Michalski ML et al. (1999) Mol Biochem Parasitol "Gender-specific gene expression in Brugia malayi."

Michalski ML, Weil GJ

[Mol Biochem Parasitol, 1999]

Brugia malayi is a mosquito-borne filarial nematode that causes lymphatic filariasis and elephantiasis in humans. The purpose of this study was to identify and characterize genes that are expressed differentially in male and female B. malayi in hopes of gaining new insight into the reproductive biology of the parasite. Two approaches were used. A 5' differential display PCR (splice leader differential display PCR, SL DD-PCR) was performed by PCR with splice leader and random primers on cDNA templates, and electronic subtraction was performed on expressed sequence tag (EST) cluster databases developed by the Filarial Genome Project (FGP). Gender-specific expression of candidate clones was confirmed by RT-PCR for six of 22 (27%) clones identified by DD and in seven of 15 (47%) clones identified by electronic subtraction. One clone was identified by both methods. Several female-specific clones had homology to known nematode genes that encode a fatty acid binding protein, a high mobility group protein, an eggshell protein, a glutamate-gated ion channel, and a collagen. However, most of the clones have no significant homology to known genes or proteins in computer databases. This project has confirmed the value of SL DD-PCR and electronic subtraction for analysis of gene expression in filariae. These two complimentary techniques may be generally applicable to the study of gender-specific (and by analogy stage specific) gene expression in other nematodes.

Michalski ML et al. (2002) Mol Biochem Parasitol "An embryo-associated fatty acid-binding protein in the filarial nematode Brugia malayi."

Michalski ML, Weil GJ, Cistola DP, Monsey JD

[Mol Biochem Parasitol, 2002]

Brugia malayi is a filarial nematode parasite that causes lymphatic filariasis, a disease that affects millions of people in the tropics. Sexual reproduction of filarial worms occurs within the lymphatic vessels of the human host and is crucial for transmission of the parasite to the mosquito vector. We have previously identified several B. malayi genes that exhibit apparent gender-specific expression. One of these had significant sequence similarity to the Ascaris suum embryo-associated fatty acid binding protein, As-p18. The full length cDNA for the B. malayi female-associated fatty acid binding protein (Bm-FAB-1) encodes a 17.8 kDa protein (excluding a signal peptide) with 70% sequence identity with mature As-p18 and significant similarity to Caenorhabditis elegans and mammalian fatty acid-binding proteins (FABPs). Antibodies raised to Bm-FAB-1 bound to developing embryos within female worms, especially around early embryo cells and the surfaces of immature worms within eggs. Functional studies showed that recombinant Bm-FAB-1 binds to several long chain fatty acids including oleate, but not retinol. Taken together, these results demonstrate that Bm-FAB-1 is a member of an unusual nematode-specific, secreted lipid binding protein family. The existence of a novel class of lipid binding proteins in nematode embryos raises the possibility that drugs targeting these proteins could be developed with broad activity against nematode parasites of medical and veterinary importance.

Michalski ML et al. (2010) PLoS Negl Trop Dis "Midgut barrier imparts selective resistance to filarial worm infection in Culex pipiens pipiens."

Bartholomay LC, Erickson SM, Michalski ML, Christensen BM

[PLoS Negl Trop Dis, 2010]

