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Hatching of parasitic nematode eggs: a crucial step determining infection

2021; Elsevier BV; Volume: 38; Issue: 2 Linguagem: Inglês

10.1016/j.pt.2021.08.008

1471-5007

Tapoka T. Mkandawire, Richard K. Grencis, Matthew Berriman, María A. Duque-Correa,

Helminth infection and control

Egg hatching is induced by a wide variety of host, environmental, and physicochemical cues. Each parasitic nematode responds to specific hatching-inducing factors, reflecting the particularities of their interactions with their host. While well described for some species, for the majority of them knowledge on the triggers and processes behind hatching remains elusive.The egg stage is often the only life-cycle stage that can be readily sampled from patients and the environment. Understanding hatching will allow us to efficiently hatch eggs, supporting existing life-cycle models and the development of new in vivo and in vitro models of infection.Novel research technologies, including high-throughput sequencing, bacterial metagenomics, and mass spectrometry, can lead to discoveries in nematode-hatching biology with translational implications for human and agricultural parasite control. Although hatching from eggs is fundamental for nematode biology it remains poorly understood. For animal-parasitic nematodes in particular, advancement has been slow since the 1980s. Understanding such a crucial life-cycle process would greatly improve the tractability of parasitic nematodes as experimental systems, advance fundamental knowledge, and enable translational research. Here, we review the role of eggs in the nematode life cycle and the current knowledge on the hatching cascade, including the different inducing and contributing factors, and highlight specific areas of the field that remain unknown. We examine how these knowledge gaps could be addressed and discuss their potential impact and application in nematode parasite research, treatment, and control. Although hatching from eggs is fundamental for nematode biology it remains poorly understood. For animal-parasitic nematodes in particular, advancement has been slow since the 1980s. Understanding such a crucial life-cycle process would greatly improve the tractability of parasitic nematodes as experimental systems, advance fundamental knowledge, and enable translational research. Here, we review the role of eggs in the nematode life cycle and the current knowledge on the hatching cascade, including the different inducing and contributing factors, and highlight specific areas of the field that remain unknown. We examine how these knowledge gaps could be addressed and discuss their potential impact and application in nematode parasite research, treatment, and control. Nematoda is a speciose phylum occupying most environmental habitats, from alpine grasslands to marine sediment, as well as colonising plants and animals, including 43 945 known vertebrate hosts [1.Dobson A. et al.Homage to Linnaeus: How many parasites? How many hosts?.Proc. Natl. Acad. Sci. U. S. 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Infections cause an enormous impact on human, animal, and plant health, with gross medical, social, and economic repercussions; for instance, annual crop production losses are estimated to be between 8.8% and 14.6%, and it has been estimated that approximately 1.45 billion people are infected with at least one soil-transmitted helminth (STH) (see Glossary), accounting for an average of 4.98 million years lived with disability (YLDs) [5.Nicol J.M. et al.Current nematode threats to world agriculture.in: Jones J. Genomics and Molecular Genetics of Plant–Nematode Interactions. Springer, 2011: 21-43Crossref Google Scholar, 6.Jourdan P.M. et al.Soil-transmitted helminth infections.Lancet. 2018; 391: 252-265Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 7.Pullan R.L. et al.Global numbers of infection and disease burden of soil transmitted helminth infections in 2010.Parasit. Vectors. 