Inhibitors of the lipoxygenase pathway block development of Brugia malayi L3 in vitro

Brugia malayi L3 molt to the L4 stage in serum-free cultures supplemented with arachidonic, linoleic, or linolenic acids and the basidiomycetous yeast Rhodotorula minuta. These fatty acids are capable of entering the eicosanoid pathway of arachidonate metabolism, the pathway responsible for generating a number of biologically active mediators, including prostaglandins, leukotrienes, and lipoxins. To determine whether this pathway was required for L3 development, we added dual inhibitors of cyclooxygenase and lipoxygenase to in vitro cultures containing B. malayi L3. These compounds significantly inhibited L3 molting. To evaluate whether 1 or both of these pathways of arachidonate metabolism were involved in molting, we tested drugs inhibiting either cyclooxygenase or lipoxygenase. Lipoxygenase inhibitors blocked L3 molting, whereas cyclooxygenase inhibitors did not. To assess whether enzymes operating downstream of lipoxygenase were also involved in L3 molting, we added inhibitors of enzymes involved in leukotriene synthesis and found they were also capable of preventing development. We tested the same inhibitor panel on Dirofilaria immitis L3. A single lipoxygenase inhibitor and inhibitors of 2 different enzymes operating downstream of lipoxygenase disrupted D. immitis development. These results demonstrate that a lipoxygenase pathway product is required for molting of the infective stage larvae of filarial parasites.     

Tetracycline inhibits development of the infective-stage larvae of filarial nematodes in vitro

In recent years, studies have linked tetracycline treatment of filaria-infected animals with reduced adult worm burdens and decreased levels of microfilaremia. These observations are believed to be attributable to clearance of Wolbachia, intracellular rickettsial-like organisms found within filarial tissues. Although maximal worm reductions were observed when treatment was initiated early in infection, it is not known whether tetracycline inhibits development of infective-stage larvae. To address this issue, we studied the effect of tetracycline on three different species of filarial nematodes, Brugia malayi, Brugia pahangi, and Dirofilaria immitis, in a serumfree in vitro system supporting molting to the fourth larval stage. Tetracycline was capable of inhibiting L3 to L4 molting within a dosage range similar to that reported for susceptible rickettsial organisms. However, Wolbachia DNA could still be detected in nematodes from tetracycline-treated cultures. In addition, three other antibiotics with anti-rickettsial and anti-chlamydial activity (chloramphenicol, erythromycin, and ciprofloxacin) failed to inhibit L3 to L4 molting. Although tetracycline is capable of completely blocking molting of infective-stage larvae, it remains possible that this effect is due to pharmacological activities unrelated to its anti-rickettsial functions.     

Evaluation of medium supplements for in vitro cultivation of Wuchereria bancrofti

Third-stage larvae (L3) of Wuchereria bancrofti molt to the fourth stage in an in vitro culture medium composed of NCTC 135 and Iscove's modified Dulbecco's medium (1:1; v/v) supplemented with 10% human serum and a mixture of anti-bacterial and anti-mycotic agents. In the present investigation this culture medium was used to examine the effects of different concentrations of human serum, medium supplements, and serum replacements on larval growth, development, and molting. Several medium supplements and serum replacements were evaluated including hemin, Nutridoma, and a mixture of soybean lipids, bovine serum albumin, and transferrin. The supplements tested could not support larval growth and development in the absence of serum and they did not have an enhancing effect on larval growth and development in combination with human serum. A medium supplement of 30% human serum resulted in molting of 80-94% of L3s and optimum growth to the mid to late fourth stage. This culture system provides an excellent alternative to experimentally infected animals as a source of larvae undergoing the third molt and fourth-stage larvae for screening potential anti-filarial compounds and for immunologic and biochemical studies.     

