Susceptibility of early larval stages of Pseudaletia unipuncta and Spodoptera exigua (Lepidoptera: Noctuidae) to the entomogenous nematode Steinernema feltiae (Rhabditida: Steinernematidae)

Early stages (neonate to 7- or 8-day-old larvae) of Spodoptera exigua and Pseudaletia unipuncta were exposed to the entomogenous nematode, Steinernema feltiae, at concentrations of 0, 10, 25, 60, 100, or 200 nematodes per larva. Larvae of both species were susceptible to nematode infections. However, neonate larvae of S. exigua were significantly less susceptible to nematode infection than 3- or 8-day-old larvae at or above 50 nematodes per larva. Mortalities of neonate larvae exposed to 50 or more nematodes ranged from 68 to 74% while mortalities of 3- and 8-day-old larvae ranged from 91 to 100%. The results with P. unipuncta showed similar trends as described for S. exigua, albeit at a lower mortality level and usually with no statistical differences. Mortalities of neonate larvae exposed to 50 or more nematodes ranged from 34 to 44% while mortalities of 7-day-old larvae ranged from 32 to 91%.     

Elaphostrongylus rangiferi: Influence of temperature,substrate, and larval age on the infection rate in the intermediate snail host, Aarianta arbustorum

The infection rate of the first stage larval nematodes, Elaphostrongylus rangiferi, was studied experimentally, using the juvenile snail Arianta arbustorum as intermediate host. The nematode showed a linear, fivefold increase in infection rate within the temperature range of 4 to 28 C. The snails were exposed to the larval nematodes on three different substrates. The highest infection rate was recorded when snails were exposed in tap water and significantly slower infection rates were obtained when either lettuce or soil was used as the substrate. First stage larvae of E. rangiferi were infective for at least 2 months when stored at 12 C. Throughout this period, the infection rate showed a significant decline, while the motility of the larvae remained unchanged.     

Infection of the Cat Flea, Ctenocephalides felis (Bouché) by Neoaplectana carpocapsae Weiser

Infection of cat flea, Ctenocephalides felis, larvae by the entomophilic nematode Neoaplectana carpocapsae was accomplished in the laboratory. The Breton strain of N. carpocapsae provided higher larval mortality at lower dosages than did the DD-136 strain. Adult nematodes were evident in the insect hemocoel after 48 h; however, no infective third-stage larvae were produced. Larval flea infection increased with an increase in the moisture content of sand from 2% to 7% and of sandy clay from 7% to 12%. Larval flea infection was also obtained on turf containing dauer larvae. Nematode penetration of cocoons with invasion of prepupal and pupal fleas was apparent.     

Parelaphostrongylus odocoilei: Life cycle in experimentally infected cervids including the mule deer, Odocoileus h. hemionus

The life cycle of a metastrongyloid nematode, Parelaphostrongylus odocoilei, was successfully completed in three members of the Cervidae: mule deer (Odocoileus h. hemionus), black-tailed deer (O. h. columbianus), and moose (Alces alces andersoni). The terrestrial gastropod, Triodopsis multilineata, was the experimental intermediate host. White-tailed deer (O. virginianus dacotensis) were refractory to infection. The prepatent period of P. odocoilei was significantly shorter in mule deer (X = 53 days) than in the black-tailed deer or moose. There was an inverse relationship between the size of the infective inoculum and the duration of the prepatent period of P. odocoilei in mule deer, but not in black-tailed deer. The duration and intensity of larval production of P. odocoilei were higher in mule deer than in the other hosts. Peak larval production in the feces (approximately 14,000 larvae/g) of mule deer was in excess of previous reports for elaphostrongyline nematodes, regardless of the size of the infective inoculum. Daily larval production, estimated at 3 to 4 × 10⁶ larvae/day, was six times higher than estimates for other elaphostrongylines. The duration of patency was not clearly established, but three mule deer and one black-tailed deer passed larvae for 12, 18, 24, and 18 months, respectively. On the basis of the reduced prepatent period and increased length and intensity of larval production, O. h. hemionus is considered the primary host of P. odocoilei.     

