated mice at day 0 and day 5 PI achieving efficacies of 98.5% and 80.0%, while efficacies of myrrh in treated mice were 80.7% and 51.5%, respectively. At days 15 and 35 post-infection, ivermectin induced significant reduction in encysted larval counts achieving efficacies of 76.5% and 54.0%, respectively, while myrrh efficacies were 76.6% and 35.0%, respectively. AST, ALT, urea, and creatinine levels were reduced, while total proteins were increased in response to both treatments compared to their values in the infected non-treated mice. Ivermectin use for controlling T. spiralis could be continued. Myrrh was effective and could be a promising drug against the Egyptian strains of T. spiralis with results nearly comparable to ivermectin.     

Characterisation of IgG(T) serum antibody responses to two larval antigen complexes in horses naturally- or experimentally-infected with cyathostomins

Cyathostomins are the most common parasitic nematodes of horses. Larval stages, which inhabit the intestinal wall, are particularly pathogenic and can cause severe colitis and colic. Despite their clinical importance, diagnostic techniques for the prepatent stages do not exist. A method that could estimate mucosal infection intensity would have a major impact on the control and diagnosis of cyathostominosis. Here, serum IgG(T) responses to two larval antigen complexes of 25 and 20 kDa were quantified in horses with experimental infections, natural infections and in horses that presented with clinical larval cyathostominosis. In experimentally-infected animals, anti-25 kDa complex IgG(T) levels correlated positively with field exposure and with early third stage larval (r(s)=0.74, P=0.015) and total mucosal parasite (r(s)=0.78, P=0.010) burdens. In naturally exposed horses whose parasite burdens were quantified upon post-mortem examination, antigen-specific IgG(T) responses were significantly higher in infected than in uninfected horses (P=0.0001 and 0.002, for anti-25 and anti-20 kDa responses, respectively). In these animals, anti-25 kDa IgG(T) levels correlated positively with mucosal and lumenal burdens (P<0.05). IgG(T) responses to the 20 kDa antigen complex correlated positively with lumenal burdens (P=0.0043). In cases of larval cyathostominosis, antigen-specific IgG(T) levels were significantly higher than in uninfected ponies (P=0.002 and 0.0035, for anti-25 and anti-20 kDa responses, respectively). These results provide evidence that these two complexes contain antigens with potential as markers for prepatent cyathostomin infection.     

Assessing the Potential of Entomopathogenic Nematodes to Control the Grape Root Borer Vitacea polistiformis (Lepidoptera: Sesiidae) Through Laboratory and Greenhouse Bioassays

Seventeen entomopathogenic nematode species and strains were evaluated for virulence to the grape root borer, Vitacea polistiformis (Harris) in laboratory and greenhouse bioassays. Heterohabditis bacteriophora strain GPS11 and H. zealandica strain X1 produced a larval mortality rate of over 85% of larvae embedded in the root cambium in laboratory bioassays. The nematode species H. marelata and H. bacteriophora strain Oswego produced mortality rates of over 75%. Of the Steinernema species tested, S. carpocapsae strain 'All' performed the best with a mortality rate of 69%. All other nematode species and strains tested, with the exception of S. bicornutum , produced some degree of larval mortality. In the greenhouse bioassays, 93% control was achieved with H. zealandica strain X1 applied at 4 ×109 infective juveniles (IJs) acre1 -1 (9.88 ×10 9 IJs ha -1 ). H. bacteriophora strain GPS11 successfully reproduced in grape root borer larvae. The numbers of IJs produced within infected larvae were related to larval size. The survival rate of neonate larvae on grape root sections was 61%, which thus provides a means to rear the neonate larvae for bioassays.     

