Mermithidae Research Articles

Survey of Mosquito Larvae for Potential Nematode Parasitic Biological Control in Alachua County

There is an obvious and considerable interest in developing novel methods to control mosquito-borne diseases due to their impacts on mammalian hosts. Biological control practices are sustainable alternatives to chemical control of mosquito populations. Some nematodes are efficient insect parasites and represent a potential source of novel biological control agents. Such beneficial nematodes are potent biological control agents that can remove harmful pests whilst also preserving non-target insect populations. One such nematode, Romanomermis culicivorax, is a parasite of mosquito larvae, inducing paralysis and eventually killing its host. The potential success of a biological control agent can be informed from the current prevalence of native populations. Thus, we conducted a survey of parasitic nematodes in mosquito larvae in Alachua County. Twenty-five samples were obtained from all around Alachua County, with assistance from the Gainesville Department of Public Works Mosquito Control Section. Subsequent visual inspection and PCR for R. culicivorax were conducted to detect the presence of mosquito and nematode DNA in each sample. Out of the twenty-five mosquito samples tested, only one sample tested positive for the presence of the beneficial nematode. This sample was found in an apartment complex in proximity to Devil’s Millhopper Geological State Park. This work demonstrates the presence of mosquito parasitic nematodes in Alachua County, but at very low levels, potentially discouraging the use of such biological control practices. In addition, the proximity of the positive sample to the natural area may indicate the need to preserve such habitats for effective biological control measures.

Genus-level evolutionary relationships of FAR proteins reflect the diversity of lifestyles of free-living and parasitic nematodes

BackgroundNematodes are a widespread and diverse group comprising free-living and parasitic species, some of which have major detrimental effects on crops, animals, and human health. Genomic comparisons of nematodes may help reveal the genetic bases for the evolution of parasitic lifestyles. Fatty acid and retinol-binding proteins (FARs) are thought to be unique to nematodes and play essential roles in their development, reproduction, infection, and possibly parasitism through promoting the uptake, transport, and distribution of lipid and retinol. However, the evolution of FAR family proteins across the phylum Nematoda remains elusive.ResultsWe report here the evolutionary relationship of the FAR gene family across nematodes. No FAR was found in Trichocephalida species and Romanomermis culicivorax from Clade I, and FAR could be found in species from Clades III, IV, and V. FAR proteins are conserved in Clade III species and separated into three clusters. Tandem duplications and high divergence events lead to variable richness and low homology of FARs in Steinernema of Clade IVa, Strongyloides of Clade IVb, and intestinal parasitic nematodes from Clades Vc and Ve. Moreover, different richness and sequence variations of FARs in pine wood, root-knot, stem, and cyst nematodes might be determined by reproduction mode or parasitism. However, murine lungworm Angiostrongylus and bovine lungworm Dictyocaulus viviparus from Clade Vd have only 3–4 orthologs of FAR. RNA-seq data showed that far genes, especially far-1 and far-2, were highly expressed in most nematodes. Angiostrongylus cantonensis FAR-1 and FAR-3 have low sequence homology and distinct ligand-binding properties, leading to differences in the cavity volume of proteins. These data indicate that FAR proteins diverged early and experienced low selective pressure to form genus-level diversity. The far genes are present in endophyte or root-colonized bacteria of Streptomyces, Kitasatospora sp., Bacillus subtilis, and Lysobacter, suggesting that bacterial far genes might be derived from plant-parasitic nematodes by horizontal gene transfer.ConclusionsData from these comparative analyses have provided insights into genus-level diversity of FAR proteins in the phylum Nematoda. FAR diversification provides a glimpse into the complicated evolution history across free-living and parasitic nematodes.

Single-molecule mechanical folding and unfolding kinetics of armless mitochondrial tRNAArg from Romanomermis culicivorax * *Project supported by the Natural Science Foundation of Guangdong Province, China (Grant No. 2017A030310085) and the Science and Technology Planning Project of Guangdong Province, China (Grant No. 2018A050506034).

