Nippostrongylus brasiliensis: factors influencing movement of males toward a female pheromone

Single, responding males of Nippostrongylus brasiliensis exhibited a dose-dependent movement toward a pheromone source that was derived from incubation or homogenization of female helminths in Tyrode's solution. Increases in female homogenate beyond an optimum concentration resulted in reduced male movement toward the pheromone source. Crowding of male N. brasiliensis prior to their exposure to female pheromone gradients diminished the males' response. However, crowding of females had no effect on response to the pheromone. The locomotory responses of males exposed to various mixed male and female pheromone sources reaffirmed earlier suggestions of a pheromone feedback system in which males influence the pheromone released from females.     

Nippostrongylus brasiliensis: Effect of cyclophosphamide treatment on worm expulsion in rats

The precise immunological mechanisms associated with expulsion of the gastrointestinal nematode Nippostrongylus brasiliensis remain controversial. In order to investigate the effects of drug-induced immunosuppression on parasite burdens and expulsion, various regimens of cyclophosphamide were administered to parasitized Wistar rats. It was observed that both the number of worms established from an infective dose of 3000 larvae and the time of expulsion were markedly increased with higher doses of cyclophosphamide. Thus, at the highest sublethal level of treatment (100 mg/kg), 82% of the infective dose was recovered at Day 9 postinfection compared with 51% in nontreated controls. Furthermore, in such treated rats expulsion was delayed in 6 days beyond that of nontreated animals. As cyclophosphamide, at the levels used in the present study, is known to primarily effect B-cell function, the results support the view that antibody-mediated responses play an essential role in worm expulsion.     

Nippostrongylus brasiliensis: Mast cell kinetics at small intestinal sites in infected rats

Mast cell kinetics during infection with the nematode Nippostrongylus brasiliensis were studied at various sites in the small bowel of rats and in heterotopically transplanted isografts of foetal small intestine placed under the kidney capsule. Infection produced an increase in the number of mast cells not only in the proximal jejunum, where most of the worms are located, but also in the distal ileum and in isografts of small intestine. However, globule leucocyte infiltration of the gut epithelium was confined to the proximal small intestine and did not occur in the distal ileum or isografts. These results show that the mast cell increase in the small bowel of N. brasiliensis-infected rats is a property of the whole organ, and is not restricted to sites of worm infection; but that in contrast, globule leucocyte infiltration of the epithelium is dependent upon the presence of worms within the bowel lumen.     

Nippostrongylus brasiliensis: Peripheral blood leucocyte response of rats,with special reference to basophils

Changes in peripheral blood leucocytes were followed in male August rats given one or two infections with the parasitic nematode, Nippostrongylus brasiliensis. During the initial infection, there was a biphasic increase in total numbers of leucocytes, lymphocytes, neutrophils, large mononuclear cells, and eosinophils. All except eosinophils fell rapidly to normal levels as the parasites were expelled, but eosinophils were elevated much longer. All these cell types increased in number to a single peak 5 days after reinfection. Basophils were detected at very low levels in uninfected rats (0.06% or $\text{1}\text{1600}$ leucocytes) and increased in number to a peak 13 days after initial infection, at which time they represented about 4.5% of total leucocytes, an 80-fold increase compared with the number in normal rats. In reinfected rats, the basophilia occurred more rapidly than in a primary infection, suggesting that the appearance of these cells in the circulation is probably an immunologically mediated event.     

Nippostrongylus brasiliensis: Indirect fluorescent antibody studies of immunity in mice