Ivermectin inhibits molting of Wuchereria bancrofti third stage larvae in vitro

The effect of ivermectin or diethylcarbamazine (DEC) on Wuchereria bancrofti molting from the third to the fourth larval stage (L3 to L4) was evaluated in vitro. L3 larvae were harvested from laboratory-reared Aedes togoi 2 wk after feeding upon a microfilaremic human volunteer. The larvae were kept in an artificial medium (Franke's NI medium) with 10% human serum under an atmosphere of 5% CO2 for 20 days. Experimental tubes also contained ivermectin (0.1-1,000 ng/ml) or DEC (0.1-10,000 ng/ml). An estimated concentration of 50 ng/ml ivermectin inhibited molting in 50% of the larvae expected to molt. For DEC, this value was roughly 1,000 ng/ml. In this in vitro culture system, ivermectin inhibited the L3 to L4 molt of W. bancrofti and was roughly 20-fold more potent in this activity than DEC.     

In vitro culture of Dirofilaria immitis third- and fourth-stage larvae under defined conditions

The objective of the present study was to define culture conditions under which larval Dirofilaria immitis would molt, grow, and survive. Third-stage larvae (L3) survived for over 3 wk with a molt rate of up to 95% in a variety of media supplemented with fetal calf serum. Bovine albumin, added to several media at concentrations of 10-30 mg/ml, also proved to be an effective culture supplement for the induction of molting and for supporting larval survival. Two gas phases were tested, 5% CO2/95% N2 and 5% CO2/air; no differences were noted in larval development based on gas phase. Larvae, maintained in media with FCS or albumin for 48 hr, were capable of completing the molting process and growing in length in unsupplemented media. If the temperature at which cultures were maintained was changed from 37 C to 27 C, L3 did not molt but did survive for several weeks. Two factors required for larval D. immitis molting and growth have been identified, temperature of approximately 37 C and the presence of albumin in the culture medium. The defined culture system developed for D. immitis L3 may provide a source for collection of excretory-secretory antigens, which could prove useful in immunodiagnosis or immunoprophylaxis as well as provide a means of studying the process and requirements of filarial larval molting.     

Brugia malayi: effects of antibacterial agents on larval viability and development in vitro

Recent studies have suggested that intracellular Wolbachia bacteria are necessary for reproduction and survival of adult filarial worms. We now report results of in vitro studies of effects of antibacterial antibiotics (tetracycline, rifampicin, chloramphenicol, azithromycin, and doxycycline) on Brugia malayi infective larvae (L3) motility and molting. All of the antibiotics tested except chloramphenicol decreased L3 motility by 50% or more at 10 days, with minimal effective concentrations (MECs) of 20-100 microg/ml. Tetracyclines, rifampicin, and chloramphenicol inhibited L3 to L4 molting by 12 days in a concentration- and time-dependent manner, with MECs in the range of 1-20 microg/ml. These studies show that antibiotics active against Rickettsiaceae inhibit B. malayi L3 molting at low concentrations in vitro; higher concentrations kill the larvae. While it is possible that antibiotics directly affect filarial L3, we believe it is more likely that the effects seen are indirect effects related to bacterial killing.     

Transglutaminase activity in equine strongyles and its potential role in growth and development.

Transglutaminases (E.C. 2.3.3.13) are a family of Ca(2+)-dependent enzymes that stabilize protein structure by catalyzing the formation of isopeptide bonds. A novel form of transglutaminase has been identified and characterized that seem to play an important role in growth, development, and molting in adult and larval stages of filarial nematodes. The aim of this study was to identify the ubiquitous nature of this enzyme in other nematodes and to measure its significance to larval growth, molting, and development. For this purpose, equine Strongylus spp. were used. Activity of this enzyme was identified in extracts of larvae and adults of Strongylus vulgaris, S. edentatus, Parascaris equorum and Cylicocyclus insigne. The significance of transglutaminase in the early growth and development of Strongylus vulgaris, S. edentatus and S. equinus was tested by adding specific inhibitors, monodansylcadaverine (MDC) or cystamine (CS), to in vitro cultures of third (L3) and fourth stage larvae (L4). The viability, molting and growth of these nematode species were affected by both inhibitors. Cystamine promoted abnormal development of Strongylus edentatus L3, resulting in an aberrant expansion of the anterior end. Addition of these inhibitors to cultures of L4 also reduced growth of the three species. The results indicated that transglutaminase is present in a wide array of nematode parasites and may be important in growth and development of their larval stages.     

