Gaseous Requirements for Postparasitic Development of Romanomermis culicivorax

The development of postparasitic stages of Romanomermis culicivorax was studied under various concentrations of oxygen and carhon dioxide. The nematode developed poorly if only nitrogen was supplied; only one-third molted and all died eventually. In the presence of 5% CO₂ - 95% N₂, development was normal; most nematodes molted and oviposited with respective mean developmental times of 32 and 50 d. Addition of 0.2% O₂ stimulated development; molting and oviposition commenced at days 18 and 41, respectively. There was an additional stimulation of development by increasing amounts of O₂ up to 1%, but concentrations greater than 1% produced no additional stimulation. Carbon dioxide was required for development after exsheathment under anaerobic conditions or O₂ concentrations less than 1%. Oxygen or CO₂ were not required for embryological development or egg hatch. It is suggested that post-parasitic stages function as facultative anaerobes,     

The Effects of Temperature on the Infectivity of Romanomermis culicivorax

The survival time (ST) of the preparasitic larvae of Rornanomermis culicivorax was determined by measuring motility at 1, 6, 12, 18, 21, 27, 30, and 37 C; the ST₅₀ at each of these temperatures was 2.3, 2.2, 2.0, 2.0, 1.7, 1.6, 0.9, and 0.7 days, respectively. About one-third of the preparasites infected first-instar larvae of Culex pipiens within 24 h at 27 C. The preparasites were infective at 12 to 33 C with the optimum infectivity at 21-33 C. Lower temperatures decreased the percent infectivity but increased the time that the nematodes remained infective. The time required for host infection increased as the preparasitic larvae aged at 15, 21, and 27 C.     

Uptake of Lipids by the Entomophilic Nematode Romanomermis culicivorax

Romanomermis culicivorax juveniles were dissected out of Aedes aegypti larvae 7 days after infection and incubated under controlled conditions in isotonic saline containing a ¹⁴C-labeled fatty acid (palmitic acid), monoacylglycerol (glycerol monoolein), or triacylglycerol (glycerol tripalmate) nutrient source. The mermithid absorbed each of these lipids from the incubation medium, the rate of uptake being greatest for glycerol monoolein. No lipase activity was detected in whole nematode homogenates or in the media in which the nematodes were incubated. It is suggested that the nematode transports complex lipid molecules across its outer cuticle intact.     

Effect of Aedes aegypti (Diptera:Culicidae) Age on Sex Ratios in Romanomermis culicivorax (Nematoda:Mermithidae)

First, second, third, early and late fourth-instar larvae, and pupae of Aedes aegypti were infected with Romanomerrais culicivorax and reared at 20, 25, and 30 C. An increase in the ratio of male to female nematodes was observed with increase in host age at the time of infection at each temperature. The number of pupal and late fourth-instar infections was low, but R. culicivorax continued to develop in adult A. aegypti. Since male nematodes were recovered from both male and female hosts infected as late fourth instars or pupae, the sex of the host did not influence the sex of the nematode.     

Teratological Development in the Cephalic Anatomy of the Nematode Romanomermis culicivorax

Adults of Romanomermis culicivorax obtained from mass cultures were examined by scanning and transmission electron microscopy to determine the organization of their anterior sense organs. The normal pattern apparently consists of two lateral amphids plus six cephalic papillae. Lateral cephalic papillae contain two sense organs, each with a cuticular pore, while subdorsal and subventral papillae have three sense organs, each with a cuticular pore. About 30% of females and 80% of males examined showed aberrant developments in these sense organs. Both cuticle and underlying cells (hypodermis and neurons) are affected; some sense organs are absent while others are incompletely formed. Few aberrant worms were found in a smaller collection of worms reared in lower population densities. Perhaps aberrant forms are examples of teratological development resulting from, or promoted by, conditions used for mass rearing of biological control agents.     

An Improved Method for Long-Distance Shipping the Mosquito Parasite Romanomermis culicivorax

To prevent losses during long-distance shipment, a new delivery system was developed for the mosquito parasite Romanomermis culicivorax. A specially designed shipping container, two types of culture media (sand and a mixture of sand, vermiculite, and charcoal [SVC]), and two types of insert (polystyrene and polyester urethane foam [PUF]) were tested. Cultures shipped in SVC/PUF insert combination produced significantly higher yields of preparasites than did the other three combinations tested, and the sand/polystyrene combination produced significantly lower yields than did either sand or SVC media shipped with the PUF insert. Yields from cultures shipped in sand/PUF and SVC/PUF combinations were significantly lower than from unshipped controls. Maximum losses (52%) occurred in cultures shipped when 13-16-wk old. Also, yields of preparasites were significantly lower from cultures shipped singly when compared to similar cultures shipped in groups of eight. Techniques and procedures developed in this study did not completely solve the loss associated with delivery of R. culicivorax. For the first time, however, the system can guarantee the delivery of a quality product. The study defines the importance of the carrier medium, age of cultures, shipment size, and the restriction of the movement of the culture medium.     