2014; 7: 37Crossref PubMed Scopus (784) Google Scholar]. Additionally, all livestock farmed with pasture grazing are exposed to gastrointestinal nematodes, and their resistance to certain classes of anthelmintics is costing the European livestock industry €38 million annually [8.Charlier J. et al.Initial assessment of the economic burden of major parasitic helminth infections to the ruminant livestock industry in Europe.Prev. Vet. Med. 2020; 182: 105103Crossref PubMed Scopus (66) Google Scholar]. Unlike Phylum Platyhelminthes, the other main phylum containing human-parasitic worms, Phylum Nematoda is unusual because the majority of members undergo life cycles that have several key transitions in common [9.Lee D.L. Life cycles.in: Lee D.L. The Biology of Nematodes. CRC Press, 2002: 61-72Crossref Google Scholar]. The canonical nematode life cycle starts with embryonation inside an egg, giving rise to a first-stage (L1) larva. The larva grows and moults through three subsequent larval stages (L2–L4), each time increasing in size and shedding its cuticle, with a final moult resulting in a sexually reproductive adult (Figure 1). Variations to this canonical scheme are seen across several species, for instance certain Xiphinema spp. only have three juvenile stages [10.Halbrendt J.M. Brown D.J. Morphometric evidence for three juvenile stages in some species of Xiphinema americanum sensu lato.J. Nematol. 1992; 24: 305-309PubMed Google Scholar,11.Robbins R.T. et al.Compendium of juvenile stages of Xiphinema species (Nematoda: Longidoridae).Russ. J. Nematol. 1996; 4: 163-171Google Scholar]. Additionally, the first moult for several plant nematodes, such as Meloidogyne spp. and Globodera spp., occurs inside the egg, as does the first two moults for Ascaris species [12.Bird A.F. Cuticle formation and moulting in the egg of Meloidogyne javanica (Nematoda).Parasitology. 1977; 74: 149-152Crossref Scopus (11) Google Scholar, 13.Chitwood D.J. Perry R.N. Reproduction, physiology and biochemistry.in: Perry R. Root-knot Nematodes. CABI, 2009: 182-200Crossref Google Scholar, 14.Baldwin J.G. Handoo Z.A. General morphology of cyst nematodes.in: Perry Roland Cyst Nematodes. CABI, 2018: 337-364Crossref Google Scholar]. However, for many species, moulting takes place post-hatching [15.Lee D.L. Cuticle, moulting and exsheathment.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 171-209Crossref Google Scholar,16.Maung M. The occurrence of the second moult of Ascaris lumbricoides and Ascaris suum.Int. J. Parasitol. 1978; 8: 371-378Crossref PubMed Scopus (20) Google Scholar]. Free-living species hatch seemingly spontaneously in the environment; however, hatching is likely still governed by a specific internal switch or external cue that is more challenging to isolate [17.Van Gundy S.D. Factors in survival of nematodes.Annu. Rev. Phytopathol. 1965; 3: 43-68Crossref Google Scholar]. Conversely, for parasitic nematodes, there is often a clear stimulus for their 'nonspontaneous' hatching. Hatching of embryonated eggs can occur either inside a host, as happens in Trichuris spp. (whipworms) and Ascaris spp. (roundworms) or in the environment, as occurs with the hookworms and threadworms that hatch and develop through to the L3 stage before infecting a new host [18.Panesar T.S. Croll N.A. The location of parasites within their hosts: site selection by Trichuris muris in the laboratory mouse.Int. J. 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CRC Press, 2002: 61-72Crossref Google Scholar]; but notable exceptions include parthenogenic reproduction from adult female root-knot nematodes (Meloidogyne spp.) and threadworms (Strongyloides ratti) [13.Chitwood D.J. Perry R.N. Reproduction, physiology and biochemistry.in: Perry R. Root-knot Nematodes. CABI, 2009: 182-200Crossref Google Scholar,22.Viney M.E. A genetic analysis of reproduction in Strongyloides ratti.Parasitology. 1994; 109: 511-515Crossref PubMed Scopus (61) Google Scholar,23.Castagnone-Sereno P. Genetic variability and adaptive evolution in parthenogenetic root-knot nematodes.Heredity (Edinb.). 2006; 96: 282-289Crossref PubMed Scopus (124) Google Scholar]. The role of the egg in the nematode life cycle is multifaceted [24.Stein K.K. Golden A. The C. elegans eggshell.WormBook. 2018; 2018: 1-36Crossref PubMed Google Scholar]. Firstly, the egg provides a contained environment in which embryonation, and occasionally moulting, can occur. Secondly, the nematode egg acts as a protective shield, and both its structure and composition contribute to this function. Specifically, the components of the eggshell form a multilayered and resilient barrier composed of lipid, chitin, vitelline layers (mainly glycoproteins) and, in some species, uterine layers (Figure 1 and Table 1) [24.Stein K.K. Golden A. The C. elegans eggshell.WormBook. 