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 effect of infection with Dirofilaria immitis (dog heartworm) on fluid secretion rates in the Malpighian tubules of the mosquitoes Aedes taeniorhynchus and Anopheles quadrimaculatus

Dirofilaria immitis, the parasitic nematode causing the disease dog heartworm, is transmitted by female mosquitoes. During their development, larval nematodes reside in the cells of the Malpighian tubules of these mosquitoes for approx 13 days. We have examined the effect of the presence of these large intracellular parasites on the main physiological function of the Malpighian tubules, i.e. fluid secretion. Rates of fluid secretion were examined in vitro using both normal and infected tubules of the mosquito species Aedes taeniorhynchus and Anopheles quadrimaculatus. Tubules of A. quadrimaculatus show changes in trasport rate during the reproductive cycle. Those of A. taeniorhynchus do not. Infection with larvae of D. immitis had no effect on the rate of fluid secretion in tubules of A. quadrimaculatus. In A. taeniorhynchus by contrast, the tubules show decline in transport with time following infection. The reduction in transport capacity is proportional to the number of worms infecting the tubule. The present paper and separate ultrastructural studies demonstrate that parallel changes in microvillar ultrastructure and epithelial transport rates occur in response to infection by the parasite. In both species examined, the survival of the mosquitoes and their vector potential are determined by factors other than the transport capacities of the infected Malpighian tubules.     

The effect of nematode parasitism on the osmolality and major cation concentration in the haemolymph of three larval mosquito species

Parasitism by a mermithid nematode, Romanomermis culicivorax, causes severe depletion of haemolymph carbohydrates and proteins in mosquito larvae. We undertook a study to determine if haemolymph osmolality and cation concentrations were affected also by mermithid parasitism. The haemolymph osmolality of R. culicivorax-infected and control Aedes taeniorhynchus and Culex pipiens fourth-instar larvae was not significantly different. However, the haemolymph osmolality decreased significantly in infected Anopheles quadrimaculatus. Each mosquito species demonstrated significant alterations in the haemolymph concentration of at least one cation when infected although the cation concentrations affected differed for each species. The changes observed were statistically significant but the magnitude of change was not great. Overall, despite the severe nutritional burden of the mermithid nematode, these species of mosquito larvae can continue to maintain osmoregulation.     

Heterorhabditis bacteriophora as a vector for introducing its associated bacterium into the hemocoel of Galleria mellonella larvae

The nematode Heterorhabditis bacteriophora serves as a vector enabling its bacterial associate to reach the hemocoel of its host, the seventh-instar larva of Galleria mellonella. At 28.5°C, the LD₅₀s of the orally introduced nematode-bacterial complex and the intrahemocoelically injected bacteria are three to six nematodes and one to two cells, respectively.     

Observations on the biology of Octomyomermis muspratti, a nematode parasite of mosquitoes

The mosquito parasite Octomyomermis muspratti was able to infect mosquitoes in diluted sea water with conductivity readings of 4000 μmho/cm and in all dilutions of organically rich tree-hole water. In contrast, the infectivity of Romanomermis culicivorax, a more extensively studied species, was adversely affected in dilutions of sea water with conductivity of 1500 μmho/cm and no infections were observed at concentrations above 3000 μmho/cm. Also, R. culicivorax failed to infect hosts at any dilution of the tree-hole water. Diet was shown to have an effect on male-female sex ratios in developing O. muspratti, and this may be employed to enhance a better male-female ratio in laboratory cultures of this nematode. When cultures of O. muspratti were flooded every 3–4 months, they continued to produce infective-stage nematodes for up to 5 years. Also, cultures that were repeatedly flooded produced infective nematodes for more than 39 floodings.     

Bioassays for comparative infectivity of mermithid nematodes (Romanomermis iyengari,Romanomermis culicivorax and Strelkovimermis spiculatus) for culicine mosquito larvae

Two improved bioassays were developed to establish infectivity baselines for selection experiments using mermithid nematode variants. Comparative infectivity of Romanomermis iyengari, Romanomermis culicivorax and Strelkovimermis spiculatus using larvae of three mosquito spp. Aedes sierrensis, Aedes aegypti and Culex pipiens were evaluated with “plate” and “tray” bioassays at selected intensity of infections. Using the “plate” bioassay, single mosquito larvae were immersed in 2 ml of water within individual depressions of 12-well, polystyrene tissue culture plates. One, three, or five preparasitic juveniles (J2) were added to each well. In the “tray” bioassay, polyethylene trays containing 500 ml water and 100 mosquito larvae were exposed to 500 (5:1, nematode:insect host) or 1000 (10:1) J2s. Percentage infection (PINF, infectivity) and intensity of infection (IINF, #nematodes per infected larvae) number were determined only after emergence of post-parasitic J3 juveniles. Under the bioassay conditions, all three species of nematodes resulted in infections in all mosquito hosts, but R. iyengari exhibited better effectiveness in the parasitism of mosquito larvae. The three species of mosquitoes presented high levels of susceptibility to each of the three species of nematodes, but in general Cx. pipiens and Ae. sierrensis were slightly more susceptible than Ae. aegypti. The “plate” bioassay was more efficient in measurement of infectivity of the mermithid species and in establishing baseline characteristics for these mosquito-parasitic nematodes. The “tray” bioassay was an effective bioassay for large cohorts of both infective juveniles and host larvae and, potential for field interactions.     