The mechanical stability of tRNAs contributes to their biological activities. The mitochondrial tRNAArg from Romanomermis culicivorax is the shortest tRNA ever known. This tRNA lacks D- and T-arms, represents a stem–bulge–stem architecture but still folds into a stable tertiary structure. Although its structure had been reported, studies on its mechanical folding and unfolding kinetic characteristics are lacking. Here, we directly measured the single-molecule mechanical folding and unfolding kinetics of the armless mt tRNAArg by using optical tweezers in different solution conditions. We revealed a two-step reversible unfolding pathway: the first and large transition corresponds to the unfolding of acceptor stem and bulge below 11 pN, and the second and small transition corresponds to the unfolding of anticodon arm at 12 pN–14 pN. Moreover, the free energy landscapes of the unfolding pathways were reconstructed. We also demonstrated that amino acid-chelated Mg2+(aaCM), which mimics the intracellular solution condition, stabilizes the bulge of mitochondrial tRNAArg possibly by reducing the topological constraints or stabilizing the possible local non-canonical base pairings within the bulge region. Our study revealed the solution-dependent mechanical stability of an armless mt tRNA, which may shed light on future mt tRNA studies.

Adaptation of the Romanomermis culicivorax CCA-Adding Enzyme to Miniaturized Armless tRNA Substrates.

The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that—when inserted into the human enzyme—confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3′-end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.

Effects of Salinity and pH on the Infective Capacity of Romanomermis iyengari and Romanomermis culicivorax in Larvae of Culex quinquefasciatus Mosquito

Abstract. Effects of salinity and pH were assessed on the infective capacity of parasitic nematodes of mosquitoes Romanomermis culicivorax Ross & Smith and Romanomermis iyengari (Welch), on larvae of southern house mosquito, Culex quinquefasciatus Say. Thirteen pH levels (3.6, 3.8, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.2, and 8.4) were evaluated. Six salinity levels (NaCl concentrations) were evaluated, with values of 0.01 M (584 mg/L), 0.02 M (1,169 mg/L), 0.03 M (1,753) mg/L), 0.04 M (2,338 mg/L), 0.05 M (2,922 mg/L), and a 0.00 M check, with three replications. Percentage of infection (of parasitized larvae) and infestation intensity (average nematodes parasitizing a mosquito larva) were recorded for both factors. To determine effects of pH and salinity on percentage of infection and infestation intensity, a sample (n = 20, IV instar) of larvae surviving at high salinity levels was extracted 5 days after application. The greatest percentage of infection statistically significant in R. iyengari...

Small but large enough: structural properties of armless mitochondrial tRNAs from the nematode Romanomermis culicivorax.

As adapter molecules to convert the nucleic acid information into the amino acid sequence, tRNAs play a central role in protein synthesis. To fulfill this function in a reliable way, tRNAs exhibit highly conserved structural features common in all organisms and in all cellular compartments active in translation. However, in mitochondria of metazoans, certain dramatic deviations from the consensus tRNA structure are described, where some tRNAs lack the D- or T-arm without losing their function. In Enoplea, this miniaturization comes to an extreme, and functional mitochondrial tRNAs can lack both arms, leading to a considerable size reduction. Here, we investigate the secondary and tertiary structure of two such armless tRNAs from Romanomermis culicivorax. Despite their high AU content, the transcripts fold into a single and surprisingly stable hairpin structure, deviating from standard tRNAs. The three-dimensional form is boomerang-like and diverges from the standard L-shape. These results indicate that such unconventional miniaturized tRNAs can still fold into a tRNA-like shape, although their length and secondary structure are very unusual. They highlight the remarkable flexibility of the protein synthesis apparatus and suggest that the translational machinery of Enoplea mitochondria may show compensatory adaptations to accommodate these armless tRNAs for efficient translation.

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 2ml 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 500ml 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.

Operons are a conserved feature of nematode genomes.

The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and “spliced leader” (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.

