Cryopreservation produces limited long-term effects on the nematode Caenorhabditis elegans

Cryopreservation, the freezing and later warming of biological samples with minimal loss of viability, is important in many scientific disciplines. For some applications, particularly those where there is limited available material, it is critical to ensure the maximal survival rates of cryopreserved materials. Most of the challenges encountered with such techniques take place after the warming process where cryodamage affects cell viability and future development. Here we have used the nematode Caenorhabditis elegans to investigate the effects of cryodamage caused by slow-freezing. We find that freezing results in the death of some worms, with an approximately 40% reduction in the number of worms that develop in the frozen populations, but that the effects on worms that survive are limited. For example, there are no differences in the lifetime fecundity or in lifespan between frozen and control worms, although early fecundity and body size was reduced in frozen worms. Similarly, analyses of body wall muscle structure and of pharyngeal function indicates that muscle development and function are not significantly affected by freezing. We do however determine that freezing increases the rates of matricidal hatching, where progeny hatch within the mother. Overall, these results indicate that, for worms that survive, cryopreservation produces limited long-term effects, but do indicate that some phenotypes could be used in further analyses of the cellular damage induced by cryopreservation.     

Toward next-generation sequencing of mitochondrial genomes — Focus on parasitic worms of animals and biotechnological implications

Helminths (worms) include parasitic nematodes (roundworms) and platyhelminths (flatworms). These worms are abundant, and many of them are of agricultural, aquacultural, veterinary and medical importance and cause substantial socioeconomic losses worldwide. The genetic characterization of parasitic nematodes using advanced molecular tools is central to the diagnosis of infections and the control of parasitism. The accurate analysis of genetic variation also underpins studies of their taxonomy, epidemiology and evolutionary history. Although the nuclear genome contains suitable genetic markers (e.g., in ribosomal DNA) for the identification of many species, the large size and high variability of the mt genome consistently provides a rich source of such markers for informative systematic and epidemiological studies both within and among species. There is significant value in establishing a practical platform for the rapid sequencing, annotation and analysis of mt genomic datasets to underpin such fundamental and applied studies of parasitic worms (= helminths). In the last decade, there have been some important advances in the mt genomics of helminths, but next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation. In this article, we provide a background on mt genomics, cover technological challenges and recent advances, and provide a perspective on future mt genome research of parasitic helminths and its fundamental scientific and biotechnological implications.     

The mucosal cellular response to infection with Ancylostoma ceylanicum

Although hookworms are known to stimulate inflammatory responses in the intestinal mucosa of their hosts, there is little quantitative data on this aspect of infection. Here we report the results of experiments conducted in hamsters infected with Ancylostoma ceylanicum. Infection resulted in a marked increase in goblet cells in the intestinal mucosa, which was dependent on the number of adult worms present and was sustained as long as worms persisted (over 63 days) but returned to baseline levels within 7 days of the removal of worms by treatment with ivermectin. Increased mast cell responses were also recorded. Levels were again dependent on the intensity of worm burdens and lasted as long as 63 days after infection. When worms were eliminated, mast cell numbers took over 2 weeks to return to normal. Paneth cell numbers fell soon after infection, the degree of reduction being dependent on the worm burden. After clearance of worms, Paneth cell numbers returned to normal within a week, but then rebounded and numbers rose to higher levels than those in control na?ve animals. The time course of the response was similar whether animals experienced a chronic low-intensity infection without loss of worms or a higher intensity infection during the course of which worm burdens were gradually reduced. Clearly, A. ceylanicum was able to induce a marked inflammatory response in its host's intestine which was sustained for over 9 weeks after infection, and which hamsters appeared able to tolerate well. Our data draw attention to the resilience of hookworms which, unlike many other nematodes, are able to survive for many weeks in a highly inflamed intestinal tract.     