Respiratory Physiology of a Facultative Anaerobe, Romanomermis culicivorax

The respiratory physiology of postparasitic larval and adult Romanornermis culicivorax was studied by manometric and polarographic methods. Endogenous respiration rates were relatively low and unaffected by postemergent development. The respiratory quotient (RQ) of larvae and young adults was 0.4 but increased to 0.7 about 3 wk after emergence. Exogenous glucose (0.02 mM) had no effect on QO₂ or RQ. Respiration of adults and larvae was completely inhibited by KCN (l mM) but not by NaN₃ (l mM) or 2,4-DNP (0.1 mM). The nematodes survived exposure to cyanide (l mM) for 18 h. Carbon dioxide (10%) inhibited respiration. The postparasitic stages of the nematode were mixed respiratory regulators-conformers. Exposure to anaerohic conditions resulted in an increased postanaerobic oxygen consumption which persisted for 3.5 h. The experiments confirmed that the postemergent stages of the nematode are facultative anaerobes.     

Glyoxylate Pathway in the Free-Living Stages of the Entomophilic Nematode Romanomermis culicivorax

Isocitrate lyase and malate synthetase, key enzymes of the glyoxylate cycle, were present in postparasites of the mermithid nematode Romanomermis culicivorax. Specific activities of enzymes were higher in adult postparasites than in newly emerged juveniles. Isocitrate lyase had a well-defined pH optimum (7.5), whereas malate synthetase functioned optimally over a broad range of alkaline pH (7.5-9.0). Substrate affinities of the two enzymes were measured.     

Susceptibility of Psorophora columbiae Larvae over Time to Parasitism by Romanomermis culicivorax

The ability of Romanomermis culicivorax preparasites to penetrate and infect Psorophora columbiae decreased substantially after ca. 28 hours. Parasitism at temperatures typical of Louisiana rice fields (i.e., 26, 29, and 32 ± 0.5 C) showed a significant linear decrease (P < 0.01) as the percentage of older larval instars increased at the times of exposure. These data emphasize the need for a synchronous field application of preparasites to challenge the rapid development of early instars of Ps. columbiae. Applications of postparasites rather than insecticide treatments to potential mosquito breeding habitats may offer greater flexibility in larval mosquito control programs.     

Permeability of the Body Wall of Romanomermis culicivorax to Lanthanum

Ultrastructural study of the body wall of preparasitic, parasitic, and postparasitic stages of Romanomermis culicivorax showed that the cuticle of all three stages was permeable to lanthanum. The cuticle of the parasitic stage was the thinnest and showed the greatest permeability. Lanthanum accumulated on the apical surfaces of the hypodermal cells but was not found intracellularly. The negative staining characteristics of lanthanum enhanced the detection of numerous smooth septate junctions in the hypodermis of the parasitic stage.     

Hemolymph pH of the larvae of three species of mosquitoes,and the effect of Romanomermis culicivorax parasitism on the blood pH of Culex pipiens

The hemolymph pHs of late fourth-instar Culex pipiens, Aedes taeniorhynchus, and Toxorhynchites amboinensis were measured with pH-sensitive glass microelectrodes and were slightly alkaline; pH 7.51, 7.62, and 7.37, respectively. The hemolymph pH remained relatively constant during the development of C. pipiens larvae through the third and early and late fourth instars. The hemolymph pH in C. pipiens larvae parasitized with the mermithid nematode Romanomermis culicivorax was unaltered. These measurements provide an approximate pH (ca 7.4) which is normal for the hemolymph of larval mosquitoes, and should be useful for further development of a culture medium for R. culicivorax.     

Mass Production of the Mosquito Parasite Romanomermis culicivorax: Effect of Density

When numbers of Romanomermis culicivorax Ross and Smith were varied in containers with a constant surface area and depth of sand, densities of 12-24 nematodes per cm² yielded significantly more preparasites than higher densities. When container surface area and numbers of nematodes were constant and sand volume and depth were varied, yields did not differ significantly. When numbers of nematodes and sand volume were constant and surface area and sand depths were varied, yields were significantly higher for a density of 24 nematodes per cm². Yields of preparasites were tripled by simply setting up three cultures, each containing 5 g of nematodes, instead of a single culture containing 15 g.     