Biological evidence for the world's smallest tRNAs

Due to their function as adapters in translation, tRNA molecules share a common structural organization in all kingdoms and organelles with ribosomal protein biosynthesis. A typical tRNA has a cloverleaf-like secondary structure, consisting of acceptor stem, D-arm, anticodon arm, a variable region, and T-arm, with an average length of 73 nucleotides. In several mitochondrial genomes, however, tRNA genes encode transcripts that show a considerable deviation of this standard, having reduced D- or T-arms or even completely lack one of these elements, resulting in tRNAs as small as 66nts. An extreme case of such truncations is found in the mitochondria of Enoplea. Here, several tRNA genes are annotated that lack both the D- and the T-arm, suggesting even shorter transcripts with a length of only 42nts. However, direct evidence for these exceptional tRNAs, which were predicted by purely computational means, has been lacking so far. Here, we demonstrate that several of these miniaturized armless tRNAs consisting only of acceptor- and anticodon-arms are indeed transcribed and correctly processed by non-encoded CCA addition in the mermithid Romanomermis culicivorax. This is the first direct evidence for the existence and functionality of the smallest tRNAs ever identified so far. It opens new possibilities towards exploration/assessment of minimal structural motifs defining a functional tRNA and their evolution.

The genome of Romanomermis culicivorax: revealing fundamental changes in the core developmental genetic toolkit in Nematoda.

BackgroundThe genetics of development in the nematode Caenorhabditis elegans has been described in exquisite detail. The phylum Nematoda has two classes: Chromadorea (which includes C. elegans) and the Enoplea. While the development of many chromadorean species resembles closely that of C. elegans, enoplean nematodes show markedly different patterns of early cell division and cell fate assignment. Embryogenesis of the enoplean Romanomermis culicivorax has been studied in detail, but the genetic circuitry underpinning development in this species has not been explored.ResultsWe generated a draft genome for R. culicivorax and compared its gene content with that of C. elegans, a second enoplean, the vertebrate parasite Trichinella spiralis, and a representative arthropod, Tribolium castaneum. This comparison revealed that R. culicivorax has retained components of the conserved ecdysozoan developmental gene toolkit lost in C. elegans. T. spiralis has independently lost even more of this toolkit than has C. elegans. However, the C. elegans toolkit is not simply depauperate, as many novel genes essential for embryogenesis in C. elegans are not found in, or have only extremely divergent homologues in R. culicivorax and T. spiralis. Our data imply fundamental differences in the genetic programmes not only for early cell specification but also others such as vulva formation and sex determination.ConclusionsDespite the apparent morphological conservatism, major differences in the molecular logic of development have evolved within the phylum Nematoda. R. culicivorax serves as a tractable system to contrast C. elegans and understand how divergent genomic and thus regulatory backgrounds nevertheless generate a conserved phenotype. The R. culicivorax draft genome will promote use of this species as a research model.

Mermithid nematodes found in adult Anopheles from southeastern Senegal

BackgroundOver two dozen mermithid nematodes have been described parasitizing mosquitoes worldwide, however, only two species were found in Africa. Mermithid nematodes kill their mosquito host upon emergence, which suggests that they could be developed as biological control agents of mosquitoes. Both Romanomermis culicivorax and Romanomermis iyengari have been reared for mass release to control numerous Anopheles species vector populations, and in one instance this may have led to reduced malaria prevalence in a human population.MethodsAnopheles mosquitoes were collected during a malaria study in southeastern Senegal. Two different adult blood fed mosquitoes had a single mermithid nematode emerge from their anus while they were being held post-capture. Primers from the 18 S rDNA were developed to sequence nematode DNA and screen mosquitoes for mermithid DNA. 18 S rDNA from the Senegalese mermithid and other mermithid entries in GenBank were used to create a Maximum Parsimony tree of the Mermithidae family.ResultsThe mermithid was present in 1.8% (10/551) of the sampled adult Anopheles species in our study area. The mermithid was found in An. gambiae s.s., An. funestus, and An. rufipes from the villages of Ndebou, Boundoucondi, and Damboucoye. Maximum parsimony analysis confirmed that the nematode parasites found in Anopheles were indeed mermithid parasites, and of the mermithid sequences available in GenBank, they are most closely related to Strelkovimermis spiculatus.ConclusionsTo our knowledge, this is the first report of mermithids from adult Anopheles mosquitoes in Senegal. The mermithid appears to infect Anopheles mosquitoes that develop in diverse larval habitats. Although maximum parsimony analysis determined the mermithid was closely related to Strelkovimermis spiculatus, several characteristics of the mermithid were more similar to the Empidomermis genus. Future mermithid isolations will hopefully allow: formal taxonomic identification, laboratory colonization, determination of life history traits and species specificity, and characterize its usefulness as a biological control agent.

