The relevance of in vitro anthelmintic screening tests employing the free-living stages of trichostrongylid nematodes

The response of the free-living stages of Nippostrongylus brasiliensis, Nematospiroides dubius, Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia ostertagi to a wide variety of antiparasitic agents in vitro was investigated. All the major broad spectrum veterinary anthelmintics showed good activity against each of these worms with EC50 values varying from about 0.0002 mg/l for certain benzimidazoles and ivermectin to about 6.5 mg/l for febantel. Of 22 known narrow spectrum anthelmintics useful only against H. contortus and/or helminths other than trichostrongyles, only 10% showed good activity at concentrations equal to or less than 10.0 mg/l. Further, only one of 15 antiprotozoal agents showed good activity in these tests at the 10.0 mg/l level. The screening test employing free-living Nippostrongylus brasiliensis was selected for an extended trial where the evaluation of 1400 miscellaneous organic chemicals was undertaken. Approximately 10% of these showed activity at concentrations equal to or less than 10.0 mg/l. It is concluded that in vitro screening tests employing the free-living stages of these five genera of nematodes afford simple yet effective means for selecting relevant compounds for further evaluation as possible leads to new broad spectrum anthelmintics for use in ruminants. However, tests using the free-living stages of these worms, including H. contortus, are unsuitable for detecting narrow spectrum 'specifics', e.g., for the treatment of haemonchiasis.     

The effect of oxygen and carbon dioxide on the development of the free-living stages of Strongyloides ratti in axenic culture

The effect of oxygen and carbon dioxide concentration on the development of the free-living stages of Strongyloides ratti in axenic culture was examined. Hatching of S. ratti eggs was inhibited at very low oxygen levels. Development of all organisms was inhibited in medium gassed with less than 4% O2 in 0.03% CO2 with the remainder N2. Between 8 and 21% O2 there was no significant difference in the percentage developing directly to filariform larvae, free-living females, or free-living males. Cultures gassed with CO2 concentrations greater than 1% in 21% O2 with the remainder N2 manifested inhibited development to filariform larvae, whereas concentrations between 5 and 7.5% enhanced development to free-living females. There was a positive effect on increasing the CO2 concentration from 0.03% to 5% on development to filariform larvae and male worms at levels of 1% and 2% oxygen in nitrogen, respectively. Direction of development, either directly to the filariform larva or to the free-living female, was influenced by CO2 and O2 concentrations encountered by the egg or the newly hatched larva of the parasitic female in axenic culture. The axenic culture system permits an important experimental approach to the study of factors modulating the differentiation of the free-living stages of Strongyloides.     

Cadmium effects on development and reproduction of Oncopeltus fasciatus (Heteroptera: Lygaeidae)

Newly hatched nymphs of the insect Oncopeltus fasciatus were exposed to various concentrations of CdCl2 administered in drinking water until the end of adult life. Significant nymphal mortalities were observed at concentrations above 30 mg Cd/l (corresponding to the LC50). The duration of the nymphal stages increased in proportion to the Cd concentration; at the lowest Cd concentration of 10 mg Cd/l, the median duration was significantly prolonged by one day, while at the highest concentration of 100 mg Cd/l it was increased by 10 days over the control group. The weight of newly emerged adults lineally decreased with Cd concentration, being reduced to half the weight of controls at 100 mg Cd/l. In addition, a proportionality between delay in development and weight reduction was found in Cd-treated adults. Survival of adult females was decreased at concentrations higher than 10 mg Cd/l, while males were only affected at 30 mg Cd/l or higher doses. Reproduction was the most affected parameter. Oviposition rate, fecundity and fertility of females exposed to 10 mg Cd/l were significantly lower than controls (73%, 58% and 55% relative to controls, respectively). Hatchability of the eggs laid by treated females was also reduced. These results show that development and reproduction of O. fasciatus are seriously impaired at sublethal Cd concentrations.     