Transport of Palmitic Acid Across the Tegument of the Entomophilic Nematode Romanomermis culicivorax

Romanomermis culicivorax juveniles, dissected out of Aedes aegypti larvae 7 days after infection, were incubated under controlled conditions in isotonic saline containing ¹⁴C-U-palmitic acid to investigate the nature of the transport mechanism(s) used by the nematode for transcuticular uptake of palmitic acid. Net uptake of the isotope by the nematode was of a logarithmic nature with respect to time. Uptake of palmitic acid was accomplished by a combination of diffusion and a mediated process which was substrate saturable and competitively inhibited by myristic and stearic acids. Both 2,4-dinitrophenol and ouabain inhibited uptake of palmitic acid and thus supported the hypothesis that the carrier system is of the active transport variety and is coupled to a Na⁺K⁺ ATPase pump.     

Effects of Host Diet on Romanomermis culicivorax,a Mermithid Parasite of Mosquitoes

When larval mosquitoes (Aedes aegypti) infected with the mermithid nematode Romanomermis culicivorax were fed on a diet low in quantity or protein content or both, the number of postparasites which emerged from the hosts decreased and host mortality increased marginally. Parasitic development was prolonged and became asynchronous in nutritionally deprived hosts. Nematodes emerged from insects infected by more than one nematode before the remaining juveniles comprising such infections had completed parasitic development; this resulted in substantial reductions in postparasite nttmbers. Host development was retarded by low protein and/or reduced diets. Postparasites emerging from second and third instars were reduced in size and in the amount of stored nutriment compared to those recovered from hosts fed on a high protein diet ad libitum. A greater proportion of the rnermithids developed into males in hosts fed on reduced diets but not in hosts fed on low protein diets.     

In Vivo Growth of Romanomermis culicivorax: Biochemical Changes During Parasitism

Biochemical analyses of total protein, lipid, carbohydrate, DNA, amino acid, and length, width, and dry weight measurements are reported for different stages of Romanomermis culicivorax cultured in the mosquito, Culex pipiens. The Bradford technique for assaying total protein was the most sensitive and reliable biochemical technique tested for assaying in vivo growth of R. culicivorax. Increases in total protein, lipid, carbohydrate, and dry weight during growth from preparasite to postparasite were greater than 6,900-fold for females and 2,300-fold for males. DNA increased 650-fold and 233-fold during development to female and male postparasites, respectively. The proportions of amino acids for preparasites were significantly different (P ≤ 0.01) from female and male postparasites for all amino acids tested, except methionine and tyrosine. Female and male postparasites were similar in protein, lipid, carbohydrate, DNA, and most amino acid proportions, but were significantly different in relative concentrations of serine, glycine, and alanine (P ≤ 0.01). Preliminary results suggest that the use of amino acid ratios from female postparasites improves the in vitro culture performance of R. culicivorax.     

Oxygen and the Infectivity of Romanomermis culicivorax

The effects on itffectivity by preparasitic Romanomermis culicivorax resulting from four exposure times to four concentrations of oxygen at three temperatures were studied . Low oxygen (1.8 and 3.1 mg /liter) slowed losses of infectivity. Losses of infectivity were similar under aerobic (6 mg /liter) and microaerobic (0.4 mg /liter) conditions for the first 24 h but thereafter were slower in the microaerobic group. N interactions between temperature and oxygen were found over the ranges tested (15-27 C; 0.4-6.0 mg /liter).     

Nutrient Composition of Romanomermis culicivorax in Relation to Egg Production and Metabolism

The nutrient composition of postparasitic females (newly emerged juveniles, newly molted adults, and spent adults) and eggs of Romanomermis culicivorax was studied. Throughout post-parasitic development, proteins increased and lipids decreased progressively as a proportion of the dry weight; the proportion of glycogen within the nematodes remained stable. The greatest decrease in the lipid moiety occurred during egg production. Eggs contained relatively low levels of lipids (12% dry weight), and ca. 20% of the dry weight of the eggs was unaccounted for by lipid, protein, and glycogen determinations. Chitin, mucoproteins, and peptides were present in the eggs. The fatty acid composition of nematodes remained constant during postparasitic development; eggs contained a similar profile of fatty acids as postparasites, with marginally higher content of unsaturated fatty acids. Radiotracer studies showed that the eggs could oxidize glucose and palmitic acid.     

Salts and the Infectivity of Romanomermis culicivorax

The effects of common inorganic ions found in freshwater on the infectivity of Romanomermis culicivorax and the survival of its host, Culex pipiens, were tested. In general, the median lethal concentrations found for R. culicivorax were greater than the reported median concentrations of these ions in freshwater but less than the reported maximum natural concentrations. The ion toxicity for R. culicivorax (on a molar basis) increased in the following order: sodium < potassium < calcium; aml chloride < carbonate = sulfate < nitrate < nitrite < phosphate. The larvae of C. pipiens were generally 20 to 75 times as tolerant of higher ion concentrations as were the preparasitic stages of R. culicivorax.