Rampant gene rearrangement and haplotype hypervariation among nematode mitochondrial genomes

Rare syntenic conservation, sequence duplication, and the use of both DNA strands to encode genes are signature architectural features defining mitochondrial genomes of enoplean nematodes. These characteristics stand in contrast to the more conserved mitochondrial genome sizes and transcriptional organizations of mitochondrial DNAs (mtDNAs) derived from chromadorean nematodes. To address the frequency of gene rearrangement within nematode mitochondrial DNA (mtDNA), mitochondrial genome variation has been characterized within a more confined enoplean taxonomic unit, the family Mermithidae. The complete nucleotide sequences of the mosquito parasitic nematodes Romanomermis culicivorax, R. nielseni, and R. iyengari mtDNA have been determined. Duplicated expanses encompassing different regions of the mitochondrial genomes were found in each of these congeners. These mtDNA shared few rRNA and protein gene junctions, indicating extensive gene rearrangement within the Romanomermis lineage. Rapid structural changes are also observed at the conspecific level where no two individual nematodes carry the same haplotype. Rolling circle amplification was used to isolate complete mitochondrial genomes from individuals in local populations of Thaumamermis cosgrovei, a parasite of terrestrial isopods. Mitochondrial DNA length variants ranging from 19 to 34 kb are observed, but haplotypes are not shared between any two individuals. The complete nucleotide sequences of three haplotypes have been determined, revealing a constant region encoding most mitochondrial genes and a hypervariable segment that contains intact and pseudogene copies of several mitochondrial genes, duplicated to different copy numbers, resulting in mtDNA size variation. Constant rearrangement generates new T. cosgrovei mtDNA forms.

Embryogenesis of Romanomermis culicivorax: An alternative way to construct a nematode

The current picture of embryonic development in nematodes is essentially shaped by Caenorhabditis elegans and its close relatives. As their pattern of embryogenesis is rather similar, it is often considered to be representative for the taxon Nematoda as a whole. Here we give for the first time a comprehensive description of embryonic development in an ancestrally diverged nematode. Romanomermis culicivorax differs strikingly from C. elegans with respect to cell division pattern, spatial arrangement of blastomeres and tissue formation. Our study reveals a number of unexpected phenomena. These include (i) unique polar interphase microtubule caps forming in early blastomeres destined to undergo asymmetric cleavages, suggesting the presence of a so far undescribed MTOC; (ii) embryonic cell lineages of reduced complexity with predominantly monoclonal sublineages, generating just a single tissue type; (iii) construction of major parts of the body from duplicating building blocks consisting of rings of cells, a pattern showing some resemblance to segmentation; (iv) prominent differences in cell fate assignment which can be best explained with a global shift affecting all somatic founder cells. In summary, our data indicate that during nematode evolution massive alterations in the developmental program took place of how to generate a juvenile.

Cellular pattern formation, establishment of polarity and segregation of colored cytoplasm in embryos of the nematode Romanomermis culicivorax

We have begun to analyze the early embryogenesis of Romanomermis culicivorax, an insect-parasitic nematode phylogenetically distant to Caenorhabditis elegans. Development of R. culicivorax differs from C. elegans in many aspects including establishment of polarity, formation of embryonic axes and the pattern of asymmetric cleavages. Here, a polarity reversal in the germline takes place already in P1 rather than P2, the dorsal–ventral axis appears to be inverted and gut fate is derived from the AB rather than from the EMS blastomere. So far unique for nematodes is the presence of colored cytoplasm and its segregation into one specific founder cell. Normal development observed after experimentally induced abnormal partitioning of pigment indicates that it is not involved in cell specification. Another typical feature is prominent midbodies (MB). We investigated the role of the MB region in the establishment of asymmetry. After its irradiation the potential for unequal cleavage in somatic and germline cells as well as differential distribution of pigment are lost. This indicates a crucial involvement of this region for spindle orientation, positioning, and cytoplasmic segregation. A scenario is sketched suggesting why and how during evolution the observed differences between R. culicivorax and C. elegans may have evolved.