Biological control of ruminant trichostrongylids by Arthrobotrys oligospora, a predacious fungus

Sheep and cattle are prey to many parasitic worms, including the trichostrongylid nematodes. Conventional control involves the use of anthelmintic drugs, but in this article Hadji Ahmad Hashmi and Roger Connan discuss the possible biological control of these nematodes by means of a 'living lasso', the predacious fungus Arthrobotrys.     

In vivo effects of putative crowding factors on development of Hymenolepis diminuta.

During in vitro incubation, Hymenolepsis diminuta secretes substances into the medium that inhibit DNA synthesis in the germinative region of freshly isolated, uncrowded worms. Of the many substances that are released by H. diminuta into the medium, earlier studies indicate that only succinate, acetate, glucosaminic acid, and cGMP are responsible for the inhibition. In the present report, effects of these putative crowding factors on worm development in vivo were examined. At 7 days postinfection the proximal end of the host's intestine was catheterized and perfused with test solution. The test solution contained 28 nM cGMP, 250 microM glucosaminic acid, 120 mM succinate, and 40 mM acetate. The solution was perfused by a peristaltic pump at a rate of 50 ml/day. At 2 wk postinfection, worms were recovered for subsequent analysis. Worms developing in the presence of crowding factors were 53% less in wet weight than control worms. Carbohydrate concentrations in worms from experimental groups were not different from those in control groups; therefore, the inhibition in growth was probably not due to carbohydrate deprivation. Worms from experimental groups had fewer immature, mature, and gravid proglottids than did worms from control groups. The results are consistent with the hypothesis that the tested substances, which inhibit DNA synthesis in H. diminuta in vitro, are a part of the cause of the crowding effect in vivo.     

Schistosoma mansoni: reversible destruction of testes by procarbazine

1. The anticancer drug procarbazine is profoundly damaging to the testes of Schistosoma mansoni when administered at 200 mg/kg or more to the mouse host. Somatic tissues appear entirely unaffected. 2. Within 2 days the meiotic process is disrupted, and primary and secondary spermatocytes and spermatids are destroyed and replaced by amorphous granular material. 3. The testes regenerate within about 15 days, apparently from surviving spermatogonial resting cells near the germinal epithelium of the testis. 4. Livers of mice treated once 7 weeks earlier have numerous egg granulomas and give rise to many miracidia, suggesting that full testis function is regained. 5. Male worms given the drug at 200 mg/kg at 19 days of age and fixed 7 weeks later have on average one testis less than control worms, indicating that about 15% of immature testes are unable to regenerate, whereas mature worms given the drug regenerate the normal number of testes. 6. The drug did not have significant antispermatogenic effects when male worms were incubated for a week with various concentrations in vitro, suggesting that a host metabolite is the active agent.     

Manipulating the manipulators: advances in parasitic helminth transgenesis and RNAi

Because tropical medicine and parasitology research has moved into the postgenomic era, an enormous amount of gene sequence information for parasitic helminths is now accumulating. These sequences undoubtedly hold information that can be used for new interventions and control. However, to exploit the new resource, methods for gene manipulation and transformation of parasitic worms are needed. Until recently, gene manipulation approaches had not been seriously addressed. This situation is now changing in response to the availability of genome sequences and other advances. In this article, we review advances in the transgenesis and gene silencing of parasitic worms.     

Distribution, behavior, and course of patency of Dracunculus insignis in experimentally infected ferrets

From 106 ferrets experimentally infected with Dracunculus insignis, 273 female worms and 42 male worms were recovered. The percentage of female worms that mated, the location of all worms, and the rate of emergence of gravid females were recorded. Of 174 mature female worms recovered, only 11% had emerged before necropsy. Eighty-one percent of the male worms and 87% of all unmated (immature) females were found on the trunk of the animal or on the skin overlying it. Gravid female worms were found on the extremities (legs) 88% of the time. Almost one-third of gravid female worms were found on the left front leg. On 4 occasions, females were found in unusual locations: the tail, the cheek, the lumbar region of the spinal cord, and the peritoneal cavity associated with the omentum. The results showed that only a small percentage of mature female worms emerge, which suggests that infected persons harbor many more gravid female worms than indicated by reports of emergent worms. More than one-third of female worms were not mated, but it is likely that these immature females would be capable of further development. Furthermore, male worms have been observed to survive for up to 330 days, suggesting that male or unmated female worms may carry over from one transmission season to the next.     