Juvenile toxicity assessment of d,l-methylphenidate in rats

BACKGROUND: The oral administration of d,l‐methylphenidate (MPH) was designed to encompass the major part of postnatal development in the rat and to evaluate potential chronic effects. METHODS: Wistar Hannover rats were cross‐fostered on postpartum day 0 (day of birth) and were administered MPH at doses of 5, 50, and 100 mg/kg/day (mpkd) on postpartum days 7 to 70. Clinical signs, body weight, food consumption, developmental, behavioral, clinical/anatomic pathology, toxicokinetic, and fertility evaluations were conducted. RESULTS: MPH‐related effects on clinical signs, body weight, and behavior tests were noted. Increased locomotor activity and cage biting/chewing occurred at ≥5 mpkd (females) and ≥50 mpkd (males) and were absent after dosing ceased. Body weight parameters were decreased at ≥50 mpkd and were comparable to controls at 5 weeks' recovery. Open field motor activity tests conducted 2 weeks after dosing ceased revealed decreased peripheral beam breaks at ≥50 mpkd. Passive avoidance tests conducted 3 weeks after dosing ceased indicated decreased females reaching learning criterion at 100 mpkd. This is considered of nominal significance as there were no effects in the water maze test or retention in passive avoidance test. After multiple doses, females exhibited higher exposures than males and exposures were reduced in all groups in comparison to those after a single dose. CONCLUSIONS: These results suggest that MPH can produce enduring behavioral effects in rats. The no‐toxicologic‐effect‐level was 5 mpkd, associated with AUC_{(0–24 h)} racemate values in males and females, respectively, of 101 and 153 ng.h/mL after chronic dosing. Birth Defects Res (Part B) © 2008 Wiley‐Liss, Inc.     

Response of pre-adult and adult stages of Trichuris muris to common anthelmintics in mice.

The common anthelmintics, oxantel, mebendazole, albendazole and pyrantel were assessed for their comparative activity against Trichuris muris in mice. Mice were infected with T. muris and the infection was maintained by a brief cortisone administration during the second week of infection. Mice carrying the infection with different life cycle stages, viz. fourth stage larvae (L4), pre-adult and adult stages were dosed with anthelminitics. The worm burdens in control infection groups varied although infection dose and other conditions were uniformly followed. With various dose regimens tested, oxantel was highly potent; it eliminated completely pre-adult and adult stages, respectively at 25 and 12.5 mg kg-1 dose levels with significant activity also against adult worms at a 1.56 mg kg-1 dose level and against pre-adults at a 6.25 mg kg-1 level. Pre-adults required twice the dose given to that of adults for complete (100%) activity. Mebendazole was the next most active; a dosage of 37.5 mg kg-1 was completely active against pre-adults whereas a dosage of 2 x 50 mg kg-1 was required for complete elimination of adult worms. In addition, about 90% of the worms were eliminated with a single dose of 150 mg kg-1. However, a significant activity was seen against adults at a 25 mg kg-1 level and pre-adults at 37.5 mg kg-1, the lowest level tested. In comparison, albendazole did not induce complete clearance of pre-adult and adult stages even when tested at dose levels as high as 150 and 2 x 75 mg kg-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility of various developmental stages of the maize weevil, Sitophilus zeamais Motschulsky (Col., Curculionidae) to methyl iodide in brown rice

Abstract:  The efficacy of methyl iodide (MI) as a fumigant against all developmental stages of the maize weevil, Sitophilus zeamais Motsch. was investigated. Tests were conducted with concentrations of 1.5, 1.8, 2.1, 2.4, 2.7 and 3.0 mg/l, for a 6-h exposure period. Values of LC₅₀, LC₉₅ and LC₉₉ of MI for immatures and adult stages were determined. The present laboratory tests showed that MI was toxic to various life stages of S. zeamais at relatively short exposure periods. At the LC₅₀ and LC₉₅ levels, the most susceptible stage was the egg stage followed by larvae, pupae and adults (1-day mortality). The egg was found to be most susceptible to MI, requiring 0.81 and 2.16 mg/l for 50 and 99% mortality, respectively, while the adult was most tolerant, requiring 2.30 and 3.02 mg/l for 50 and 99% mortality, respectively, based on 1-day mortality count. Pupae were less susceptible to MI than egg and larvae, requiring 1.47 and 3.19 mg/l for 50 and 99% mortality, respectively. Based on the present toxicity tests, MI has the potential for use as a fumigant to control all developmental stages of the maize weevil, S. zeamais .     