Mosquitoes in the Culex pipiens complex thrive in temperate and tropical regions worldwide, and serve as efficient vectors of Bancroftian lymphatic filariasis (LF) caused by Wuchereria bancrofti in Asia, Africa, the West Indies, South America, and Micronesia. However, members of this mosquito complex do not act as natural vectors for Brugian LF caused by Brugia malayi, or for the cat parasite B. pahangi, despite their presence in South Asia where these parasites are endemic. Previous work with the Iowa strain of Culex pipiens pipiens demonstrates that it is equally susceptible to W. bancrofti as is the natural Cx. p. pipiens vector in the Nile Delta, however it is refractory to infection with Brugia spp. Here we report that the infectivity barrier for Brugia spp. in Cx. p. pipiens is the mosquito midgut, which inflicts internal and lethal damage to ingested microfilariae. Following per os Brugia exposures, the prevalence of infection is significantly lower in Cx. p. pipiens compared to susceptible mosquito controls, and differs between parasite species with <50% and <5% of Cx. p. pipiens becoming infected with B. pahangi and B. malayi, respectively. When Brugia spp. mf were inoculated intrathoracically to bypass the midgut, larvae developed equally well as in controls, indicating that, beyond the midgut, Cx. p. pipiens is physiologically compatible with Brugia spp. Mf isolated from Cx. p. pipiens midguts exhibited compromised motility, and unlike mf derived from blood or isolated from the midguts of Ae. aegypti, failed to develop when inoculated intrathoracically into susceptible mosquitoes. Together these data strongly support the role of the midgut as the primary infection barrier for Brugia spp. in Cx. p. pipiens. Examination of parasites recovered from the Cx. p. pipiens midgut by vital staining, and those exsheathed with papain, suggest that the damage inflicted by the midgut is subcuticular and disrupts internal tissues. Microscopic studies of these worms reveal compromised motility and sharp bends in the body; and ultrastructurally the presence of many fluid or carbohydrate-filled vacuoles in the hypodermis, body wall, and nuclear column. Incubation of Brugia mf with Cx. p. pipiens midgut extracts produces similar internal damage phenotypes; indicating that the Cx. p. pipiens midgut factor(s) that damage mf in vivo are soluble and stable in physiological buffer, and inflict damage on mf in vitro.

Ohba K et al. (1984) Journal of Pesticide Science "A nematode, C. elegans, as a test organism for nematicide evaluation."

Ohba K, Ishibashi N

[Journal of Pesticide Science, 1984]

Juveniles of Caenorhabditis elegans were transformed to dumpy in the media containing methomyl (10 ug/ml) or aldoxycarb (500 ug/ml), but not in the media containing methylisothiocyanate (MITC). The dumpy did not recover to its normal structure even after being transferred to fresh medium, but underwent to adulthood with a lower reproduction rate. The progeny from the dumpy forms was normal in structure, however, its reproductivity was as low as one-third of normal ones. Longevity of the dumpy form and its progeny were longer than those of normal worms. Methomyl (1 ug/ml), MITC (1 ug/ml) and aldoxycarb (10 ug/ml) did not suppress population growth, but MITC (10 ug/ml) did for the first two weeks. The population growth was markedly suppressed at 100 ug/ml of methomyl, 20 ug/ml of MITC and 1000 ug/ml of aldoxycarb.

Polli JR et al. (2020) Sci Total Environ "Quantitative analysis of PAH compounds in DWH crude oil and their effects on Caenorhabditis elegans germ cell apoptosis, associated with CYP450s upregulation."

Pan X, Lish L, Lewis L, Rushing BR, Selim MI, Polli JR

[Sci Total Environ, 2020]

The Deepwater Horizon (DWH) oil spill marked the largest environmental oil spill in human history, where it was estimated a large amount of the polycyclic aromatic hydrocarbons (PAHs) were released with crude oil into the environment. In this study, common PAH compounds were quantitatively determined in crude oil from the DWH spill by gas chromatography-mass spectroscopy (GC-MS). Twelve PAH compounds were identified and quantified from a 100x dilution of DWH crude oil: naphthalene (7800ng/mL), acenaphthylene (590ng/mL), acenaphtehen (540ng/mL), fluorene (2550ng/mL), phenanthrene (2910ng/mL), anthracene (840ng/mL), fluoranthene (490ng/mL), pyrene (290ng/mL), benzo(k) fluoranthene (1050ng/mL), benzo(b)fluoranthene (1360ng/mL), dibenz(a,h)anthracene (2560ng/mL), and benzo(g, h, i) perylene (630ng/mL). Toxicity assays using the nematode, Caenorhabditis elegans (C. elegans), indicated a single PAH compound naphthalene, exposure increased C. elegans germ cell apoptosis which may adversely affect progeny reproduction. The number of apoptotic germ cells significantly increased from 1.4 to 2.5 when worms were treated with 10g/mL of naphthalene and from 1.3 to 2.5 and 3.5 cells in presence of 1g/mL and 5g/mL of benzo(a)pyrene, respectively. Five CYP450 genes (CYP14A3, CYP35A1, CYP35A2, CYP35A5, and CYP35C1) were significantly upregulated following 500x dilution of dispersed crude oil exposure (p<0.05). These results suggest that CYP450s may play a role in bioactivation of PAHs in crude oil, resulting in DNA damage related germ cell apoptosis.