Faecal egg counts and nemabiome metabarcoding highlight the genomic complexity of equine cyathostomin communities and provide insight into their dynamics in a Scottish native pony herd

Understanding the composition of gastrointestinal nematode communities may help to mitigate or exploit parasite adaptations within their host. We have used nemabiome deep amplicon sequencing of internal transcribed spacer-2 (ITS-2) ribosomal DNA to describe the temporal and host species composition of gastrointestinal nematode communities following sampling of six Scottish ponies across 57 months. In the absence of parasite control, each horse showed seasonal trends of increases and decreases in faecal egg counts, consistent with the epidemiology of equine strongylid parasites, however, the composition of parasites within individuals changed over time. Sixteen presumptive strongylid species were identified in each of the horses, 13 of which were distributed in a complex clade together with small numbers of amplicon sequences which could not be classified beyond the Cyathostominae subfamily level. Egg shedding of seven trichostrongylid species, which had previously been identified in co-grazed Soay sheep, was identified during the early spring. Faecal egg counts and the percentage of amplicon sequences assigned to each gastrointestinal nematode species were combined to describe their relative abundance across both host and time. Significant differences in species diversity between horses and between months were observed, being greatest from March to May and least from October to December. The magnitude of the individual horse effect varied between months and, conversely, the magnitude of the seasonal effect varied between individual horses. The most abundant gastrointestinal nematode in each of the horses was Cylicostephanus longibursatus (46.6% overall), while the abundance of the other strongylid species varied between horses and relative to each other. Patent C. longibursatus infections over the winter months might represent a genetic adaptation towards longer adult worm survival, or a lower rate of developmental arrest in the autumn. This study provides insight into highly complex phylogenetic relationships between closely related cyathostomin species; and describes the dynamics of egg shedding and pasture contamination of co-infecting equine gastrointestinal nematode communities. The results could be applied to determine how climatic and management factors affect the equilibrium between hosts and their parasites, and to inform the development of sustainable gastrointestinal nematode control strategies for different host species.     

Description and genetic characterisation of Hysterothylacium (Nematoda: Raphidascarididae) larvae parasitic in Australian marine fishes

Nematodes belonging to the genus Hysterothylacium (family Raphidascarididae) infect various species of marine fish in both the larval and adult stages. Humans can be accidentally infected upon eating infected seafood. In spite of their importance, relatively little is known of their occurrence and systematics in Australia. An examination of various species of marine teleosts in Australian waters revealed a high prevalence of Hysterothylacium larval types. In the present study, seven previously undescribed Hysterothylacium larval morphotypes (V to VII and IX to XII) were discovered. In total we found 10 different morphotypes and we genetically characterised nine morphotypes identified. A morphological dichotomous identification key has been established to differentiate these morphotypes. Since some larvae of Hysterothylacium from marine fishes cannot be differentiated morphologically from other nematode larvae, such as Paraheterotyphlum, Heterotyphlum, Iheringascaris and Lapetascaris, the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of these larvae were characterised to confirm their taxonomic status. This genetic characterisation implied that some distinct morphotypes belong to different developmental stages of the same species. In addition, it revealed that some morphotypes can comprise distinct genotypes. No match was found between ITS-1 and ITS-2 sequences obtained from larvae in the present study and those from adults available in the GenBank, highlighting the lack of knowledge on occurrence of adult nematodes infecting Australian fish.     

In vitro bioassay of sheep gastrointestinal mucus for nematode paralysing activity mediated by substances with some properties characteristic of SRS-A

A bioassay is described for determining the inhibition of nematode larval migration from agar by substances exerting a paralysing action.In the assay, larval migration was completely inhibited by the anthelmintic levamisole (25 μg/ml) whereas biogenic amines, and prostaglandins E₁ and E₂ at 50 μg/ml, were without effect. Mucus from the gastrointestinal tract of sheep resistant to nematode infection inhibited larval migration by up to 93% whereas mucus from heavily infected sheep or sheep reared helminth free did not significantly inhibit larval migration. Mucus from sheep resistant to Trichostrongylus colubriformis inhibited the migration of larvae of other nematode species.The larval migration inhibitory (LMI) activity of mucus from resistant sheep was associated with components having some properties of slow reacting substance of anaphylaxis (SRS-A).Faecal samples from resistant sheep possessed significantly more LMI activity than faecal samples from heavily infected sheep or sheep reared helminth free. The level of LMI activity in the faeces of sheep undergoing challenge infection may be a useful indicator of the sheep's resistance status.The presence of the larval migration inhibitory activity in sheep mucus is discussed in relation to resistance to infection.     