Antibodies against Nippostrongylus brasiliensis were demonstrated by the indirect fluorescent antibody technique in four pools of sera from mice 3 weeks after one or more exposures to this parasite. These antibodies combined with antigens in sections of N. brasiliensis and included IgG, IgM, and IgA classes of immunoglobulins. Antibodies of the first two classes produced intense fluorescence in many areas of the parasites, whereas IgA usually evoked only minimal reactions at most of these same sites. Results produced by all pools of antisera were similar, while pools of sera from nonparasitized mice were essentially negative.The principal site of fluorescence in sections treated with antisera was in the cellular cytoplasm and in the microvillar layer of the intestinal epithelium. The nuclei in these cells did not fluoresce. The cuticle, lateral canals, body wall musculature, and reproductive organs fluoresced to a lesser extent. In this group of structures, the most notable fluorescence occurred in the cytoplasm of developing ova, in shells of completed eggs, and in muscle cells of the vagina. Slight autofluorescence was observed in the cuticle, cytoplasm of cells of the lateral canals, substances in the lumina of the lateral canals, and in the extracellular substance of the excretory glands.     

Nippostrongylus brasiliensis: Peripheral leukocyte responses and correlation of basophils with blood histamine concentration during infection in rats

Rats infected on Day 0 with 3000 infective L₃ larvae of Nippostrongylus brasiliensis, and uninfected controls, were monitored daily through Day 23 postinfection for changes in peripheral leukocytes and blood histamine concentrations. A generalized leukocytosis was observed between Days 7 and 18, the period leading up to and immediately following the time of expulsion of adult worms from the small intestine. The total number of lymphocytes was elevated between Days 11 and 17 post-infection; however, there was no change in the percentage of lymphocytes relative to other white blood cell types. The total number and percentage of monocytes were no different from controls, with the exception of Day 5 postinfection. On that day, there was a significant elevation in the number (614/mm³ blood in infected rats, as compared to 160/mm³ blood in controls) and relative proportion (2.7% of total leukocytes in infected animals, compared to 0.8% in controls) of monocytes, coinciding with the termination of the pulmonary migration of larvae. A period of moderate neutrophilia occurred between Days 7 and 12, but this was not accompanied by any changes in the proportion of neutrophils. A biphasic eosinophil response was observed. An early elevation of eosinophils occurred between Days 3 and 5, corresponding to the period of larval migration through the lungs. A second period of eosinophilia began on Day 11, when worm expulsion was beginning, and continued through Day 19, i.e., beyond the period of worm expulsion. Basophilia was observed as early as Day 6 after infection, rising to a peak on Day 13 (6.8% of total leukocytes in the infected animals, as compared to 0.5% in controls), and declining thereafter, but remaining above control levels until termination of the experiment on Day 23. The histamine content of blood samples, as determined by an enzymic-isotopic assay, closely paralleled the development and decline of basophilia; histamine levels also peaked on Day 13 postinfection (422.5 pg histamine/mm³ blood in infected rats, compared to 66.0 pg histamine/mm³ blood in controls). As basophilia progressed during the course of infection, there was a decline in the amount of histamine per basophil. In uninfected rats and during the first week after infection, basophils contained about 1.5–2.0 pg histamine per cell. In the third week of infection, there was about 0.6 pg histamine per basophil. The time course of the basophilia suggests that these cells may be involved in the expression of immunity to N. brasiliensis.     

Trichinella spiralis: Immunization of rats with an antigen fraction enriched for allergenicity

Trichinella spiralis whole muscle larval extract was fractionated by gel filtration and anion-exchange chromatography, and the protein fractions were assayed for allergenicity by a footpad-swelling test in mice; IgE antibody levels in rats immunized with the fractions were determined by passive cutaneous anaphylaxis test in rats. By these methods, an allergenic fraction from T. spiralis was isolated. The fraction, F1-b, was shown to be monodisperse by analysis with SDS-PAGE, IEP, and isoelectric focusing, indicating that it is a single protein moiety with a molecular weight of approximately 45,000 and a pI of approximately 5.1. The Schiff-periodate test showed Fl-b to be a glycoprotein. Rats immunized with Fl-b had significantly fewer intestinal worms than did nonimmunized controls at 24 hr and 7 days after oral challenge with T. spiralis larvae.     