Lu NC et al. (1983) Nematologica "A study of mineral requirements in C. elegans."

Briggs GM, Cheng AC, Lu NC

[Nematologica, 1983]

The quantities of Mg+2, Na+, K+, Mn+2, Ca+2 and Cu+2 required by the free-living nematode C. elegans were determined. An individual mineral deficieny was developed by deleting the mineral from the basal medium. Quantitative requirements of individual minerals were determined respectively by adding each mineral at various concentrations to the depleted medium. Serial subcultures and biological pre-treated media were used for the development of Mn+2, Ca+2 and Cu+2 deficiencies. It was found that most C. elegans were supported at 73 ug/ml Mg+2, 300 ug/ml Na+, 530 Ug/ml K+, 6.3 ug/ml Mn+2, 1500 ug/ml Ca+2 and 7.2 ug/ml Cu+2.

Lu NC et al. (1993) Nematologica "Carbohydrate requirement of Caenorhabditis elegans and the final development of a chemically-defined medium."

Goetsch KM, Lu NC

[Nematologica, 1993]

The carbohydrate requirement in the free-living nematode Caenorhabditis elegans was investigated. Glucose, fructose, sucrose, trehalose and glycogen were tested individually at concentrations of 0, 1.3, 6.5, 32.5, and 162.5 mg/ml as the energy source in a chemically defined medium containing C . briggsae Maintenance Medium (CbMM without glucose), 50 .mu.g/ml cytochrome c and 50 .mu.g/ml .beta.-sitosterol. Potassium acetate, used as the energy source in other studies, was not added to the medium. Therefore, carbohydrate was the major energy source for the nematode. At 32.5 mg/ml, glucose was found to support the maximal population at 80,000 nematodes/ml (100%), followed by glycogen (96%) and trehalose (73%). Population was significantly reduced when fructose (46%) or sucrose (26%) was the carbohydrate source. Toxicity was shown at 162.5 mg/ml for four carbohydrates tested, except glycogen. These results suggested that all five carbohydrates can be utilized as energy sources by C . elegans ; however, the degree of utilization of each carbohydate by C . elegans varied. Since glucose was best utilized by the nematode at 32.5 mg/ml, this concentration is recommended for future use in preparation in CbMM. Based on this study, the chemically defined medium that has been used for cultivation of C . elegans can also be modified to: CbMM (1x, with 32.5 mg/ml glucose), 50 .mu./ml cytochrome c and .mu.g/ml .beta.-sitosterol. Glucose (at 32.5 mg/ml) can be used as the major energy source in a chemically defined medium for the axenic cultivation of C . elegans .

David MA et al. (2016) Int J Parasitol Drugs Drug Resist "In silico analysis of the binding of anthelmintics to Caenorhabditis elegansP-glycoprotein 1."

Orlowski S, Lespine A, Prichard RK, Hashem S, David MA, Andre F

[Int J Parasitol Drugs Drug Resist, 2016]