周期型马来丝虫感染期幼虫在人卵巢癌细胞系中的体外培养

周期型马来丝虫感染期幼虫(L_3)在三种含人卵巢粘液性囊腺癌细胞系(OMC_(685))的RPMI1640培养系统中均能蜕皮发育为L_4,幼虫最长存活66天,蜕皮率和完成蜕皮率可分别达57.1％和89.3％。在不含细胞系的培养液中,幼虫最长存活14天,基本上不蜕皮。本实验结果提示OMC_(685)细胞系可能产生某些有利于周期型马来丝虫L_3体外生存和发育的物质。     

Dirofilaria immitis: the moulting of the infective larva in vitro

A number of in vitro culture systems were tested for their ability to support the development of Dirofilaria immitis infective larvae to the fourth larval stage. In cultures of medium ML-15 containing a feeder layer of Dog Sarcoma (DS) cells larvae successfully moulted and showed a small but significant increase in length. Ultrastructural observations demonstrated that the fourth-stage cuticle was synthesized in vitro and in some larvae was fully formed by 60 hours of culture. The hypodermis of moulting larvae contained numerous multi-vesicular bodies. It is concluded that the moult in vitro is a true moult and not an atypical response of the larvae to the conditions of culture.     

The in vitro effect of albendazole, ivermectin, diethylcarbamazine, and their combinations against infective third-stage larvae of nocturnally subperiodic Brugia malayi (Narathiwat strain): scanning electron microscopy.

Scanning electron microscopy (SEM) was employed to observe the effects of ivermectin (IVM), diethylcarbamazine (DEC), and albendazole (ALB) alone, and the drugs in combination (ALB+IVM and ALB+DEC) against infective third stage larvae (L3) of nocturnally subperiodic (NSP) Brugia malayi (Narathiwat strain) in vitro. IVM, at a concentration of 10(-4) M, killed L3 within 1-2 h. The SEM data showed damage to the L3 surface and loss of regular cuticular annulations. The cuticles were grooved in the middle region of the body. In comparison with normal L3 before treatment with IVM, the cuticular surface showed transversed striations with periodic annulations. The result demonstrated that IVM showed a larvicidal activity against L3 of NSP B. malayi cultivated in vitro. Compared with those larvae in the control group, the treated larvae had no morphological changes in the cuticular surface at the head, body, and tail regions after cultivation with all drugs alone, and in their combinations at a concentration of 10(-5) M for 7 d. In this system, and at that concentration, only the larvae cultured with ALB alone remained highly motile. Although no morphological changes had been observed by SEM, those drugs used alone (IVM and DEC) and in combinations (ALB+IVM and ALB+DEC), reduced larval motility throughout the experiments at a concentration of 10(-5) M. The minimum lethal concentration (MIC) of IVM against NSP B. malayi was 10(-4) M.     

Requirements for molting of the crochet epidermis of the tobacco hornworm larva in vivo and in vitro

Summary In the tobacco hornworm,Manduca sexta, the epidermis which underlies the larval crochets is the first tissue to become independent of the prothoracic glands (PG) in a larval molt. In each successive larval molt, crochet forming cells increase in size, form hooks at their distal ends and, finally, secrete cuticle. This paper examines the endocrine requirements for competence to molt and describes parallel cultures in vivo and in vitro to define the hormonal control of crochet molting. When implanted into a fourth instar host larva prior to initiation of the last larval molt, competent crochet epidermis molted, forming crochets synchronously with its host. In the fourth instar, competence to form crochets is attained slowly during the first two days following ecdysis from the third instar. During the feeding phase of the fifth (last) instar, the crochet epidermis remains competent to molt (to form an extra sixth instar set of crochets) until the larva attains a weight of about 4.5 gm. Then, concurrent with the decline in the titer of juvenile hormone (JH) in the hemolymph, competence to form crochets declines. A similar loss of competence did not occur when fourth instar crochet epidermis was exposed to a declining JH titer by culture in either fourth instar isolated abdomens for 72 h or in fifth instar host larvae between 4 and 7 gm. Responses of crochet epidermis cultured in vitro also were examined. Competent fourth instar crochet epidermis formed crochets following 3–6 h exposure to ecdysone in vitro. Six ×10^–7M -ecdysone was required for 50% response, whereas a 10–50-fold higher concentration of -ecdysone was necessary. Although formation of morphologically complete crochets in vitro proceeded with similar time course to that in situ, no molt-induced growth occurred in vitro. When crochet epidermis was exposed to ecdysone in vitro immediately after explantation, exogenous JH was not required for molting. But when tissue was first cultured for 72 h without hormones, subsequent molting in vitro could not be elicited, although molting still could occur when the tissue subsequently was implanted into a fourth instar host. Exposure to corpora allata or to JH during the 72 h of culture in vivo partially prevented the loss in capacity to respond to ecdysone in vitro, suggesting that JH may be one factor involved directly or indirectly in maintenance of tissue responsiveness.Preliminary presentation of some of this work given at the December, 1973 Meeting of the American Society of Zoologists (Fain and Riddiford, 1973)     