2018; 2018: 1-36Crossref PubMed Google Scholar]. The arrangement of these layers makes the egg resistant to stress but selectively permeable [25.Wharton D. Nematode egg-shells.Parasitology. 1980; 81: 447-463Crossref PubMed Scopus (202) Google Scholar,26.Quilès F. et al.In situ characterisation of a microorganism surface by Raman microspectroscopy: the shell of Ascaris eggs.Anal. Bioanal. Chem. 2006; 386: 249-255Crossref PubMed Scopus (35) Google Scholar]. 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Finally, the egg serves as a vehicle for the larvae to reach the site of hatching and infection [25.Wharton D. Nematode egg-shells.Parasitology. 1980; 81: 447-463Crossref PubMed Scopus (202) Google Scholar]. This is evidenced by observations of specific activation of hatching in response to the correct niche. Indeed, mouse infections with Trichuris muris have revealed that T. muris hatches in response to caecal but not ileal contents, and selectively infects the caecum and proximal colon [18.Panesar T.S. Croll N.A. The location of parasites within their hosts: site selection by Trichuris muris in the laboratory mouse.Int. J. Parasitol. 1980; 10: 261-273Crossref PubMed Scopus (17) Google Scholar].Table 1Summary of nematode egg composition across representative species from major nematode cladesCladeaPhylogenetic clades as defined by Blaxter et al. [102].OrderSpeciesUterine layerVitelline layerChitinous layerLipid layerRefsPresent (+) or absent (–)ITrichuridaTrichuris suis–+++[95.Beer R.J. Studies on the biology of the life-cycle of Trichuris suis Schrank, 1788.Parasitology. 1973; 67: 253-262Crossref PubMed Scopus (69) Google Scholar,96.Wharton D.A. Jenkins T. Structure and chemistry of the egg-shell of a nematode (Trichuris suis).Tissue Cell. 1978; 10: 427-440Crossref PubMed Scopus (38) Google Scholar]IIIOxyuridaEnterobius vermicularis++++[44.Inatomi S. A study on the structure of egg shell of Enterobius vermicularis (Linnaeus, 1758) Leach, 1853, with the electron microscope.Acta Med. Okayama. 1957; 11: 18-22Google Scholar,97.Jacobs L. et al.Studies on oxyuriasis. XXI. The chemistry of the membranes of the pinworm egg.Proc. Helminthol. Soc. Wash. 1939; 6: 57-60Google Scholar]AscarididaAscaris lumbricoides++++[98.Meng X.Q. et al.The membranous structure of eggs of Ascaris lumbricoides as revealed by scanning electron microscopy.Scan. Electron Microsc. 1981; : 187-190PubMed Google Scholar]IVTylenchidaGlobodera rostochiensis–+++[99.Clarke A.J. et al.The chemical composition of the egg shells of the potato cyst-nematode, Heterodera rostochiensis Woll.Biochem. J. 1967; 104: 1056-1060Crossref PubMed Scopus (45) Google Scholar,100.Bird A.F. McClure M.A. The tylenchid (Nematoda) egg shell: structure, composition and permeability.Parasitology. 1976; 72: 19-28Crossref Scopus (96) Google Scholar]VStrongylidaHaemonchus contortus–+++[101.Waller P.J. Structural differences in the egg envelopes of Haemonchus contortus and Trichostrongylus colubriformis (Nematoda: Trichostrongylidae).Parasitology. 1971; 62: 157-160Crossref PubMed Scopus (14) Google Scholar]a Phylogenetic clades as defined by Blaxter et al. [102.Blaxter M.L. et al.A molecular evolutionary framework for the phylum Nematoda.Nature. 1998; 392: 71-75Crossref PubMed Scopus (1448) Google Scholar]. Open table in a new tab The hatching process broadly follows the following cascade: (i) induction via intrinsic and extrinsic factors (explained in the next section) that promote changes in the eggshell and volume of the eggs; (ii) changes in larval behaviour and activity; and (iii) larval eclosion (Figure 2) [31.Perry R. Hatching.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 147-170Crossref Google Scholar]. Induction of hatching has been investigated primarily in parasitic nematodes in which host cues lead to an ion-mediated exchange of water and sugars across the eggshell, altering osmotic pressure, the flexibility of the eggshell, and the size of the egg (Figure 2) [31.Perry R. Hatching.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 147-170Crossref Google Scholar]. For instance, in the potato-cyst nematode Globodera rostochiensis calcium mediates trehalose and water exchange across the eggshell during hatching induction [31.Perry R. Hatching.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 147-170Crossref Google Scholar,32.Duceppe M.