Heterorhabditis sp. (Nematoda: Heterorhabditidae): A Nematode Parasite Isolated from the Banded Cucumber Beetle Diabrotica balteata

A nematode identified as Heterorhabditis sp. was discovered in June 1982 in larval cadavers of the banded cucumber beetle, Diabrotica balteata, in soil on wooded land. Effective beetle control (over 95%) was obtained when larvae were exposed to potted soil containing infective stage nematode juveniles or infected larval cadavers. The nematode was propagated in vivo on larvae of D. balteata, Diaphania nitidalis (the pickleworm), and Galleria mellonella (the greater wax moth). This Heterorhabditis sp. has promising potential as a biocontrol agent for the banded cucumber beetle.     

Shortened egg reappearance periods of equine cyathostomins following ivermectin or moxidectin treatment: morphological and molecular investigation of efficacy and species composition

Macrocyclic lactones have been the most widely used drugs for equine parasite control during the past four decades. Unlike ivermectin, moxidectin exhibits efficacy against encysted cyathostomin larvae, and is reported to have persistent efficacy with substantially longer egg reappearance periods. However, shortened egg reappearance periods have been reported recently for both macrocyclic lactones, and these findings have raised several questions: (i) are egg reappearance period patterns different after ivermectin or moxidectin treatment? (ii) Are shortened egg reappearance periods associated with certain cyathostomin species or stages? (iii) How does moxidectin’s larvicidal efficacy affect egg reappearance period? To address these questions, 36 horses at pasture, aged 2–5 years old, were randomly allocated to three treatment groups: 1, moxidectin; 2, ivermectin; and 3, untreated control. Strongylid fecal egg counts were measured on a weekly basis, and the egg reappearance period was 5 weeks for both compounds. Strongylid worm counts were determined for all horses: 18 were necropsied at 2 weeks post-treatment (PT), and the remaining 18 at 5 weeks PT. Worms were identified to species morphologically and by internal transcribed spacer-2 (ITS-2) rDNA metabarcoding. Moxidectin and ivermectin were 99.9% and 99.7% efficacious against adults at 2 weeks post treatment, whereas the respective efficacies against luminal L4s were 84.3% and 69.7%. At 5 weeks PT, adulticidal efficacy was 88.3% and 57.6% for moxidectin and ivermectin, respectively, while the efficacy against luminal L4s was 0% for both drugs. Moxidectin reduced early L3 counts by 18.1% and 8.0% at 2 or 5 weeks, while the efficacies against late L3s and mucosal L4s were 60.4% and 21.2% at the same intervals, respectively. The luminal L4s surviving ivermectin treatment were predominantly Cylicocyclus (Cyc.) insigne. The ITS-2 rDNA metabarcoding was in good agreement with morphologic species estimates but suggested differential activity between moxidectin and ivermectin for several species, most notably Cyc. insigne and Cylicocyclus nassatus. This study was a comprehensive investigation of current macrocyclic lactone efficacy patterns and provided important insight into potential mechanisms behind shortened egg reappearance periods.     

Anthelmintic therapy of equine cyathostomin nematodes – larvicidal efficacy,egg reappearance period,and drug resistance