Biological Control of the Leafminer Liriomyza trifolii (Burgess): Implications for Intraguild Predation between Diglyphus begini Ashmead and Steinernema carpocapsae (Weiser)

Studies were conducted on the combined use of the eulophid parasitoid wasp Diglyphus begini Ashmead and the entomopathogenic nematode Steinernema carpocapsae (Weiser) for control of the leafminer Liriomyza trifolii (Burgess) on chrysanthemums. Several factors indicated that these two agents were suitable for combined use: adult D. begini were not susceptible to nematode infection, leafminer larvae parasitized by the wasp were less susceptible to nematode infection, adult wasps detected and tended to avoid ovipositing on nematode-infected leafminer larvae, nematode-infected larvae served as host-feeding sources for the adult wasps, and nematodes showed equal orientation toward paralyzed/parasitized leafminer larvae and healthy leafminer larvae. However, interspecific interference and intraguild predation (IGP) between the agents were found. Infection of D. begini larval stages by nematodes was seen in petri dishes and in intact leaf mines. The presence of nematodes in mines with wasp eggs decreased the chance of wasp survival to adulthood. IGP may be minimized through proper timing of natural enemy releases.     

Culex pipiens affected by joint infection of a mosquito iridescent virus and Strelkovimermis spiculatus

Dual infections with a mosquito iridescent virus (MIV) and the mermithid nematode, Strelkovimermis spiculatus were recorded in natural Culex pipiens populations around La Plata city, Argentina. S. spiculatus was detected in 82% of samples that were positive for MIV infection. Dissected larvae of Cx. pipiens with patent MIV infection presented 42% infection with S. spiculatus. Larvae of Cx. pipiens exposed to MIV and S. spiculatus under laboratory conditions produced a high joint infection rate (82.5%) while no infection was recorded on larvae exposed to virus suspension only. Field and laboratory results suggest a strong association between S. spiculatus and MIV in natural populations of Cx. pipiens, in which S. spiculatus could be a mode of entry for the virus into the mosquito hemocele.     

Intracellular melanization of the larvae of Dirofilaria immitis in the malpighian tubules of the mosquito, Aedes sollicitans

Frequent melanization of larvae of the nematode Dirofilaria immitis parasitizing the Malpighian tubules of the mosquito, Aedes sollicitans, has been observed. Melanized and nonmelanized larvae in the Malpighian tubules were examined using light and electron microscopy. The results indicate that the pattern of melanin deposition and the ultrastructural characteristics of the pigment around the worms are identical to that observed on nematodes which have undergone humoral melanization in other dipteran insects. In the Malpighian tubules, no contact between the intracellular melanized nematodes and the hemolymph or hemocytes was observed. The results suggest that the Malpighian tubules of this species of mosquito are capable of inducing a melanotic response to invading nematode parasites. It is proposed that this is an example of “humoral” melanization at an intracellular site.     

Caenorhabditis elegans: Decay of isocitrate lyase during larval development

Changes in the levels of isocitrate lyase, malate synthase, catalase, fumarase, and NADP⁺-isocitrate dehydrogenase have been investigated during larval development of the free-living soil nematode Caenorhabditis elegans in the presence and absence of Escherichia coli. The specific activities of isocitrate lyase, malate synthase, and catalase are maximal at the time of egg hatching and, thereafter, decline during larval development when larvae feed on E. coli, whereas in the absence of E. coli specific activities of the same enzymes increase for 12 hr and subsequently remain constant. There is, however, no change in specific activity of fumarase or NADP⁺-isocitrate dehydrogenase during the same developmental period, in either case. Cycloheximide at 100 μM arrests the decline of isocitrate lyase during development of feeding larvae but has no effect upon the appearance of isocitrate lyase during starvation. The latter is true also for 15 mM itaconate. There is inactivation of isocitrate lyase in crude extracts of frozen worms in comparison to that in analogous extracts prepared from freshly harvested nematodes.     

Resistance of Cucumis spp. to the Root-knot Nematode,Meloidogyne incognita acrita

The nature of resistance in Cucumis ficifolius and C. metuliferus to the root-knot nematode, Meloidogyne incognita acrita, was studied under greenhouse conditions. Although as many larvae penetrated the roots of these species as those of the susceptible C. melo, few developed to the adult female stage. Resistance in C. ficifolius and C. metuliferus was associated with hindrance of larval development beyond the second stage, delayed development of larvae to adults and stimulation toward maleness. Tissue necrosis or hypersensitivity was not associated with larval penetration. Comparisons of the histopathology of 26-day-old infections of C. melo and C. metuliferus roots showed no observable differences in the type of giant cell development in regions of roots associated with adult females. However, in C. rnetuliferus immature nematodes were associated with small giant cells which were limited to a few cells near the head of the nematode.     