On the mutagenicity of methadone hydrochloride. Induced dominant lethal mutation and spermatocyte chromosomal aberrations in treated males

The mutagenicity of methadone hydrochloride was tested in male mice using the dominant lethal mutation technique and the spermatocyte test of treated mice. Male mice of C3H inbred strain received one of the following doses, 1, 2, 4 or 6 mg/kg body weight once a day for 3 consecutive days. Another group of mice served as control and received saline instead. Treated males were then mated to virgin females at 3-day intervals for a period of 45 days. Pregnant females were dissected at mid-term and the corpora lutea and intrauterine contents were recorded. The spermatocytes of treated males were examined 45-50 d after treatments with methadone and abnormal pairing configurations were scored. The methadone treatment was found to increase the rate of preimplantation deaths consistently in all post-meiotic stages with all doses used. In addition, the higher doses, 4 and 6 mg, affected spermatogonia stages. Quantitatively, the dose-response relationship cannot be demonstrated though the spectrum of effect increased with higher doses as more spermatogenesis stages became more sensitive to the treatment. In many cases the frequency of live implants showed a positive correlation with preimplantation deaths in contrast with the frequency of early deaths which showed only sporadic variation. The mutation indices based on total embryonic death indicate that methadone hydrochloride affected several stages of germ-cell maturation namely, spermatozoa (M.I. 14-35), late spermatids (M.I. 15-48), early spermatids (M.I. 14-50), late spermatocytes (M.I. 15-43) and spermatogonial stages (M.I. 12-63). Chromosome analysis at diakinesis-metaphase 1 revealed significant increase in the frequency of sex chromosome and autosome univalents with different doses of methadone. The smallest dose applied was quite effective and the data represent direct dose-response relationship. Of the multivalent configuration, the most frequent type was chain quadrivalents. The frequencies of total translocations per cell were estimated as 0.1, 0.16 and 0.2 for the 4 applied doses illustrating a dose-response relationship for the doses: 1, 2 and 4 mg, whereas with the higher dose, 6 mg, an abrupt decrease was apparent (0.05). This study calls for concern regarding the possible genetic hazards this drug may impose upon human populations.     

Review of the Order Strepsiptera

The morphology, biology and life history of the immature stages, the free-living and the neotenic females (of the suborders Mengenillidia and Stylopidia respectively) and the free-living males of the order Strepsiptera are discussed. Strepsiptera are entomophagous parasitoids and are known to parasitize seven orders and thirty-five families of Insecta. The morphological and physiological changes they cause to the host insect are outlined. The classification of the order is revised; the geographical distribution, phylogenetic system and keys to the families, subfamilies and genera (when possible) are given. As the sexes are dimorphic, separate keys are provided for adult males and neotenic females.     

The influence of aging on the normal oral glucose tolerance

381 glucose intake normal curves were studied according to the Diabetes Data Group new classification in healthy persons between 10 and 80 years in order to assess the influence of the age upon the normal glucose tolerance. Such an influence, which was evident in all the subjects, turned out to be more important in women with respect to me. In fact, males showed an increase, per decade, of about 1 mg/dl in fasting glycemic levels, of about 6 mg/dl at 60', of 4 mg/dl at 120', while in females there was an increase of about 2 mg/dl in fasting glicemic values, of about 6 mg/dl at 60' and of about 5 mg/dl at 120'. No meaningful correlation between age and insulinemic values was found at all considered points, either in males or in females. The reasons of the decreased glucose tolerance with aging and of its different behavior in the two sexes are discussed.     

Physical fitness of the Czechoslovak population between the ages of 12 and 55 years. Pulmonary ventilation.