-O. et al.Analysis of survival and hatching transcriptomes from potato cyst nematodes, Globodera rostochiensis and G. pallida.Sci. Rep. 2017; 7: 3882Crossref PubMed Scopus (17) Google Scholar]. T. muris and Tylenchorhynchus maximus eggs swell in the early stages of hatching (Figure 2) [33.Panesar T.S. Croll N.A. The hatching process in Trichuris muris (Nematoda: Trichuroidea).Can. J. Zool. 1981; 59: 621-628Crossref Google Scholar,34.Bridge J. Hatching of Tylenchorhynchus maximus and Merlinius icarus.J. Nematol. 1974; 6: 101-102PubMed Google Scholar]. Surprisingly, the factors that induce hatching in C. elegans are not known; however, the eggs of C. elegans exhibit weakening and digestion of the eggshell during hatching [31.Perry R. Hatching.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 147-170Crossref Google Scholar,35.Hall D.H. et al.WormAtlas Embryo Handbook – Introduction to C. elegans Embryo Anatomy, 6 Hatching. WormAtlas, 2017Google Scholar]. Larval movement and metabolic activity increase as hatching progresses, following distinct patterns of widespread or local exploration (Figure 2) [31.Perry R. Hatching.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 147-170Crossref Google Scholar]. Widespread exploration, or unfocussed movement, includes head waving and whole-body rolling and occurs well before eclosion. For example, in T. maximus continuous movement is observed for up to 3 days before hatching [34.Bridge J. Hatching of Tylenchorhynchus maximus and Merlinius icarus.J. Nematol. 1974; 6: 101-102PubMed Google Scholar]. Local exploration immediately precedes eclosion, and consists of targeted and localised thrusting of the larval head or tail, secretions from the pharyngeal glands, and propulsion of the larval's stylet to cut through the shell, strategies employed by Aphelenchus avenae, and Meloidogyne spp. [31.Perry R. Hatching.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 147-170Crossref Google Scholar,36.Taylor D.P. Effect of temperature on hatching of Aphelenchus avenae eggs.Proc. Helminthol. Soc. Wash. 1962; 29: 52Google Scholar,37.Curtis R.H.C. et al.Hatch and host location.in: Perry R. Root-knot Nematodes. CABI, 2009: 139-162Crossref Google Scholar]. In G. rostochiensis, local exploration is used to both propel and guide the stylet to create a perforated slit in one region of the eggshell [31.Perry R. Hatching.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 147-170Crossref Google Scholar,34.Bridge J. Hatching of Tylenchorhynchus maximus and Merlinius icarus.J. Nematol. 1974; 6: 101-102PubMed Google Scholar]. In some nematode species the larva also produces enzymes to weaken the integrity of the eggshell. These enzymes are released through activation of the pharyngeal glands [31.Perry R. Hatching.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 147-170Crossref Google Scholar]. Pharyngeal pumping has been observed in A. avenae, G. rostochiensis, and C. elegans, and enzymes including chitinases, lipases, and proteases have been found in the perivitelline fluid or hatching fluid of Ascaris lumbricoides, Haemonchus contortus, and Ancylostoma ceylanicum [31.Perry R. Hatching.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 147-170Crossref Google Scholar,36.Taylor D.P. Effect of temperature on hatching of Aphelenchus avenae eggs.Proc. Helminthol. Soc. Wash. 1962; 29: 52Google Scholar,38.Rogers W.P. Physiology of the hatching of eggs of Ascaris lumbricoides.Nature. 1958; 181: 1410-1411Crossref PubMed Scopus (40) Google Scholar, 39.Doncaster C.C. Shepherd A.M. The behaviour of second-stage Heterodera rostochiensis larvae leading to their emergence from the egg.Nematologica. 1967; 13: 476-478Crossref Scopus (28) Google Scholar, 40.Abriola L. et al.Development and optimization of a high-throughput screening method utilizing Ancylostoma ceylanicum egg hatching to identify novel anthelmintics.PLoS One. 2019; 14e0217019Crossref PubMed Scopus (4) Google Scholar, 41.Chen Q. et al.Crystal structure and structure-based discovery of inhibitors of the nematode chitinase CeCht1.J. Agric. Food Chem. 2021; 69: 3519-3526Crossref PubMed Scopus (4) Google Scholar, 42.Rogers W.P. Brooks F. The mechanism of hatching of eggs of Haemonchus contortus.Int. J. Parasitol. 1977; 7: 61-65Crossref PubMed Scopus (46) Google Scholar]. The last step of the hatching process is eclosion in which, occasionally, the whole eggshell degrades, as has been observed in A. lumbricoides [38.Rogers W.P. Physiology of the hatching of eggs of Ascaris lumbricoides.Nature. 