Effects of a fat body extract on larval midgut cells and growth of lepidoptera

Treatment with fat body extract (FBX) from pupae of the tobacco hornworm, Manduca sexta, caused mortality in larvae of two pest lepidopterans, the gypsy moth, Lymantria dispar, and the cotton leafworm, Spodoptera littoralis. In FBX-treated larvae, the feeding rate was depressed, causing reduced weight gain and then larval death. Their midgut showed formation of multicellular layers of midgut epidermis, indicating stem-cell hyperplasia. Hence, the integument of FBX-treated larvae had a double cuticle, indicating induction of premature molting. But radioimmunoassay measurements confirmed that the amount of ecdysteroids in FBX was too low to be responsible for the molt-inducing effects observed after treatment with FBX. With midgut stem cell cultures in vitro, addition of FBX to the culture medium stimulated cell proliferation and differentiation in a concentration-dependent manner. This effect was compared with those of insect molting hormones, ecdysone and 20-hydroxyecdysone; an ecdysteroid agonist, RH-2485; and a purified protein from FBX (multiplication factor). This article describes the mode of action of FBX and possible interplay between fat body factor(s) and insect hormones in the development and metamorphosis of the insect midgut.     

Development of Onchocerca volvulus (Filarioidea: Onchocercidae) in the West African black fly Simulium yahense (Diptera: Simuliidae) in Liberia.

Simulium yahense black flies infected with microfilaria of Onchocerca volvulus were kept in a defined insectary environment in Liberia, West Africa. A daily sample of infected flies was dissected for larvae developing in the thoracic muscles and examined for growth in stadial development. Microfilariae ingested by black flies transformed to the L1 larval stage without molting. Successive larval development included molting to the L2 stage and, finally, to the L3 stage, which was infective in humans. The cephalic cap, consisting of a laterally located hook and central stoma, occurs in the first larval stage. The caudal appendix and the laterally located anal opening are apparent in the L1 larva. In the L2 stage, the cephalic cap is lost and the large circular stoma becomes surrounded with elevated flaps. The caudal appendix was lost after larvae molted to the L3 stage, and in its place, 3 terminal papillae developed. Sense organs, such as 2 opposing phasmids and 8 papillae that were arranged into 2 circles, developed in the cephalic region of the L3 larva. The evidence of pathological consequences due to the presence of the L3 larva in the fly host are illustrated and discussed.     

Effects of Glyptapanteles liparidis (Hym.: Braconidae) parasitism, polydnavirus, and venom on development of microsporidia-infected and uninfected Lymantria dispar (Lep.: Lymantriidae) larvae

Effects of parasitism, polydnavirus, and venom of the endoparasitoid Glyptapanteles liparidis on Lymantria dispar larvae infected with the microsporidium Vairimorpha sp. and uninfected hosts were studied. We tested the impact on growth and development of hosts, as well as on microsporidian infection. Both parasitism and polydnavirus/venom treatment alone caused a slight increase in growth rate and relative growth rate in uninfected fourth instar hosts. This effect was more pronounced with the addition of Vairimorpha infection. With no parasitism, however, infection reduced host growth markedly. Microsporidiosis delayed larval molts of L. dispar, and additional polydnavirus/venom treatment or parasitization induced significantly earlier molting. Polydnavirus/venom treatment of uninfected L. dispar resulted in prolonged larval development due to supernumerary molts and in higher pupal mortality. Infected larvae treated with polydnavirus/venom died earlier than infected larvae that were not treated and produced more Vairimorpha spores per unit fresh mass of the host.     