Examination of ventilation function in an average population showed that maximum minute ventilation in adults fell with advancing age. Males had higher values than females, the highest values being attained, in both sexes, at 18 (117 and 83 1/min respectively). Tidal volume during loading, expressed as a percentage of vital capacity, rose with advancing age in all groups of males and females (from 53% at 18 to 64% at 55 in males and from 51% to 59% in females). The ventilation equivalents for O2 and CO2 attained optimum values at a load of 100 W in males and at 50 W in females. On increasing the load they rose in both sexes--in females more than in males. Percentual utilization of O2 from the inspired air was greatest in males at a load of about 100 W (4.8--5%) and in females at about 50 W (4.3--4.8%). At higher loads it fell faster in females than in males. In males, the respiratory quocient attained the value 1 at a load of about 2 W/kg body weight and in females at 1.5 W/kg. At maximum loading it was over 1 in all the age categories. No differences between males and females were observed, but males, in maximum loading, gave the greatest absolute and the greatest relative performance.     

Metaparasitylenchus hypothenemi n. sp. (Nematoda: Allantonematidae), a parasite of the coffee berry borer, Hypothenemus hampei (Curculionidae: Scolytinae)

Metaparasitylenchus hypothenemi n. sp. (Nematoda: Allantonematidae) is described from the coffee berry borer, Hypothenemus hampei (Ferrari) (Curculionidae: Scolytinae), in Chiapas, Mexico. This species differs from other members of the genus by its small size, annulated cuticle, lateral fields with 3 ridges, free-living stages with an excretory pore located between the pharyngeal gland orifices, a distinct stylet with basal swellings in free-living females, a postvulval uterine extension, a thin stylet lacking basal swellings in males, 2 separate spicules, a gubernaculum, and a peloderan bursa. Parasitic females are white, with a straight or slightly curved body and are ovoviviparous. Third-stage juveniles emerge from parasitized beetles and molt twice before reaching the adult stage. Because the coffee berry borer is the most important pest of coffee throughout the world and this parasite partially or completely sterilizes female beetles, it is worthy of further investigation as a potential biological control agent.     

Toxicity of Selected Insecticides to the Spined Soldier Bug, Podisus maculiventris (Heteroptera: Pentatomidae)

Susceptibility of Podisus maculiventris to the insect growth regulator teflubenzuron, the carbamate methomyl, the pyrethroid deltamethrin, and the bioinsecticide Bacillus thuringiensis subsp. kurstaki (Bactospeine) was evaluated in the laboratory. Newly-moulted fourth instars and seven-day-old female adults were exposed to formulated materials of each compound via ingestion for 48 h. Teflubenzuron was highly toxic to fourth instars (LC 50 = 14.7 mg a.i. l -1 ) of the predator, but proved to be harmless to female adults. Both fourth instars and females suffered the highest mortality when they were exposed to methomyl (LC 50 = 5.4 and 10.6 mg a.i. l -1 , respectively). Deltamethrin was relatively safe for the predator in either stage; however, fourth instars were more tolerant to the pyrethroid than females (LC 50 = 158.8 and 43.4 mg a.i. l -1 , respectively). Neither fourth instars nor females were affected by B. thuringiensis at 10 000 mg formulated material l -1 . Reproductive capacity of the predator was also examined when applying field concentrations of teflubenzuron (200 mg a.i. l -1 ) or deltamethrin (12.5 mg a.i. l -1 ) via ingestion. Treatment with deltamethrin only prolonged preoviposition period. A marked decline in egg hatch was observed when the insects were exposed to teflubenzuron compared with the control (7 vs 63%, respectively). Fourth and fifth instars that had ingested sublethal doses of teflubenzuron demonstrated longer developmental times than did those of the control. Ingestion experiments suggest that use of deltamethrin and B. thuringiensis may be compatible with releases of P. maculiventris . In contrast, populations of the predator may be harmed when methomyl or teflubenzuron are applied.     

The toxicity of textile reactive azo dyes after hydrolysis and decolourisation

The toxicity of C.I. Reactive Black 5 and three Procion dyes, as found in textile effluents, was determined using the bioluminescent bacterium Vibrio fischeri. Hydrolysed Reactive Black had a slightly greater toxicity than the parent form (EC(50) 11.4+/-3.68 and 27.5+/-4.01 mg l(-1), respectively). A baffled bioreactor with anaerobic and aerobic compartments was used to decolourise hydrolysed Reactive Black 5 in a synthetic effluent. Decolourisation of hydrolysed Reactive Black resulted in an increased toxicity (EC(50) 0.2+/-0.03 mg l(-1)). Toxicity was not detectable when decolourised Reactive Black 5 was metabolised under aerobic conditions. No genotoxicity was detected after the decolourisation of either the parent or the hydrolysed reactive dyes, either in vitro or in the bioreactor. The toxicity and genotoxicity of decolourised C.I. Acid Orange 7 was due to the production of 1-amino-2-naphthol (EC(50) 0.1+/-0.03 mg l(-1)).     