Multiple near-identical genotypes of Schistosoma japonicum can occur in snails and have implications for population-genetic analyses

We genotyped (using 16 or 17 microsatellite loci) numerous adult Schistosoma japonicum raised in rabbits exposed to pooled cercariae from small numbers of naturally infected snails from several localities in China. As expected, duplicate multi-locus genotypes (MLGs) were found among these worms. Additionally, many more MLGs, often near-identical, were found than snails used as sources of cercariae. Explanations for these results include (i) genotyping errors, (ii) development within each infected snail of multiple sibling miracidia and (iii) somatic mutation producing genetically varied cercariae from a single miracidium. To control for genotyping errors we re-analysed samples from many individual worms, including repeating the initial PCR. Explanations invoking the development of multiple sibling miracidia within a single snail are not likely to be correct because almost all duplicate MLGs fell within same-sex clusters in a principal coordinates analysis. We would expect both sexes to be represented in a multi-miracidium infection. In addition, we exposed several snails to infection by a single miracidium. One such snail, via an experimentally infected mouse, yielded 48 adult worms. The presence of at least nine near-identical MLGs among these worms was confirmed by re-genotyping. We regard somatic mutation as the most likely explanation for our results. The implications of multiple MLGs for population-genetic studies in S. japonicum are discussed.     

Epilogue – one hundred years of nemertean research: Bürger (1895) to the present

The past one hundred years of research on nemerteansis reviewed. Scientific methodology and philosophyhave advanced considerably during this period, yetnemerteans remain one of the least well known ofinvertebrate groups; nemerteans are not just membersof a minor phylum, they constitute a neglected yethighly successful assemblage of animals. Despite manyworkers in the last hundred years having madesignificant contributions to our knowledge about thisphylum of worms, many aspects of their biology arestill virtually, if not totally, unknown, and theenormous advances in many areas of scientificknowledge have not been paralleled by comparable gainsin our understanding of nemerteans.     

水丝蚓对太湖沉积物有机磷组成及垂向分布的影响

以太湖常见底栖动物——水丝蚓为研究对象,借助室内流动培养装置和31P核磁共振技术(phosphorus-31 nuclearmagnetic resonance spectroscopy)(31P-NMR),研究生物扰动对太湖沉积物有机磷组成及垂向分布的影响。结果表明:短时间内水丝蚓扰动对沉积物有机磷化合物种类组成影响并不显著,但会引起上层沉积物中稳定性较差的有机磷化合物磷脂和DNA含量出现显著降低,同时沉积物中总磷和有机磷的垂向分布亦发生明显改变。此外,水丝蚓扰动下沉积物含水率、孔隙率和碱性磷酸酶活性显著增加,使活性较高的有机磷化合物分解加速,最终导致表层沉积物中的磷脂与DNA含量降低。     

The pervalence of caecal threadworms (Trichostrongylus tenuis) in red grouse (Lagopus lagopus scoticus)

Summary Ninety percent of wild red grouse examined carried threadworms in their intenstinal caeca. Old birds had 30 times as many worms as young birds. Some infections were as high as 30,000 worms. The occurrence of worms in old grouse conformed to the negative binomial distribution.     