Recovery, distribution, and development of Acanthocheilonema viteae third- and early fourth-stage larvae in adult jirds

Living third- and fourth-stage larvae (L3 and L4) of Acanthocheilonema viteae were recovered quantitatively from adult Meriones unguiculatus within the first 10 days after subcutaneous inoculation of 60 arthropod-derived larvae (mL3). The average recovery of the inoculated larvae was about one third (28.5%), and the majority (87.7%) were found in muscular tissues. Seventy-two hours after inoculation, larvae could be isolated from all body locations, although the majority still was found near the site of inoculation. Morphological and biometrical data indicated that, at least until molting, the development of the larval population was not synchronous, with molting occurring over a period of 48 hr on days 7 and 8 postinoculation. The stomatal rings of postinvasive L3's and L4's were distinguishable structurally and could be used as stage-specific determinants. Immediately after infection, L3's showed a linear growth in diameter; rapid longitudinal growth started after the molt, leading to a doubling in the length of L4's within 4 days. The time course of shedding was reconstructed in detail using isolated L3/L4 intermediates.     

Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone

Dopa decarboxylase (DDC) which converts dopa to dopamine is important for cuticular melanization and sclerotization in insects. An antibody to Drosophila DDC was found to precipitate both DDC activity and a 49-kDa polypeptide synthesized by the epidermis of molting Manduca larvae. Using the Drosophila DDC gene, we isolated the Manduca DDC gene which on hybrid selection produced a 49-kDa translation product precipitable by the Drosophila DDC antibody. The 3.1-kb DDC mRNA appeared 12 hr after head capsule slippage (HCS) and reached maximal levels 7 hr later. Peak expression was twofold higher in melanizing allatectomized larvae and could be depressed to normal levels by application of 0.1 micrograms juvenile hormone I at HCS. Infusion of 1 microgram/hr 20-hydroxyecdysone (20-HE) for 18 hr beginning 2 hr after HCS or addition of 1 microgram/ml 20-HE to the culture medium for 24 hr prevented the normal increase in DDC mRNA. When Day 2 fourth instar epidermis was explanted before the molting ecdysteroid rise and cultured with 1-3 micrograms/ml 20-HE for 17 hr and then for 24 hr in hormone-free medium, DDC expression was three- to fourfold higher than that in epidermis cultured in the absence of hormone. Twelve or more hours of incubation with 20-HE was required for an increase in DDC mRNA, but continuous exposure to 20-HE prevented the increase. In all cultures an initial rapid increase in DDC mRNA was observed which decayed with time in vitro and apparently was associated with the wound response. Thus, ecdysteroid during a larval molt is necessary to program the later expression of DDC, but the subsequent decline of the ecdysteroid is required for this expression to occur.     

Anisakis simplex: CO(2)-fixing enzymes and development throughout the in vitro cultivation from third larval stage to adult

We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).     

The influence of host sex on the L3 to L4 molt of Brugia malayi

Immunocompetent male mice are more susceptible to experimental infection with Brugia spp. than are females. Because permissive male SCID mice (severe combined immunodeficient mice), which lack T and B cells, also possess higher worm burdens, the mechanism is not solely immune mediated. Recovery of fewer adult worms from the female SCID mouse suggests that females do not provide sufficient nutrients for larval growth. This study assessed the potential of the female SCID mouse to support the L3 to L4 molt of Brugia malayi. Unexpectedly, worms grown in females molted at earlier time points of recovery than those harvested from males. This suggests that the early stage of development of B. malayi is delayed in the male murine host. To determine whether the effect of host sex on molting may be similar in humans, worms were cultured in media supplemented with serum from male or female donors. Worms grown in serum obtained from female donors exhibited a significantly higher percentage of complete molts over those cultured with serum from males. Host-derived molecules required for the L3 to L4 molt may be more abundant in the female, perhaps allowing the worms to survive a vulnerable developmental stage in a less permissive environment.