Cyathostomins are ubiquitous in grazing horses across the world, and anthelmintic resistance has been reported with increasing levels over past decades. The aims of the present study were (i) to investigate the efficacy against encysted larval stages of moxidectin (0.4?mg/kg) and fenbendazole (10?mg/kg daily for five consecutive days) and compare these regimens at 2 and 5?weeks post-treatment, (ii) to investigate individual cyathostomin species associated with shortened egg reappearance periods, and (iii) to document species exhibiting decreased susceptibility to the evaluated compounds. Thirty-six ponies were allocated to treatment groups with half euthanatized 2?weeks post-treatment, and the remainder necropsied after 5?weeks. Luminal and mucosal worm counts were conducted and strongyle egg counts were determined at weekly intervals. At 2?weeks, mean reductions of early L3s were 50.4% and 73.8% for fenbendazole and moxidectin, respectively. At 5?weeks, the respective efficacies were 51.3% and 71.8%. Two week efficacies against late L3s and L4s (LL3s/L4s) were 70.8% and 74.6% for fenbendazole and moxidectin, respectively, whereas very low numbers were found in all three groups at 5?weeks. None of the mucosal counts were significantly different between treatment groups. Fenbendazole and moxidectin reduced luminal worm counts by 93.2% and 98.3% at 2?weeks following administration, with moxidectin group adult counts being significantly lower than the other two groups (P?<?0.0001). Both treatment groups had increased counts 3?weeks later (P?=?0.0415). A moxidectin ERP of 4?weeks was associated with surviving luminal L4s, and adult species contributing to this were Cyathostomum catinatum, Cylicostephanus longibursatus, Cylicocyclus ashworthi and Cylicocyclus nassatus. This study documented (i) larvicidal efficacy of fenbendazole much lower than historical standards, (ii) survival of luminal immatures (L4) following moxidectin administration, and (iii) new information about cyathostomin species associated with these phenomena.     

Susceptibility of the Carrot Weevil (Coleoptera: Curculionidae) to Steinernema feltiae,S. bibionis,and Heterorhabditis heliothidis

Larvae, pupae, and adults of the carrot weevil (Listronotus oregonensis) were infected and killed by the three entomophagous nematodes (Steinernema feltiae, S. bibionis, and Heterorhabditis heliothidis) under controlled conditions. Third-stage larvae were more susceptible than pupae or adults. S. feltiae and S. bibionis were the most aggressive nematode species, causing larval mortality after 24-48 hours in both continuous and 2-hour contact with nematode suspension. The nematodes multiplied sufficiently in all insects at all stages of development; however, production of infective-stage larvae per host cadaver was variable.     

The location of parasites within their hosts: Factors affecting longitudinal distribution of Trichinella spiralis in the small intestine of mice

Inocula of encysted larvae, excysted larvae or infected meat all resulted in aggregated distributions of adults in the anterior small intestine. However, implantation studies indicated that the larvae could establish anywhere between the duodenum and the colon. The site of larval establishment determines the subsequent location of adult T. spiralis; the site of establishment was due largely to influences such as intestinal motility, size of inoculum and the size of the vehicle of infection. Predominantly female inocula were dispersed similarly to heterosexual inocula while male populations were dispersed more widely.     

Nematode larvae (order Spirurida) in gastric tissues of Australian anurans: a comparison between the introduced cane toad and sympatric native frogs

The outcomes of host-parasite interactions depend heavily on the host's immune response, which, in turn, is governed by previous interactions between the host and parasite, both over the host's life time and over evolutionary time. In the case of species introductions, such as the cane toad (Bufo marinus) to Australia, parasites that are benign to native species of the introduced range may present a major challenge to the introduced species. Stomachs of introduced cane toads and seven species of sympatric native frogs were examined for parasites, and their pathology and biology were compared. Cane toads were host to eight species of third-stage spirurid larvae, six of which also occurred in the stomach wall of four native frog species. In general, encysted nematode larvae attained higher prevalence and species richness in introduced cane toads than in sympatric native frogs. This trend was largely explained by differences in body sizes: larger anurans were more likely to possess infections, and cane toads are inherently larger than native frogs. Encysted larvae in cane toad stomachs provoked a marked pathologic response. All larvae (physalopterine and Physocephalus spp.) were surrounded by concentric layers of dense, fibrous tissue, with considerable cellular infiltration characterized by lymphocytes and polymorphs. Many cysts were invaded by cells and exudate, which, in more advanced cases, became calcified. Some larvae appeared viable; most were in various stages of destruction, and some smaller Physocephalus spp. were mummified. Conversely, pathologic response observed in native frogs was minimal, with little fibrotic reaction surrounding the cysts, and no cellular infiltration. Presumably, the contrast in pathology between introduced and native hosts reflects the long evolutionary association between these nematode larvae and native frogs, whereas the recent exposure of introduced toads to these helminths provokes a severe reaction.     

