The picture becomes clearer if the above interpretation 

 is applied. The membrane immediately enclosing the 

 embryo is the vitelline membrane, fibrous membrane, or 

 lipoidal layer (s). It corresponds to membranes "D" 

 of Zawadowsky. The hard refractive membrane sur- 

 rounding this is the shell proper or homogeneous mem- 

 brane, corresponding to the ■membrana lucida (the middle 

 layers) described by Zawadowsky. The outer covering is 

 generally designated the albuminous membrane; it has 

 been called the proteinaceous membrane by Jacobs and 

 Jones. The latter is a more satisfactory term in view 

 of its chemical nature, but for the sake of brevity it 

 will hereafter be referred to as the protein membrane. 



Ditylcnchus dipsaci has been described by Chitwood as 

 having only two membranes, the outer membrane being- 

 lacking. 



Only a few authors have concerned themselves with 

 the question of the origin of the various membranes. 

 Faure-Fremiet (1912a, 1912b, 1913) and Wottge (1937) 

 have described the formation of the shell proper and 

 the vitelline memibrane following fertilization. Two con- 

 secutive series of vacuoles appear in the egg cell and 

 move towards the surface. The first series transports 

 glycogen to the surface where it is converted into gluco- 

 samine and finally laid down as chitin in the shell 

 proper. The second series of vacuoles carries saponified 

 lipoidal constituents of the ovum to the surface where 

 they are transformed and deposited as the vitelline mem- 

 brane. There is disagreement between the two workers 

 (Wottge and Chitwood) who have studied the origin of 

 the outermost protein membrane. Wottge, on the basis 

 of the presence of this covering on unfertilized eggs, 

 expresses the opinion that it originally exists as a sort 

 of "ectoplasm" and is raised away from the egg by the 

 secondary secretion of the other two membranes. The 

 presence of rugose markings {Ascaris, etc.) and spirals 

 (Pgeudotrymus) in the protein membrane could easily be 

 explained by the assumption that regional secretion of 

 the protein takes place on the surface of the egg cell. 

 Chitwood, on the other hand, has found Asearis eggs 

 without this layer in the upper part of the uterus and 

 with advancing stages in the deposition of this covering 

 over the shell proper on eggs nearer the vagina. He 

 suggests that the outermost membrane is a uterine se- 

 cretion, added after the two inner membranes have been 

 formed. Support for this view is found in the report 

 of Wharton (1915) who obtained eggs without, the 

 protein membrane from female ascarids which oviposited 

 for several days in vitro in Kronecker's solution. He 

 attributed the absence of the membrane to "some physiol- 

 ogical condition which prevents formation and deposition 

 of the required substance by the uterine glands." 



The outermost protein membrane generally exists as 

 a coating around individual eggs. In some forms, how- 

 ever, it composes the matrix of the egg mass. Chitwood 

 has described this condition in the egg masses of Hetero- 

 dera marioni and the author has seen a somewhat similar 

 condition in the eggs of Enterobius vermicnlaris. In the 

 latter case the eggs are deposited in a mass in which 

 the eggs are held together by the adhesive properties 

 of the protein membrane, but they can be separated 

 mechanically and each egg will retain its coat. The 

 protein membrane has been studied chemically by Yo- 

 shida and Takano (1923), Wottge (1937), Chitwood 

 (1938) and Jacobs and Jones (1939), on the eggs of 

 Ascaris, Diocfophyma, Heterodera, and Enterobius. In 

 all cases it has been shown to be a complex protein. 

 It gives positive xanthoproteic. Millon's and ninhydrin 

 reactions; is digested by artificial gastric and pancreatic 

 juices; often swells and either wholly or partially dis- 

 solves in dilute mineral acids and alkalies, dilute acetic 

 acid, and five percent NaOCl. Wottge assumes that 

 some lipoidal material is present in this membrane on 

 Parascaris eqnorum eggs because of the absorption of 

 fat-soluble stains; Jacobs and Jones could not demonstrate 

 lipoids in this layer on Enterobius vermicularis eggs. 

 Chitwood obtained a positive Molisch reaction on an 

 extract of the "gelatinous mass" of Heterodera marioni 

 eggs and suggests the possibility that it is a glucoprotcin. 

 It may safely be assumed that there is variation in the 

 composition of this membrane in the eggs of different 

 forms, but that a fundamental protein base is present. 