Drpiwi-1 is essential for germline cell formation during sexualization of the planarian Dugesia ryukyuensis

A piwi homolog is required for the regulation of stem cells, formation and maintenance of germline stem cells, and gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, both asexually and sexually, and develop and maintain germ cells and sexual organs. They have many pluripotent stem cells (neoblasts) that can differentiate into both somatic and germline stem cells. Thus, we searched for a piwi subfamily in the planarian Dugesia ryukyuensis. Four piwi homologs, identified as Drpiwi-1, -2, -3, and -4, were expressed in sexually reproductive worms. We then selectively destroyed the neoblasts by irradiating the worms with X-rays. In such worms, Drpiwi-1, -2, and -3 were not expressed at all, whereas Drpiwi-4 was expressed to the same degree as that in non-irradiated controls, indicating that Drpiwi-1, -2, and -3, but not Drpiwi-4, are expressed in neoblasts. During the regeneration process, Drpiwi-2(RNAi) and -3(RNAi) worms failed to regenerate after ablation, but Drpiwi-1 and -4(RNAi) worms regenerated. During the sexualizing process, Drpiwi-1(RNAi) worms failed to develop ovaries and testes, but somatic sexual organs were unaffected. Germ cell development was normal in Drpiwi-4(RNAi) worms. Therefore, Drpiwi-2 and -3 may be related to the regulation of neoblasts important for maintaining homeostasis, and Drpiwi-1 is essential for the development of germ cells but not somatic sexual organs. DrPiwi-1 is localized in the cytoplasm of stem cells and germline cells and may be involved in regulating some gene expression. We suggest that planarian Piwi controls germline formation via RNA silencing mechanisms.     

Chaetae and mechanical function: tools no Metazoan class should be without

To address the functional contributions of capillary chaetae in the maldanid polychaete Clymenella torquata, we compared irrigation efficiency and tube structure for animals with intact and trimmed capillary chaetae. We measured pumping rates for worms before and after they were anaesthetized and subjected either to capillary trimming or mock trimming, i.e. handling without trimming. Worms with trimmed chaetae were significantly less effective at moving water through their tubes than those with intact chaetae. There were no significant differences in the ability of control worms to move water within their tubes. No significant changes in rates of peristalsis were observed among experimental or control groups. These data strongly suggest that body musculature and capillary chaetae work in concert to hold worms in position within tubes during peristaltic pumping. When chaetae are shortened, the body musculature must contract to a greater degree, increasing the functional diameter of the worm to achieve the necessary traction with the tube wall, resulting in less efficient irrigation. We also compared the inner diameters of original field tubes to tubes built by control worms or worms after capillary trimming. The inner diameters of new tubes built by worms with shortened chaetae were larger than their original tubes, while those of both control groups were not. One possible explanation is that the chaetae have a sensory role and shortened chaetae send the false message that the nascent tube walls are farther away than they are, the body contracts in compensation and the tube is widened, however this idea has not been tested.     

Schistosoma mansoni: In vitro schistosomicidal activity and tegumental alterations induced by piplartine on schistosomula

Schistosomiasis is one of the most important parasitic infections in humans that occur in many tropical and subtropical countries. Currently, the control of schistosomiasis rests with a single drug, praziquantel, which is effective against adult worms but not the larval stages. Recent studies have shown that piplartine, an amide isolated from plants of the genus Piper (Piperaceae), reveals interesting antischistosomal properties against Schistosoma mansoni adult worms. Here, we report the in vitro antischistosomal activity of piplartine on S. mansoni schistosomula of different ages (3h old and 1, 3, 5, and 7days old), and examine alterations on the tegumental surface of worms by means of confocal laser scanning microscopy. Piplartine at a concentration of 7.5μM caused the death of all schistosomula within 120h. The lethal effect occurred in a dose-dependent manner and was also dependent on the age of the parasite. Microscopy observation revealed extensive tegumental destruction, including blebbing, granularity, and a shorter body length. This report provides the first evidence that piplartine is able to kill schistosomula of different ages and reinforce that piplartine is a promising compound that could be used for the development of new schistosomicidal agent.     