Age-dependent plasticity of sex pheromone response in the moth, Agrotis ipsilon: Combined effects of octopamine and juvenile hormone

Male moths use sex pheromones to find their mating partners. In the moth, Agrotis ipsilon, the behavioral response and the neuron sensitivity within the primary olfactory centre, the antennal lobe (AL), to sex pheromone increase with age and juvenile hormone (JH) biosynthesis. By manipulating the JH level, we previously showed that JH controls this age-dependent neuronal plasticity, and that its effects are slow (within 2 days). We hypothesized that the hormonal effect might be indirect, and one neuromodulator candidate, which might serve as a mediator, is octopamine (OA). Here, we studied the effects of OA and an OA receptor antagonist, mianserin, on behavioral and AL neuron responses of mature and immature males during stimulation with sex pheromone. Our results indicate that, although OA injections enhanced the behavioral pheromone response in mature males, OA had no significant effect on behavior in immature males. However, mianserin injections decreased the behavioral response in mature males. AL neuron sensitivity increased after OA treatment in immature males, and decreased after mianserin treatment in mature males. Determination of OA levels in ALs of immature and mature males did not reveal any difference. To study the possible interactive effects of JH and OA, the behavioral pheromone response was analyzed in JH-deprived mature males injected with OA, and in immature males injected with fenoxycarb, a JH agonist, and mianserin. Results show that both JH and OA are necessary to elicit a behavioral response of A. ipsilon males to sex pheromone.     

Factors affecting pheromone secretion and oviposition by fertilized females of Culicoides nubeculosus (Diptera: Ceratopogonidae)

The effect of age and a blood meal on pheromone secretion by virgin and fertilized females of Culicoides nubeculosus was studied. In virgin females, the secretion varies in relation to age, the blood meal, and oviposition. In fertilized females, rate of pheromone secretion is very low after fertilization and does not seem to vary subsequently in relation to the above factors. Mating was found to accelerate oviposition. The role of seminal fluid in the decrease in rate of pheromone secretion by fertilized females is discussed.     

Regulation of mating in the budding yeast Saccharomyces cerevisiae by the zinc cluster proteins Sut1 and Sut2

The zinc cluster proteins Sut1 and Sut2 play a role in sterol uptake and filamentous growth in the budding yeast Saccharomyces cerevisiae. In this study, we show that they are also involved in mating. Cells that lack both SUT1 and SUT2 were defective in mating. The expression of the genes NCE102 and PRR2 was increased in the sut1 sut2 double deletion mutant suggesting that Sut1 and Sut2 both repress the expression of NCE102 and PRR2. Consistent with these data, overexpression of either SUT1 or SUT2 led to lower expression of NCE102 and PRR2. Furthermore, expression levels of NCE102, PRR2 and RHO5, another target gene of Sut1 and Sut2, decreased in response to pheromone. Prr2 has been identified as a mating inhibitor before. Here we show that overexpression of NCE102 and RHO5 also reduced mating. Our results suggest that Sut1 and Sut2 positively regulate mating by repressing the expression of the mating inhibitors NCE102, PRR2 and RHO5 in response to pheromone.     

Sound production in Scolytidae: Female sonic stimulus of male pheromone release in two Dendroctonus beetles

The hypothesis that female sonic stimulus may evoke male pheromone release in a behavioural interaction analogous to the known male sonic stimulus of female pheromone release, was confirmed in Dendroctonus pseudotsugae, and also in D. brevicomis. In both species known male-produced substances collected from males stimulated by recorded female stridulation were identified by coupled gas chromatography/mass spectrometry. In a second test with D. pseudotsugae, male pheromone release during recorded female stridulation was evident in the change of male stridulation from the simple attractant chirp to the interrupted chirp, which is known to result from a medium concentration of 3,2-MCH. Also, the D. pseudotsugae male attractant chirp was synthesised with an electronic pulse generator and used to evoke pheromone release. It is concluded that the antiaggregative pheromone of this species is released by each sex at the sonic stimulus of the other sex.     