Role of the surface coat of Romanomermis culicivorax in immune evasion

Abstract Interactions of the mermithid nematode Romanomermis culicivorax with the immune system of mosquito larvae were examined by scanning electron microscopy. The host immune system rapidly recognised invading parasites, as granulocytes and discharged granules were observed attached to parasitic nematodes within 5 min. Melanin deposition was infrequently observed. As a counter measure, the parasites secreted and shed an extracellular surface coat which aided immune evasion. During the first 4 days of infection, when parasite growth was limited, the coat served as a disposable, renewable barrier between parasite and host that was intermittently shed to cleanse the nematode of adhering host immune products. In the later infection phase the parasite grew rapidly and was beyond the effect of the depleted host immune response. The broad host range of R. culcivorax within culicines may be partly a function of the nonspecific defence it mounts against the host immune system. In summary, shedding of the surface coat is an adaptive counter response by R. culicivorax to the mosquito immune reaction to infection and provides a classic example of host-parasite coevolution.

Parasitoid-induced behavioral alterations of Aedes aegypti mosquito larvae infected with mermithid nematodes (Nematoda: Mermithidae)

A wide range of parasites are known to cause behavioral changes in their hosts and parasitized insects are especially amenable to the study of such changes. The majority of studies addressing parasite-induced behavioral alterations have focused on parasites with complex life cycles and the adaptive nature of such changes. Behavioral changes caused by parasitoids, single-host parasites that kill their host upon emergence, have been studied less and the adaptive nature of these changes is likely to be different than those in complex life cycles. I investigated behavioral alterations in Aedes aegypti mosquito larvae infected with parasitoid nematodes (family Mermithidae). I conducted several experiments in which I tested the following hypotheses: 1) Mermithid nematodes induce behavioral changes in mosquito larvae and the changes are density dependent. 2) Different species of mermithid nematodes induce similar changes in mosquito larvae behavior. 3) Behavioral alterations vary with mermithid developmental stage. 4) Mosquito larvae infected with mermithid nematodes behave similarly to uninfected food-deprived mosquito larvae. I found that 4th instar Ae. aegypti infected with Romanomermis culicivorax or Strelkovimermis spiculatus exhibited resting behaviors significantly more often than uninfected controls but that intensity of infection did not affect activity levels. In earlier instars, infected mosquito larvae were more active than uninfected control larvae in some behaviors associated with feeding. There was no significant difference between infected and uninfected food-deprived mosquitoes in nine of the ten behaviors observed. The decrease in activity of late instar Ae. aegypti larvae infected with mermithids may be a parasitoid adaptation that reduces the risk of predation and thus increases host and parasitoid survival. The increase in feeding activity in earlier instars as well as the similarity between uninfected food-deprived and infected Ae. aegypti behavior may indicate that these behaviors are adaptive for the parasitoid, increasing nutritional acquisition for successful parasitoid development.

Cryopreservation and low temperature storage of the pre-parasitic juveniles of Romanomermis culicivorax