The function of parapodial setae in a nereidid polychaete moving on two different substrata

Active polychaetes of the family Nereididae possess a large number of compound setae originating in seta-bundles at the distal end of the parapodium. Here we investigate the functional role of the setae in Nereis diversicolor by comparing the locomotor performance of neuropodial seta-ablated worms with normal (control, non-ablated) worms moving on sand and mud. No differences between worms walking on sand and mud were found, except that they were more submerged in the latter substratum. On both substrata, a similar decrease in forward speed and increase in wave speed and slip were found in seta-ablated worms. However, this was not associated with any changes in gait, since both amplitude and wavelength were similar for seta-ablated and control worms. This suggests that the setae are not adapted to a specific habitat. The same parameters were compared between seta-ablated and control worms swimming over the substratum, but no significant differences were found, indicating that the setae are not as important during swimming as previously thought.     

Pattern recognition molecules and innate immunity to parasites

Recent pioneering advances in understanding how plants, insects and worms eliminate pathogens has led to the realization that innate immunity plays a vital role in protecting humans from infection. This comprehensive review examines the molecules involved in innate immune responses, how they act to control parasites and if their engagement can explain many immune features characteristic of parasitic infections.     

Stem cells from innate sexual but not acquired sexual planarians have the capability to form a sexual individual

Planarian species may harbor as many as three populations with different reproductive strategies. Animals from innate asexual (AS) and innate sexual (InS) populations reproduce only by fission and cross‐fertilization, respectively, whereas the third population switches seasonally between the two reproductive modes. AS worms can be experimentally sexualized by feeding them with minced InS worms; we termed the resulting animals “acquired sexual” (AqS) worms. Both AqS and InS worms exhibit sexualizing activity when used as feed, suggesting that they maintain their sexual state via endogenous sexualizing substances, although the mechanisms underlying determination of reproductive strategy and sexual switching in these metazoans remain enigmatic. Therefore, we compared the endogenous sexualizing activity of InS worms and AqS worms. First, we amputated mature worms and assessed if they could re‐enter a sexual state. Regenerants of InS worms, but not AqS worms, were only sexual, indicating that sexual state regulation comprises two steps: (1) autonomous initiation of sexualizing substance production and (2) maintenance of the sexual state by continuous production of sexualizing substances. Next, InS neoblasts were characterized by transplantation, finding that they successfully engrafted, proliferated, and replaced all recipient cells. Under such conditions, the AS recipients of InS worm neoblasts, but not those of AqS worms, became sexual. These results clearly show that there is a neoblast‐autonomous determination of reproductive strategy in planarians. Mol. Reprod. Dev. 79: 757–766, 2012. © 2012 Wiley Periodicals, Inc.     

Weshalb hemmen Würmer Allergien? Parasiten als Immunologen

Parasitic worms survive within their immunocompetent hosts by modulating their immune system and by inhibiting inflammatory responses directed against the parasites. This immunomodulation has a spill over effect and also inhibits inflammatory responses originating from other causes. For this reason, persons who are infected with certain species of worms show a lower rate of allergic diseases as compared to persons who are free of parasites. In the same line, studies in mouse models revealed that many inflammatory diseases can be treated by worm infections. This effect is among others owing to specific proteins that are released by the worms. Such secreted immunomodulators, shaped by co‐evolution between parasites and their hosts, could become lead compounds for the development of new therapies against allergic and inflammatory diseases.     

Adult worm tegumental damage and egg-granulomas in praziquantel-resistant and -susceptible Schistosoma mansoni treated in vivo

The effect of treatment with praziquantel (PZQ) on the tegument of adult Schistosoma mansoni worms and on liver egg-granulomas has been examined in mice infected with PZQ-resistant and -susceptible parasite isolates. Two PZQ-resistant S. mansoni isolates, one selected by passage in the laboratory under drug pressure and one from Senegal established from eggs excreted by an uncured patient, were compared with PZQ-susceptible control isolates. Scanning electron microscopic observations on the tegument of Schistosoma adult worms treated in vivo with PZQ showed that more severe damage was inflicted by PZQ on susceptible worms than on drug-resistant worms. Observations on the pathology of Schistosoma egg-granulomas in the liver of infected mice after treatment with PZQ indicated that eggs from susceptible control isolates were more sensitive to PZQ than those from drug-resistant isolates.