Secretion of anti-parasite substances and leukotrienes from ovine gastrointestinal tissues and isolated mucosal mast cells

The presence of larval migration inhibitory (LMI) compounds in the gastrointestinal mucus of nematode resistant sheep has been shown previously to be associated with increased numbers of gastrointestinal mucosal mast cells (MMC) and globule leukocytes (GL). This experiment was designed to determine if LMI compounds were secreted by MMC/GL in response to nematode antigenic challenge and if so, could secretion account for levels observed in mucus. Rommey sheep were immunized by repeated cycles of infection with Trichostrongylus colubriformis or Haemonchus contortus larvae and anthelmintic treatment. After slaughter, gastrointestinal tissue was taken for examination of histology and mucus anti-parasite activity. Segments of small intestine were ligatured to form sacs which were incubated with exsheathed nematode larvae or larval excretory/secretory antigens. Tissue slices from small intestine or abomasum were also incubated with nematode larvae or antigens. After homologous challenge, levels of leukotrienes secreted into small intestinal tissue sacs were significantly higher than levels in heterologously challenged sacs or unimmunized sheep intestinal sacs challenged with larvae of any nematode species (279.4±33.7, 141.0±27.8 and 39.5±15.2 ng h⁻¹ respectively). Tissue slices gave a similar pattern of leukotriene secretion. LMI activity was also significantly elevated in intestinal sacs from immunized sheep challenged homologously with nematode larvae or antigen (64±10 and 68±14% respectively cf. heterologous challenge 32±10% and unimmunized sheep sacs 15±6%). Histological examination of abomasal and small intestinal sections showed that immunized sheep had significantly greater numbers of MMC/GL than unimmunized sheep. MMC/GL isolated and purified from immunized sheep secreted leukotrienes and compounds having LMI activity when cultured with homologous nematode larvae or antigens. Secretion of leukotrienes and molecules having LMI activity from MMC/GL could account for the levels of these substances observed in small intestinal mucus.     

Cloning and preliminary characterization of a novel cuticular antigen from the filarial parasite Dirofilaria immitis

We have described here the cloning and partial characterization of a cDNA encoding a cuticular antigen of Dirofilaria immitis. A 48-h third-stage larval D. immitis cDNA library was immunoscreened with sera raised in mice against third-stage larval cuticles (mouse anti-L3 cuticle antisera). A strongly immunoreactive clone (L3MC4) was isolated. Sequence analysis of L3MC4 showed that it was a partial length cDNA. The missing 5′ end of the clone was amplified by PCR from D. immitis adult female first-strand cDNA using the nematode 22-base splice leader sequence and a L3MC4-specific antisense primer. The composite cDNA sequence comprised 616 bases (nDiL3MC4) encoding a full-length protein of 146 amino acids (DiL3MC4). GenBank analysis showed that DiL3MC4 shared some homology to an unknown C. elegans gene product (31%) at the amino acid level. However, there were no related filarial expressed sequence tags in the current GenBank™ database. Antibodies to recombinant DiL3MC4 (rDiL3MC4) identified a 19-kDa native antigen in the adults and in the L3 and L4 larval stages of D. immitis. In addition, the antibodies bound to the cortical layers of the L3 cuticle, as revealed by immuno-gold electron microscopy. The native protein was not detected in larval and adult excretory–secretory products. Immunoblot analysis showed that serum from a rabbit that was repeatedly injected with a small number of D. immitis third stage larvae reacted with rDiL3MC4. Thus, DiL3MC4 is a novel cuticular antigen of a filarial parasite.     