AbstractThe effects of low temperature storage and cryopreservation on the survival and infectivity of Romanomermis culicivorax were studied in the laboratory. When pre-parasitic juveniles of R. culicivorax were stored at-2 to 2°C, more than 90% survived for 9 days and 50% were motile for 13 days. The infective ability of the pre-parasitic juveniles for mosquitoes remained high (85%) after 5 days of cold storage and the infectivity was reduced only moderately (50 to 78%) after storage for 6 to 10 days. Various cryoprotectants were investigated to develop a cryopreservation procedure for the infectious pre-parasitic juveniles of R. culicivorax. After suspension in 1, 2.5 or 5% dimethylsulphoxide (DMSO), and cryopreservation with a two-step cooling sequence prior to storage in liquid nitrogen, approximately half of the pre-parasitic juveniles of R. culicivorax, cryopreserved in 2.5 and 5% DMSO, regained motility when thawed quickly after storage for 7 and 125 days. However, revived pre-parasitic juveniles were unable to infect mosquito larvae. Pre-parasitic juveniles treated with ethanediol, hydroxyethyl starch, and polyvinylpyrrolidone as cryoprotectants did not survive the cryopreservation procedure. Similar results were obtained with the pre-parasitic juveniles (PPJ) of Romanomermis yunanensis.

Susceptibility of Aedes aegypti larvae to parasitism by Romanomermis culicivorax in laboratory and field conditions in Oaxaca, Mexico

In June 1996 in the state of Oaxaca, Mexico, larvae of the mosquito species Aedes aegypti were exposed to infective preparasites of the nematode Romanomermis culicivorax, Ross and Smith, in the laboratory and in the field. For the laboratory experiments larvae in instars I-IV were used; they had been collected in natural reservoirs. The laboratory experiments were carried out in triplicate, with 100 larvae of each larval stage per experiment. Three preparasite application dosage ratios were tested: 5, 10, or 15 preparasites per mosquito larva. For the field studies 13 A. aegypti outdoor breeding sites were used, with larvae in instars I-IV and a 15:1 preparasite dosage ratio. With the laboratory experiments, an increase was observed in the average infestation of the larvae as the preparasite application ratio was increased from 5:1 to 15:1. With a 10:1 ratio, the rates of parasitism were 100%, 100%, 85%, and 74% in the larvae in instars I, II, III, and IV, respectively; for the 15:1 preparasite ratio, parasitism rates were 100%, 100%, 90%, and 79%, respectively. The field tests with the 15:1 preparasite dosage ratio in the 13 outdoor reservoirs produced parasitism rates of 80% to 98%, thus demonstrating the susceptibility of this species of mosquito to parasitism by R. culicivorax in Oaxaca, Mexico.

Reduction of mosquito larval densities in natural sites after introduction of Romanomermis culicivorax (Nematoda:Mermithidae) in Cuba.

Romanomermis culicivorax Ross & Smith became established in 4 of 6 treated sites and produced high levels of infection in Anopheles albimanus Wiedemann, Culex atratus Theobald, Psorophora confinnis Lynch-Arribálzaga, and Uranotaenia sapphirina Oster-Sacken mosquitoes for 9 wk in 1993. Mean levels of southern house mosquito parasitism ranged from 82 to 83% for Culex quinquefasciatus Say, at 2 sites, from 89 to 95% for An. albimanus at 4 sites, and from 78 to 80% at 2 sites for Cx. atratus, Ps. confinnis, and Un. sapphirina. Results indicated that R. culicivorax has potential use as biological control agent against mosquitoes in several different habitats in Cuba.

Similar evolutionary patterning among repeated and single copy nematode mitochondrial genes

Ten of 12 mitochondrial protein-coding genes and the large (16S) mitochondrial rRNA gene have been identified and mapped within the Romanomermis culicivorax mitochondrial genome. This transcriptional map differs from other nematode mitochondrial DNAs (mtDNAs) with respect to gene order and transcriptional orientation of some genes. Several of these coding regions are components of a 3.0-kilobase mtDNA repeating unit, allowing a direct comparison of nucleotide and amino acid sequence composition for repeated and single copy genes. Analysis of protein-coding regions representing repeated (ND3, ND6) and single copy genes (ATPase 6, cyt.b, COI, COIII, ND1, ND4, ND5), and four repeat-associated open reading frames (ORFs) with unassigned function have revealed striking similarities in nucleotide composition, amino acid frequencies, and codon biases. Although we anticipated that reiterated protein coding regions might be evolving under relaxed selection, our results indicate that both repeated and unique mitochondrial genes appear subject to similar functional constraints.