Histopathology of Nomuraea rileyi in Plathypena scabra larvae

Green cloverworm larvae. Plathypena scabra, were inoculated with Nomuraea rileyi by “tumbling” larvae in a vial of conidia. The ontogeny of the pathogen was followed by using standard histological techniques. N. rileyi conidia germinated on green cloverworm integument within 12 hr after inoculation. Germ tubes penetrated larval cuticle 36 hr after inoculation, then grew parallel to endocuticular laminae. After hyphal penetration of the epidermis ca. 4.5 days after inoculation, hyphal bodies were produced and were transported throughout the hemocoel. Hyphal bodies and hemocytes cohabited the hemocoel, but gut epithelial and muscle tissues were not invaded by Day 5. Hemocytes lysed and mycelia completely ramified throughout all larval tissues by 7 days after inoculation. Death of larvae was followed by conidiogenesis ca. 7.5 days after inoculation.     

Phocanema decipiens: Pathology in seals

Infective larvae of the anisakine nematode, Phocanema decipiens from cod (Gadus morhua), were fed to harbor seals (Phoca vitulina) and gray seals (Halichoerus grypus). At termination of the experimental infections, 6 of 8 harbor seals were infected with 34(6–64) P. decipiens, while the remaining 2 seals had relatively heavy infections of 250 and 547 nematodes, respectively; 10 of 11 gray seals were infected with 128 (68–229) P. decipiens, but only 2 nematodes were recovered from the 11th seal. Larvae and adults of P. decipiens occurred throughout the gastrointestinal tract, but mainly in the fundic portion of the stomach; the anterior extremities of the nematodes were embedded in mucosa and submucosa. Clusters of adult P. decipiens were associated with ulcerous gastric lesions in harbor seals and raised inflammatory areas in stomachs of gray seals. Singly occurring larvae from challenge transmissions were associated with raised inflammatory areas in the stomachs of both host species. Histological examinations revealed that the lesions and inflammatory areas were eosinophilic granulomata. Anisakis sp. larvae from the viscera of cod were also fed to 1 of the gray seals. Eighty of these larvae were subsequently found in association with a general inflammation in the cardiac area of the stomach in this seal. Natural anisakine infection were surveyed in 16 harbor and 53 gray seals from the Nova Scotia mainland. The natural incidence of P. decipiens was 62(5–177) in harbor seals and 577(11–1694) in gray seals. Clusters of adult P. decipiens were found in association with gastric lesions in 2 juvenile harbor seals; however, in gray seals, the nematodes neither occurred in clusters nor in association with gastric lesions.     

Control of Larval Northern Corn Rootworm. (Diabrotica barberi) with Two Steinernematid Nematode Species

The entomogenous nematodes Steinerema feltiae and S. bibionis did not penetrate the roots of corn, Zea mays, to infect larval northern corn rootworm (NCR), Diabrotica barberi, feeding within. Laboratory bioassays against first instar NCR indicated that S. feltiae, Mexican strain (LD₅₀ = 49 nematodes/insect) is more virulent than S. bibionis (LD₅₀ = 100). Numbers of NCR larvae in a grain corn crop were reduced by both nematode species applied at corn seeding time at the rate of 10,000 infective-stage juveniles per linear meter of corn row. The chemical insecticide fonofos provided significantly better control than either nematode species.     

Instar Susceptibility of Simulium vittatum (Diptera: Simuliidae) to the Entomogenous Nematode Neoaplectana carpocapsae

Laboratory bioassays showed that the susceptibility of Simulium vittatum to Neoaplectana carpocapsae increased with successive larval instars. First, second, and third instar larvae were resistant to infection, while seventh instars were highly susceptible. Significant differences in intra-instar susceptibility were also evident, as mortality ranged from 58% for the smallest seventh instar larvae to 97% for the largest. Dissections revealed that the basis for the resistance of early instars was physical exclusion of the comparatively large nematodes. The principle factor regulating the susceptibility of mid and late instars was injury to nematodes caused by larval mouthparts during ingestion. Differences in intra-instar susceptibility were similarly related to nematode injury.     

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70 min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30 min, nematodes were observed adhered after 40 min and many were captured by the typical fungus traps after 70 min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.