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).     

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.     

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.     

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.     

Carbohydrate-mediated Recognition of Larval Mosquito Hosts by Romanomermis culicivorax

Proteases, glycosidases, and lectins were tested and the results supported a role in host recognition for glycoproteins containing β-glucose and α-mannose on the cuticular surface of host and parasite. Carbohydrates containing α-glucose, galactose, fucose, or N-acetylglucosamine residues apparently are not involved in nematode attachment. Chitin or a related N-acetylglucosamine polymer was found in R. culicivorax preparasites. Treatment of preparasites with neuraminidase, which hydrolyzes sialic acids, increased nematode attachment to Anopheles freeborni larvae.     

Caudal Papillae in Romanomermis culicivorax

Distribution of caudal papillae in adult Romanomermis culicivorax was determined by scanning electron microscopy. Ninety eight caudal papillae, each containing one pore, were present in males but absent in females. Papillae were arranged in three longitudinal rows, one ventral, two ventrolateral; the middle ventral row bifurcated anterior to the spicule. The appearance of the papillae was different anterior and posterior to the spicule. The role of the caudal papillae in mediating copulatory behavior was discussed.     

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.     

Polyamine Synthesis by the Mermithid Nematode Romanomermis culicivorax

The polyamine and amino acid composition of the mermithid nematode, Romanomermis culicivorax, and its host, Aedes aegypti, was determined. Putrescine, spermidine, spermine, cadaverine and two acetylated spermidine derivatives were present in parasitic juveniles, newly-emerged post-parasites, and eggs of R. culicivorax. Whole insect homogenates of fourth-instar A. aegypti contained the same array of polyamines, except that the putrescine:spermidine ratio was the inverse of that in parasitic R. culicivorax. Polyamines and amino acids were in greater concentrations in the nematode eggs than in other developmental stages investigated. Both the host and nematode possess the biosynthetic capacity for polyamine biosynthesis, as evidenced by measurable activities of ornithine decarboxylase in the host''s tissues and the nematode''s free-living stages.     

Role of Catecholamines in the Reproduction of Romanomermis culicivorax

The relative concentrations of catecholamine in the nervous system of the entomophilic nematode Romanomermis culicivorax were measured under different experimental conditions by a glyoxylic acid-induced fluorescence procedure. A greater concentration of catecholamine was recorded in the nervous system of adult males and females than in postparasitic juveniles. A higher concentration of catecholamine occurred in adults maintained in physical contact with the opposite sex than in those maintained in isolation. Adult males maintained with females in the same aqueous medium but physically separated by a barrier displayed a greater concentration of catecholamine in their nervous systems than did males maintained in isolation, but the catecholamine fluorescence intensity of such males was less than in males allowed physical contact with females. In adult males, the fluorescence intensity of catecholamine declined progressively during and after copulation. In adult females, the intensity of catecholamine remained constant before, during, and after copulation. Catecholamine(s) may play a role in regulating copulatory behavior, egg production, or oviposition.     

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.     

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.     

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.     

Mitochondrial DNA Sequence Divergence among Meloidogyne incognita,Romanomermis culicivorax,Ascaris suum,and Caenorhabditis elegans

Mitochondrial DNA sequences were obtained from the NADH dehydrogenase subunit 3 (ND3), large rRNA, and cytochrome b genes from Meloidogyne incognita and Romanomermis culicivorax. Both species show considerable genetic distance within these same genes when compared with Caenorhabditis elegans or Ascaris suum, two species previously analyzed. Caenorhabditis, Ascaris, and Meloidogyne were selected as representatives of three subclasses in the nematode class Secernentea: Rhabditia, Spiruria, and Diplogasteria, respectively. Romanomermis served as a representative out-group of the class Adenophorea. The divergence between the phytoparasitic lineage (represented by Meloidogyne) and the three other species is so great that virtually every variable position in these genes appears to have accumulated multiple mutations, obscuring the phylogenetic information obtainable from these comparisons. The 39 and 42% amino acid similarity between the M. incognita and C. elegans ND3 and cytochrome b coding sequences, respectively, are approximately the same as those of C. elegans-mouse comparisons for the same genes (26 and 44%). This discovery calls into question the feasibility of employing cloned C. elegans probes as reagents to isolate phytoparasitic nematode genes. The genetic distance between the phytoparasitic nematode lineage and C. elegans markedly contrasts with the 79% amino acid similarity between C. elegans and A. suum for the same sequences. The molecular data suggest that Caenorhabditis and Ascaris belong to the same subclass.     

Effect of Storage at Low Temperatures on Development and Survival of Postparasitic Juveniles of Romanomermis culicivorax (Nematoda: Mermithidae)

Development of postparasites and adult females of Romanomermis culicivorax Ross and Smith, 1976 may be retarded by keeping cultures at 10 or 15 C for 15 wk. Storage at 5 C resulted in high mortality of postparasites. A storage temperature of 10 C is suitable if development of postparasites, young adults, and gravid females is to be greatly retarded. None of the females were gravid when postparasites (1-6 days old) were kept at 10 C for 15 wk. Development resumed after the cultures were returned to 26 C and no significant mortality occurred. A storage temperature of 15 C is suitable if development is to be only moderately retarded. After a 15-wk storage period, only a few weeks were required to return the cultures to full production status.     

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.     

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.     

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.     

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,     

Reproductive Isolation and Taxonomic Differentiation of Romanomermis culicivorax Ross and Smith, 1976 and R. communensis Galloway and Brust, 1979

The infertility of hybrid progeny of Romanomermis communensis and R. culicivorax supports their retention as distinct species. Their taxonomic separation on the basis of morphometric data and possession of a cone-shaped spicule guide is rejected. However, differences in the enzyme patterns of peptidase and phosphoglucomutase and the restriction fragment length differences in repetitive genomic DNA provide sensitive diagnostic characters that confirm the differentiation into two species.