Side-effects of three neem (Azadirachta indica A. Juss) products on the predator Macrolophus caliginosus Wagner (Het., Miridae)

The side-effects of three neem formulations (Neem-Amin EC, Stardoor and B.P. 20/S) were tested on the mirid predator Macrolophus caliginosus Wagner in the laboratory. Direct toxicity tests on 1st instar nymphs exposed to fresh dry residues on glass plates at different doses demonstrated that all the products are harmful to the insects with LD₅₀ values much lower than the maximum recommended rate (1.217, 0.264, 1.083 mg a.i./l instead of 15, 31.5 and 80 mg a.i./l for Neem-Amin EC, Stardoor and B.P. 20/S, respectively). Moreover a reduction of fecundity of the surviving females was assessed with Neem-Amin EC and B.P. 20/S. Persistence tests were carried out on sharp pepper plants treated at the maximum recommended rate. High mortality was recorded when the insects were introduced onto the plants just after the treatment, but no significant differences compared with the controls were observed 5 days after the treatment and any consequence on the fecundity of surviving females was detected. Our experiments showed how azadirachtin can be noxious to M. caliginosus , but the short persistence makes this active ingredient a promising solution in integrated pest management programmes, when a lapse of time is guaranteed between the treatment and the introduction of the predator.     

Antifertility effect of busulfan and procarbazine in male and female coyotes

Antifertility effects of two cytostatic agents, busulfan and procarbazine, were evaluated using 43 captive breeding pairs of adult coyotes. Nineteen pairs served as untreated controls. Only the male or female of remaining pairs was treated. Females received either 8 mg busulfan/kg or 6 mg procarbazine/kg just prior to onset of the breeding season. Males were treated once with either 8 mg busulfan/kg just before onset of breeding or with 4 mg busulfan/kg or 6 mg procarbazine/kg about 1 mo before onset of the breeding season. Uterine implantation sites were counted in females of all breeding pairs postpartum via laparotomy. Busulfan given to males at 4 mg/kg or to either females or males at 8 mg/kg significantly reduced implantation sites compared to untreated controls. Thus, busulfan may be successful in controlling population in coyotes in the field where both the male and female of a breeding pair may ingest the compound. However, multiple doses at a lower rate would be preferred because dosages greater than 10 mg/kg resulted in mortality. Although procarbazine has a mode of action similar to busulfan, doses of 6 mg procarbazine/kg did not reduce implantation sites or disrupt normal spermatogenesis. Increased doses need to be evaluated before effectiveness of procarbazine for coyote population control can be determined.     

Reproduction, growth and bionomics of Dynamene bidentata (Crustacea: Isopoda)

Laboratory and field investigations show that there are five distinct marsupial stages and eight post-marsupial stages in the development of the isopod Dynamene bidentata (Adams). Extensive sexual dimorphism is apparent, beginning at stage 6, and females undergo extensive tissue reduction at the moult to the eighth stage. Young stages inhabit and feed on intertidal algae, and the non-feeding adults (stage 7 and 8 females, and stage 8 males) shelter in crevices or empty barnacle tests. Average brood numbers are fairly high (c. 90) and the average marsupial mortality rate is about 36%. Release of young takes place in May/June, after which the females die, and a resident population of males remains in the crevices to be joined by newly maturing females and males from August onwards each year. The occurrence of two breeding seasons in males, but not in females, explains how the sex ratio of males to females as they leave the algal habitat (1:9) is about half the ratio of males to females in the reproductive habitat (1:4).     

The dominant lethal effect of dietary triethylenemelamine.