1958; 181: 1410-1411Crossref PubMed Scopus (40) Google Scholar]. More commonly, nematode species exit the egg head-first, some simply by pushing their head against the eggshell, such as C. elegans, where distortion of the eggshell is observed around the head prior to eclosion [35.Hall D.H. et al.WormAtlas Embryo Handbook – Introduction to C. elegans Embryo Anatomy, 6 Hatching. WormAtlas, 2017Google Scholar]. Some species identify specific eclosion sites: Capillaria and Trichuris species, in addition to utilising a stylet, exit the egg through the polar plugs, and Enterobius vermicularis egresses through a thinned region of the eggshell [31.Perry R. Hatching.in: Lee D.L. The Biology of Nematodes. Taylor & Francis, 2002: 147-170Crossref Google Scholar,33.Panesar T.S. Croll N.A. The hatching process in Trichuris muris (Nematoda: Trichuroidea).Can. J. Zool. 1981; 59: 621-628Crossref Google Scholar,43.Wehr E.E. Studies on the Development of the Pigeon Capillarid, Capillaria Columbae. U.S. Department of Agriculture, 1939Google Scholar,44.Inatomi S. A study on the structure of egg shell of Enterobius vermicularis (Linnaeus, 1758) Leach, 1853, with the electron microscope.Acta Med. Okayama. 1957; 11: 18-22Google Scholar]. Finally, an alternative method, to date observed only in a few species, is posterior-driven eclosion. A. ceylanicum and Ancylostoma tubaeforme occasionally perforate the shell with their tail before reversing to emerge head first [45.Matthews B.E. The influence of temperature and osmotic stress on the development and eclosion of hookworm eggs.J. Helminthol. 1985; 59: 217-224Crossref PubMed Scopus (12) Google Scholar]; and H. contortus and Heterodera iri utilise their sharp tail to perforate the shell and emerge tail-first [42.Rogers W.P. Brooks F. The mechanism of hatching of eggs of Haemonchus contortus.Int. J. Parasitol. 1977; 7: 61-65Crossref PubMed Scopus (46) Google Scholar,46.Silverman P.H. Campbell J.A. Studies on parasitic worms of sheep in Scotland. I. Embryonic and larval development of Haemonchus contortus at constant conditions.Parasitology. 1959; 49: 23-38Crossref PubMed Scopus (59) Google Scholar,47.Laughlin C.W. et al.Observations on the emergence of Heterodera iri from the egg.J. Nematol. 1974; 6: 100-101PubMed Google Scholar]. Hatching in nematodes is traditionally thought of as 'spontaneous' or 'nonspontaneous' [17.Van Gundy S.D. Factors in survival of nematodes.Annu. Rev. Phytopathol. 1965; 3: 43-68Crossref Google Scholar], and while the process of hatching is broadly conserved, there are a wide variety of factors that induce hatching and each species responds to specific ones (Table 2). These factors can be split into two large categories: intrinsic and extrinsic factors.Table 2Hatching-inducing factors and larval responses of C. elegans and key parasitic species from major nematode cladesCladeaPhylogenetic clades as defined by Blaxter et al. [102].SpeciesHostIntrinsic factors and larval responsesHost factorsPhysicochemical/environmental factorsITrichuris murisMus musculusLarval movement aids hatching. Larvae use stylet to perforate polar plug [33.Panesar T.S. Croll N.A. The hatching process in Trichuris muris (Nematoda: Trichuroidea).Can. J. Zool. 1981; 59: 621-628Crossref Google Scholar]Eggs hatch in the host caecum and proximal colon where host gut microbiota is required to induce hatching [18.Panesar T.S. Croll N.A. The location of parasites within their hosts: site selection by Trichuris muris in the laboratory mouse.Int. J. Parasitol. 1980; 10: 261-273Crossref PubMed Scopus (17) Google Scholar,56.White E.C. et al.Manipulation of host and parasite microbiotas: Survival strategies during chronic nematode infection.Sci. Adv. 2018; 4eaap7399Crossref PubMed Scopus (52) Google Scholar]Incubation of eggs with Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus induces hatching in vitro via accumulation around polar plugs [75.Hayes K.S. et al.Exploitation of the intestinal microflora by the parasitic nematode Trichuris muris.Science. 