The shell proper is the first structure of the egg- to 

 have received attention. Apparently Krakow (1892) was 

 the first to note the interesting fact that while the 



cuticle of nematodes is not chitin, their egg shells are 

 composed of this substance. Early tests for chitin de- 

 pended mainly on the insolubility of the substance in hot 

 concentrated KOH. Faure-Fremiet's descriptions of the 

 origin of the shell proper indicate that he had chemical 

 proof of its composition. Schulze (1924) apparently 

 applied more specific tests in identifying chitin in the 

 eggs of Ascaris sp. Later workers have made use of the 

 van Wisselingh tests for chitin which are discussed by 

 Kunike (192.^^) Kiihnelt (1928) and Campbell (1929). 

 The procedure is superheating the substance with con- 

 centrated KOH under pressure, any chitin being converted 

 into chitosan by this process. Chitosan so produced turns 

 brown on treatment with iodine-potassium iodide solution 

 and then reddish-violet upon the addition of dilute 

 sulphuric acid. It is soluble in dilute (3 percent) acetic acid 

 and can by recrystallized from the acetic acid solution 

 as minute sphaerocrystals by 1 percent sulphuric acid. It is 

 soluble in 75 percent sulphui-ic acid from which it can be 

 recrystallized by dilution. The sphaerocrystals thus 

 produced are stained red by 0.1 percent Rose Bengal. 

 These tests distinguish the substance as chitin; cellulose, 

 the only other organic skeletal substance which will 

 withstand the KOH treatment, does not stain with iodine- 

 dilute acid, does not dissolve in dilute acetic acid, and 

 does not yield the sphaerocrystals. Chitin is probably a 

 general constituent of the egg shell throughout the class 

 Nematoda. 



Faure-Fremiet, Wottge, Zawadowsky, Chitwood, and 

 Jacobs and Jones have identified the innermost membrane 

 as a sterol. The first two named authors are agreed 

 that this membrane in Ascaris eggs is cholesterol. It is 

 soluble in absolute alcohol, ether, chloroform, acetone, 

 benzene, and xylene. It is not darkened by osmic acid, 

 nor does it absorlb fat-soluble stains such as Sudan III 

 or Nile blue sulphate. It is dissolved slowly by saturated 

 sodium sulphide and is insoluble in 10 percent NaOH, HCl, 

 and acetic acid. It does not rotate the plane of polarized 

 light. Wottge obtained a positive test for cholesterol on 

 the membrane by the use of the Liebermann-Burchard re- 

 action. In Enterobius, the solubilities of the membrane 

 are the same as in Ascaris. According to Zawadowsky's 

 results on the eggs of Nematodirus spathiger and of four 

 species of Triehostrnngylidae, the lipoidal membrane in 

 these forms is slightly different in that it dissolves only 

 partially in absolute alcohol and hardly dissolves in ether. 



The knowledge of the chemistry of the egg membranes 

 is of practical importance in relation to the control of 

 nematode infections by destruction of the eggs. Early 

 workers had repeatedly noted the difficulty of fixing 

 nematode eggs even with the most rapid fixatives. Be- 

 sides incidental studies on the permeability of the egg 

 membranes to gases in relation to the requirements of 

 development [Jammes and Martin (1907a, b; 1910), 

 Faure-Fremiet (1925), Szweikowska (1928), Dvrdowska 

 (1931)], Zawadowskv and his co-workers (1914, 1928, 

 1929a, b, c) and Wottge (1937) have attacked this 

 problem directly. Their data allow the conclusion that 

 although the membranes are very permeable to many 

 gases, the vitelline membrane is semi-permeable to liquids 

 and protects the embryo from damage by chemical agents; 

 the protein membrane is an auxiliary chemical defense; 

 and the shell proper serves as protection from mechanical 

 forces. Huff (1936) has described a five-fold increase 

 in oxygen consumption of developing Ascaris eggs after 

 removal of the protein membrane. Wottge noted in his 

 experiments that liooid-dissolving substances did not 

 penetrate to the vitelline membrane after the complete 

 formation of the chitin shell. This difficulty of penetra- 

 tion was probably due not to the presence of the chitin 

 but to the presence of the protein membrane which is 

 to be found on eggs in that part of the uterus where 

 eggs with well-developed shells are present. Jacobs 

 and Jones have also pointed out that lipoid-dissolving 

 substances can not reach the vitelline membrane until 

 the protein membrane has been removed; if this outer 

 coat is dissolved off, solutions can easily rea:h the 

 vitelline membrane. They concluded that an effective 

 ovicide must dissolve proteins and lipoids in order to 

 reach the embryo. This statement should be modified 

 to include substances which will dissolve or be dissolved by 

 proteins and lipoids. 



Recent work of Jones and Jacobs (1939) indicates 

 that the protein membrane may also serve to inhibit 

 desiccation of eggs subjected to unfavorable tempera- 



178 