Triethylenemelamine (TEM) was administered in the diet to adult male mice at doses of 0.1, 0.3, 1, 10 or 50 mg/kg body weight for 45 days or at doses of 0.1 or 0.3 mg/kg b.w. for 10 days. As a comparison, male mice were treated intraperitoneally with 5 daily doses of 0.25 or 0.5 mg TEM/kg b.w. At the end of the treatment period, males were mated sequentially with 2 untreated virgin females each for 2 or 3 weeks. Near mid-pregnancy the number of implantation sites and fetal deaths were determined. TEM, administered in the diet at 10 or 50 mg/kg b.w. for 45 dyas, was lethal to male mice. Surviving males from the 1 mg/kg level failed to impregnate any females during the two matings. TEM, given in the diet at 0.1 or 0.3 mg/kg for 10 or 45 dyas, decreased fertility and increased dominant lethal mutations in a dose and time dependent manner. These results were comparable to those obtained from males treated i.p. with TEM at 0.25 or 0.5 mg/kg b.w.     

The induced spawning of roach, Rutilus rutilus (L.), with the antioestrogens clomiphene and tamoxifen

The induced spawning of gravid roach was investigated using the antioestrogens clomiphene and tamoxifen. Three dose levels ofeach were used: 0·1, l or 10 mgkg^-1, given twice with a 4-day interval to groups of eight fish with saline controls. Running males were randomly distributed. Tamoxifen, when injected at a rate of l mg kg^-1 was found to be most successful. This treatment induced ovulation in five of the six females, and profuse spawning on 3 consecutive days, 4 days after the first injection. Clomiphene induced ovulation and spawning in one of six females at 2 × 10 mg kg^-1, and two of eight females at 2 × l mg kg^-1, respectively 6 and 7 days after the first injection. The eggs produced showed normal devel-opment. No control fish ovulated or spawned. Both drugs probably act by indirect mechanisms, blocking sex steroid feedback inhibition of gonadotropin (GtH) secretion at the pituitary, thereby inducing a plasma GtH surge. The results of this experiment suggest that tamoxifen may be an effective substitute for pituitary preparations in the induced spawning of fish.     

The male genital tract and the nipples of male and female offspring of rats given the non-steroidal antiandrogens DIMP and Sch 13521, during pregnancy.

Male and female offspring of rats given antiandrogens, the steroidal BOMT or the non steroids DIMP or Sch 13521, daily during the last third of pregnancy were studied. Detailed examinations were made of the genital tract of male, and of the nipples of male and female offspring. A) Male offspring. 1) Genital tract of newborn and 31-91 day old males : Modifications of the development of accessory sexual tissues were found in all treatment groups. As indicated by the severity of deviations from normal (morphology and weight of sex accessories), the antiandrogenic effect of the preparations, in the doses given to the mother rats, increased from BOMT (50 or 75 mg/day) via Sch 13521 (30 mg/day) and DIMP (50 or 60 mg/day) to Sch 13521 (60 mg/day). 2) Nipples of 10-60 day old males : Whole mount preparations were made unilaterally of the row of 6 mammary glands with nipples. The number of intact and abnormal nipples, respectively, was recorded. The relation between intact and abnormal nipples served as indicator of the efficiency of the antiandrogenic substances studied. The result showed that the antiandrogenic effect increased from BOMT to Sch 13521, 60 mg, in the same order as that arrived at from studies of the genital tract. The combined results obtained from the male offspring indicated that the tissues of the genital region, the growth and differentiation of which was most readily impaired by antiandrogens, were the same as those known from other work to be stimulated most easily in female rat fetuses by testosterone. B) Female offspring. Nipples of 31-60 day old females were judged from whole mount preparations and recorded as in the males. The nipples of adult virginal females were examined macroscopically. The same procedure was applied to lactating females, but the results were controlled in consecutive lactational periods and at autopsy. The 3 groups of females showed uniformly that 1) offspring of rats given BOMT during pregnancy had many (about 50 per cent) malformed nipples and 2) the treatment of mother rats with DIMP or Sch 13521 did not influence the development of nipples in female offspring. The result was assumed to be due to an androgenic effect of the steroidal antiandrogen, BOMT, on the nipple anlage.