Nippostrongylus brasiliensis and Haemonchus contortus: Function of the excretory ampulla of the third-stage larva

An improved technique for measuring the water content of nematodes is described using an electronic interferometer. Changes in phase of a laser beam passing through a known pathlength of the nematode have been used to measure the refractive index and hence the water content and relative volume of the animal. Third-stage larvae of Nippostrongylus brasiliensis and Haemonchus contortus which possess an excretory ampulla, differed from second-stage larvae lacking this ampulla in requiring a greater fall in the osmolarity of artificial tap water before there was a significant increase in their water content. Increases in the pulsation frequency of the ampulla also occurred in less hypotonic solutions than those required to increase the water content of the third-stage larvae. The ampulla pulsation frequency of third-stage larvae of N. brasiliensis increased after locomotor activity in hypotonic tap water and locomotory wave frequency of third-stage larvae of N. brasiliensis was independent of the extent of hypotonicity for a range of solutions that reduced wave propogation by its second-stage larva. The results suggest that the ampulla is an adaptation to hypotonic conditions favouring a volume homeostasis that is required for optimal locomotor activity of the third-stage infective larvae of these nematodes.     

Litomosoides carinii: Effect of splenectomy on the ability of naive Mastomys natalensis to accept transplanted adults

Fifty-eight male rats Mastomys natalensis, were used in each of two experiments to study the effect of splenectomy on the rejection of transplnted nematodes, Litomosoides carinii. The rats were divided into five groups: splenectomized recipients (SR), normal recipients (NR), splenectomized (S), normal (N) and donor (D). Each group had 12 animals except for the D group which had 10. After patency of the D group (tank infected with Litomosoides carinii), groups SR and S were splenectomized. Fourteen days later, (three to five L. Carinii derived from the D group) were surgically transplanted into the peritoneal cavity of the SR and NR groups. In the first experiment, weekly sacrifices were made starting at Day 3. In the second experiment, all groups were sacrificed at Day 32. Worms transplanted into the SR group were accepted while those transplanted into the NR group were rejected. Sequentially examined antibody titers after Day 3 fell into two groups, those that were recipients of transplants (SR and NR) and those that were not (NR and SR). After becoming positive on Day 3, the microfilaremia of the SR group rose by Day 31 while that of the NR group fell to near 0. It was concluded that the spleen is necessary for the rejection of transplantedL. carinii by naive M. natalensis.     

Phocanema decipiens: Growth,reproduction, and survival in seals

Captive harbor seals (Phoca vitulina) and gray seals (Halichoerus grypus) were fed infective larvae of Phocanema decipiens, an anisakine nematode from the flesh of Atlantic cod (Gadus morhus). Ova of P. decipiens were first detected in the feces of harbor seals 21(17–30) days after exposure; the patency period was 15 to 45 days. In gray seals, the prepatent period was 19(16–23) days; patency 20–60 days. By the sixth week of infection in harbor seals, mean body lengths of adult females and males of P. decipiens were 60.8(40.8–76.2) and 54.3(45.5–60.8) mm, respectively; mean fecundity of female nematodes was 156,000 ova. In infections of similar duration in gray seals, females and males of P. decipiens were 82.1(69.7–104.3) and 64.4(53.8–72.7) mm in length, respectively; mean fecundity of females was 366,000 ova. In sensitizing infections in harbor seals, 28% of P. decipiens survived to early patency (Days 25–30) while only 9% of the nematodes survived to midpatency (Days 35–45). In sensitizing infections in gray seals, 56% of P. decipiens survived to early patency (Days 20–30) and 48% survived to midpatency (Days 35–50). Seals with existing or recent P. decipiens infections resisted reinfection; <50% of the nematodes in challenge infections in gray seals survived to Day 3 and <10% survived to patency. Growth of the nematodes, however, was not retarded in the challenge infections and resistence to reinfection subsided when seals were maintained anisakinefree for 2–6 months after loss of prior natural or experimental infections. Natural anisakine infections were surveyed in 16 harbor and 53 gray seals from the Nova Scotia mainland. The mean incidence of P. decipiens was 62(5–177) in harbor seals and 577(11–1694) in gray seals; incidence varied seasonally and with age of host. Adult females of P. decipiens from harbor seals were 64.0(49.2–79.8) mm in length and contained 1.68(0.87–2.73) × 10⁵ ova; females from gray seals were 78.3(62.3–92.1) mm in length and contained 2.39(0.69–4.39) × 10⁵ ova.     