Macrocyclic lactones (ML) are important anthelmintics used in animals and humans against parasite nematodes, but their therapeutic success is compromised by the spread of ML resistance. Some ABC transporters, such as p-glycoproteins (Pgps), are selected and overexpressed in ML-resistant nematodes, supporting a role for some drug efflux proteins in ML resistance. However, the role of such proteins in ML transport remains to be clarified at the molecular level. Recently, Caenorhabditis elegans Pgp-1 (Cel-Pgp-1) has been crystallized, and its drug-modulated ATPase function characterized invitro revealed Cel-Pgp-1 as a multidrug transporter. Using this crystal structure, we have developed an in silico drug docking model in order to study the binding of ML and other anthelmintic drugs to Cel-Pgp-1. All tested ML bound with high affinity in a unique site, within the inner chamber of the protein, supporting that ML may be transported by Cel-Pgp-1. Interestingly, interacting residues delineate a ML specific fingerprint involving H-bonds, including T1028. In particular, benzofurane and spiroketal moieties bound to specific sub-sites. When compared with the aglycone ML, such as moxidectin and ivermectin aglycone, avermectin anthelmintics have significant higher affinity for Cel-Pgp-1, likely due to the sugar substituent(s) that bind to a specific area involving H-bonds at Y771. Triclabendazole, closantel and emodepside bound with good affinities to different sub-sites in the inner chamber, partially overlapping with the ML binding site, suggesting that they could compete for Cel-Pgp-1-mediated ML transport. In conclusion, this work provides novel information on the role of nematode Pgps in transporting anthelmintics, and a valuable tool to predict drug-drug interactions and to rationally design new competitive inhibitors of clinically-relevant nematode Pgps, to improve anthelmintic therapeutics.

Patel MR (1997) Bios "Effects of ivermectin on eggs and first-stage larvae of nematodes."

Patel MR

[Bios, 1997]

Isolated eggs of the free-living nematode Caenorhabditis elegans and parasitic nematode Haemonchus contortus were tested for susceptibility to ivermectin, a proven antiparasitic agent. Ivermectin inhibited hatching of C. elegans eggs at the highest concentration tested (400 ug/ml). At an intermediate concentration (100 ug/ml), hatching was permitted, but all of the newly hatched first-stage larvae were killed by the drug. At lower concentrations (25 ug/ml and 6.25 ug/ml), the drug permitted hatching and survival of more than 85% of the first-stage larvae. The control eggs and first-stage larvae displayed a similarly high viability. A replicate experiment using the parasitic nematode H. contortus showed similar results, but a somewhat greater sensitivity to the drug. At the highest level tested (400 ug/ml), the eggs did not embryonate and so did not hatch. At 100 ug/ml, they embryonated but did not hatch. At a concentration of 25 ug/ml, hatching was slightly inhibited in comparison to the controls and motility of the newly hatched first-stage larvae was judged to be impaired. Eggs exposed to the lowest concentration (6.25 ug/ml), like the control eggs, had a hatching rate of greater than 85%, with no locomotor impairment. Thiabendazole, used as a positive control, caused complete blockade of embryonation.

Harrington LA et al. (1988) Mech Ageing Dev "Effect of vitamin E on lifespan and reproduction in Caenorhabditis elegans."

Harrington LA, Harley CB

[Mech Ageing Dev, 1988]

Vitamin E extends the lifespan of many animals, including the nematode Caenorhabditis elegans. Our results confirm previous studies that 200 micrograms/ml vitamin E significantly prolonged C. elegans survival (17-23%, P less than 0.05) when added from hatching to day 3, while continuous exposure, either at hatching or from 4 days prior to hatching, had little additional effect. Treatment with 100 or 400 micrograms/ml vitamin E, or with other antioxidants (80 micrograms/ml vitamin C, either alone or in combination with vitamin E, or 120 micrograms/ml N,N'-diphenyl-1,4-diphenylenediamine (DPPD] did not significantly affect lifespan. All treatments with 200 micrograms/ml vitamin E moderately reduced fecundity (total progeny) and increased the mean day of reproduction. At 400 micrograms/ml, vitamin E had severe effects, while DPPD, vitamin C, and 100 micrograms/ml vitamin E had slight effects on both these parameters of reproduction. These data suggest that vitamin E increases lifespan in C. elegans in part by slowing development in the same manner that metabolic-depressant or mildly cytotoxic drugs increase lifespan, decrease fecundity, and delay the timing of reproduction.