2010; 328: 1391-1394Crossref PubMed Scopus (200) Google Scholar]Hatching occurs at 37°C [33.Panesar T.S. Croll N.A. The hatching process in Trichuris muris (Nematoda: Trichuroidea).Can. J. Zool. 1981; 59: 621-628Crossref Google Scholar]Incubation with sodium hypochlorite for 2 h at 37°C with 5% carbon dioxide, followed by incubation in RPMI media at 37°C with 5% carbon dioxide results in hatching after 4–5 days [56.White E.C. et al.Manipulation of host and parasite microbiotas: Survival strategies during chronic nematode infection.Sci. Adv. 2018; 4eaap7399Crossref PubMed Scopus (52) Google Scholar]Trichuris suisSus domesticusEggs hatch in response to mucosal scrapings from the host gastrointestinal tract [57.Vejzagić N. et al.In vitro hatching of Trichuris suis eggs.Parasitol. Res. 2015; 114: 2705-2714Crossref PubMed Scopus (7) Google Scholar]Embryonation is impaired below 5°C and above 40°C.Hatching induced by stirring eggs with glass beads and HCl. Hatching occurs at 37°C [57.Vejzagić N. et al.In vitro hatching of Trichuris suis eggs.Parasitol. Res. 2015; 114: 2705-2714Crossref PubMed Scopus (7) Google Scholar,64.Vejzagić N. et al.Temperature dependent embryonic development of Trichuris suis eggs in a medicinal raw material.Vet. Parasitol. 2016; 215: 48-57Crossref PubMed Scopus (8) Google Scholar]Trichuris trichiuraHomo sapiensOxygen is continually taken up during embryonation, and rapid changes in its availability or low starting concentrations impede development [63.Nolf L.O. Experimental studies on certain factors influencing the development and viability of the ova of the human Trichuris as compared with those of the human Ascaris.Am. J. Epidemiol. 1932; 16: 288-322Crossref Scopus (17) Google Scholar]ICapillaria obsignatabCapillaria obsignata is also referred to as Capillaria columbae.Gallus gallus domesticusColumba livia domesticaL1 have a distinct stylet used during hatching [43.Wehr E.E. Studies on the Development of the Pigeon Capillarid, Capillaria Columbae. U.S. Department of Agriculture, 1939Google Scholar,62.Wakelin D. Experimental studies on the biology of Capillaria obsignata Madsen, 1945, a nematode parasite of the domestic fowl**.J. Helminthol. 1965; 39: 399-412Crossref Scopus (18) Google Scholar]Eggs hatch in the host small intestine [43.Wehr E.E. Studies on the Development of the Pigeon Capillarid, Capillaria Columbae. U.S. Department of Agriculture, 1939Google Scholar,103.Tiersch K.M. et al.The role of culture media on embryonation and subsequent infectivity of Capillaria obsignata eggs.Parasitol. Res. 2013; 112: 357-364Crossref PubMed Scopus (9) Google Scholar]Chemical solutions influence rate of embryonation and affect larval fitness/infectivity. For example, incubation in sulphuric acid results in more embryonated eggs than incubation in water [103.Tiersch K.M. et al.The role of culture media on embryonation and subsequent infectivity of Capillaria obsignata eggs.Parasitol. Res. 2013; 112: 357-364Crossref PubMed Scopus (9) Google Scholar].In addition, potassium dichromate increases embryonation, but the infectivity of eggs embryonated in this way is low [103.Tiersch K.M. et al.The role of culture media on embryonation and subsequent infectivity of Capillaria obsignata eggs.Parasitol. Res. 2013; 112: 357-364Crossref PubMed Scopus (9) Google Scholar]IIIAscaris lumbricoidesH. sapiens'Hatching fluid' collected from eggs contains enzymes [38.Rogers W.P. Physiology of the hatching of eggs of Ascaris lumbricoides.Nature. 1958; 181: 1410-1411Crossref PubMed Scopus (40) Google Scholar]Carbon dioxide, sodium bicarbonate, and a reducing agent such as sodium dithionite are the minimum requirement for hatching [38.Rogers W.P. Physiology of the hatching of eggs of Ascaris lumbricoides.Nature. 1958; 181: 1410-1411Crossref PubMed Scopus (40) Google Scholar]Ascaris suumS. domesticusLarvae need to be sufficiently embryonated to survive hatching [19.Geenen P.L. et al.The morphogenesis of Ascaris suum to the infective third-stage larvae within the egg.J. Parasitol. 1999; 85: 616-622Crossref PubMed Scopus (79) Google Scholar]Bile has a positive effect on hatching at 5% concentration, at concentrations above 20% it is detrimental [58.Han Q. et al.Effects of bile on the in vitro hatching, exsheathment, and migration of Ascaris suum larvae.Parasitol. Res. 2000; 86: 630-633Crossref PubMed Scopus (15) Google Scholar]High carbon dioxide concentration and mechanical stimulation induces hatching [58.Han Q. et al.Effects of bile on the in vitro hatching, exsheathment, and migration of Ascaris suum larvae.Parasitol. Res. 2000; 86: 630-633Crossref PubMed Scopus (15) Google Scholar]IVGlobodera rostochie