Role of oligosaccharides in the immune response of sheep vaccinated with Lucilia cuprina larval glycoprotein, peritrophin-95

The larvae of the fly Lucilia cuprina cause a cutaneous myiasis in mammalian hosts, particularly sheep. The glycoprotein, peritrophin-95, isolated from Lucilia cuprina larval peritrophic matrix, is a candidate vaccine antigen. This protein induced an immune response in vaccinated sheep that inhibited larval growth. Recombinant forms of peritrophin-95 were produced in bacteria and baculovirus-infected insect cells. The bacterial protein was not glycosylated and incorrectly folded whereas the insect cell-expressed protein was glycosylated and probably correctly folded. Sheep immunised with purified native peritrophin-95 generated strong larval growth inhibitory activity in their sera, whereas sheep immunised with either recombinant form of peritrophin-95 generated only relatively weak inhibitory activity. Ingested ovine antibodies to native peritrophin-95 mediated the anti-larval growth activity and this was independent of the presence of ovine complement. The activity was associated with IgG(1) and IgG(2) but not IgM. There were strong antibody responses to both the correctly folded native peritrophin-95 polypeptide and the oligosaccharides present on this glycoprotein. Immuno-affinity isolation of antibody to the peritrophin-95 polypeptide and antibody to peritrophin-95 oligosaccharides demonstrated that the larval growth inhibitory activity resided with both antibodies. Lectin blots and ELISA data showed substantial differences between the oligosaccharides attached to native peritrophin-95 and insect cell-expressed recombinant peritrophin-95. It was concluded that the oligosaccharides attached to native peritrophin-95 and its unique polypeptide structure are essential for the induction of larval growth inhibitory activity in the sera of sheep vaccinated with this antigen.     

Control of Codling Moth,Cydia pomonella (Lepidoptera: Tortricidae), with Steinernema carpocapsae: Effects of Supplemental Wetting and Pupation Site on Infection Rate

Infection of cocooned codling moth (cydia pomonella) larvae by the entomopathogenic nematode Steinernema carpocapsae was studied in three field experiments. Factors that varied within or between experiments included method of application, type of substrate containing cocooned larvae, time when nematodes were applied, seasonal effects, and supplemental wetting before or after nematode application. Conventional air-blast sprayer applications of 0.5–5.0 million infective juveniles (IJs)/tree in fall resulted in ca. 30% mortality of larvae in cardboard trap bands, whereas hand-gun application (2 million IJs/tree) produced mortality of ca. 70%. Application in the evening caused higher larval mortality than application in the morning when no supplemental wetting was used after treatments. Morning and evening applications caused equivalent larval mortality when a postwetting treatment was included. In a trial conducted in midsummer, supplemental wetting, either before or after hand-gun application of 1 million IJs/tree, enhanced nematode-produced mortality. Mortality approached 100% if both pre- and postwetting was used. Larvae in exposed cocoons on apple wood were infected at a higher rate (86%) than those on wood in less exposed positions (73%) or in nonperforated cardboard (72%). Mortality rates for larvae in perforated cardboard were intermediate (77%). Application volumes used to deliver nematodes slightly enhanced infection rate of larvae in some substrates but not others. In one trial, parasitism of codling moth by the wasp Mastrus ridibundus (Ichneumonidae) was negatively correlated with nematode infection of codling moth larvae. Dissections showed that ca. 10% of larvae infected by nematodes had been attacked by the wasp.     

The effect of parasitism by the mermithid Romanomermis culicivorax on the dry weight and hemolymph soluble protein content of three species of mosquitoes

The dry weight, hemolymph soluble protein composition, and content of three species of mosquitoes, Culex pipiens, Aedes taeniorhynchus, and Anopheles quadrimaculatus were examined to determine the effects of parasitism by the mermithid nematode Romanomermis culicivorax. The dry weights of infected fourth-stage larvae of all three species were significantly lower than controls. The differences in weight found between infected early and late C. pipiens and A. quadrimaculatus larvae were attributed to the weight of the parasite itself. This difference was not noticeable in A. taeniorhynchus larvae. Hemolymph proteins were severely depleted in all three mosquito species during parasitism by R. culicivorax. Analysis of protein composition by PAGE showed that these depletions were accompanied by a reduction in the number of proteins. Differences between protein composition concentrations were evident between early and late fourthstage control larvae of C. pipiens and A. quadrimaculatus. The concentration of some low-molecular-weight proteins (below 68,000) remained constant between infected and control samples of all three mosquito species.