Oesophagostomum radiatum: Glucose metabolism of larvae grown in vitro and adults grown in vivo

Larval stages of Oesophagostomum radiatum grown in vitro and adults grown in vivo were incubated in complex media or in a simple salt solution containing radioactive glucose. Glucose disappearance and end product accumulation of third-stage larvae in a simple salt solution indicated that they excreted CO₂ and acetic, propionic, and lactic acids. Larvae in third molt, fourth stage, and adults all excreted CO₂, acetic, propionic, and lactic acids at twice the rate of third-stage larvae plus an additional product, methylbutyric acid. Carbon dioxide arose primarily from the 3 or 4 carbons of glucose. An anaerobic atmosphere (95% N₂:5% CO₂) had no apparent effect on metabolism. When incubation was done in complex media, isobutyric and 3-methylbutyric acids were seen as major excretion products (10 and 24%, respectively). However, these acids were quantitatively minor when incubations took place in simple salts-glucose medium (1 and 0–3%, respectively).     

Female sex pheromone components of the cotton bollworm, Heliothis armigera

Detailed examination of abdominal tip extracts from adult female Heliothis armigera revealed the presence of two components which elicit electroantennographic responses from the male moth. These olfactory stimulants have been fully identified as (Z)-11-hexadecenal (I) and (Z)-11-hexadecen-1-ol(II), and detected in airborne volatiles from a ‘calling’ female moth. A third olfactory stimulant was detected only in female tip extracts from some moths of Malawi origin, and this was tentatively identified as (Z)-9-hexadecenal (III). No other olfactory stimulants could be found, although hexadecenal (IV) and 1-hexadecan-1-ol (V) were detected by gas chromatography. In field tests in Malawi, (Z)-11-hexadecenal (I) attracted a few male H. armigera moths to traps but was very much less attractive than the virgin female moth. The attractiveness of (I) was not consistently affected by addition of alcohol (II), aldehydes (III) and (IV), or (E)-11-hexadecenal. Significant numbers of male Earias biplaga moths were found to be attracted to (Z)-11-hexadecenal (I).     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Nippostrongylus brasiliensis and Nematodirus battus: Changes in numbers and weight during the course of infection

Nematodirus battus has been shown to be subject to a “self-cure” mechanism when lambs 6 weeks of age are infected with 60,000 larvae. It is proposed that this self-cure phenomenon is an immune response similar to that which occurs in rats infected with Nippostrongylus brasiliensis. Changes in the weight of adult Nematodirus sp. and Nippostrongylus sp. occur over the period of an infection in their hosts. Females of both species from high-dose infections increased in weight up to that time postinfection where most adult worms were present, then decreased in weight with the onset of rejection from the host. Male Nematodirus sp. showed a similar pattern of weight change to that of females but male Nippostrongylus sp. maintained a steady weight throughout the infection. The consequences of these changes in weight are discussed with relevance to expression of enzyme activities of the nematodes on a weight of individual nematode basis.     

Heligmosomoides polygyrus: Simple recovery of post-infective larvae from mouse intestines

Mice infected orally with third-stage larvae of Heligmosomoides polygyrus were killed at various times after infection. Their small intestines were removed, tied at each end and incubated at 37 C in dilute culture medium. When intestines were taken from mice infected for a period of between 1 and 7 days, a number of developing larvae comprising up to 20% of the infective dose emerged within 60 min through the intestinal wall into the medium. The recovery of emergent larvae was highest using intestines from mice infected 36 to 120 hr previously. The proportion of parasites emerging from the intestines of 48-hr-infected mice was similar for doses of 100 to 2400 larvae. Significantly fewer larvae emerged from the intestines of mice resistant to reinfection and challenged with third-stage larvae 36–72 hr before necropsy.