The Eggs and Embryology of Some New Zealand Blepharoceridae (Diptera, Nematocera) with Reference to the Embryology of Other Nematocera

Transactions of the Royal Society of New Zealand : Zoology, Volume 8, Issue 18, 22 March 1967, Page 191

The Eggs and Embryology of Some New Zealand Blepharoceridae (Diptera, Nematocera) with Reference to the Embryology of Other Nematocera

D. A. Craig,

By

Zoology Department, University of Canterbury.

f ßeceived by the Editor, 24 February 7966.]

Abstract

The eggs of Neocurupira campbelli, N. chiltoni, N. hudsoni and N. tonnoiri are figured and described. An egg of Edwardsina australiensis is figured. The following are described for the embryonic development of N. chiltoni and less fully for N. campbelli :—germband formation, segmentation, formation of nervous system and mouthparts, blastokinesis, formation of gut and body appendages, late embryonic development and eclosion. The effect of temperature on the rate of embryonic development of N. chiltoni is also described. Comparisons are made between the embryology of N. chiltoni and N. campbelli and that of certain chironomids, culicids and simuliids. It is concluded that the similarities existing between the embryology of the simuliids and blepharocerids may indicate phylogenetic affinities.

Introduction

In the Nematocera a considerable amount of detailed embryological research has been carried out on the Chironomidae, Culicidae and Simuliidae. However, the accounts of Blepharoceridae eggs and embryology are fragmentary. Campbell (1921) described the oviposition and prehatching stages of N. chiltoni (Campbell). Tillyard (1922) and Tonnoir (1923) mentioned the oviposition of E. australiensis Tillyard and provided a description of the eggs. Tonnoir also recorded the time of segmentation in the embryo. Mannheims (1935) described eggs, oviposition and some of the early developmental changes in the eggs of Liponeura spp. Recently, Alexander (1963) has reviewed the observations of Campbell and of Mannheims; and Giudicelli (1964) has described the oviposition of Apistomyia elegans Bigot and Liponeura bischoffi Edwards.

Material: The majority of N. chiltoni eggs used in this study were collected from the field, the remainder being obtained from laboratory-reared adults. Other New Zealand blepharocerid eggs described in this paper were collected from the field. Eggs of E. australiensis were loaned by the Entomology Division, D.5.1.R., Nelson.

Methods : Developing eggs were kept in small Petri dishes lined with damp filter paper, or in small water-filled vials. Various fixatives were tried. Bouin’s fluid, hot and cold, tended to distort the eggs, but Carnoy’s fluid was found to give adequate fixation of unpunctured eggs within 5-10 minutes. Eggs fixed for this period of time were stored without deterioration in 70% alcohol. Telford (1957) experienced difficulty with Bouin’s fluid though Gambrell (1933) and Geyer-Dusznska (1956) used this fixative with success.

The chorion of mature eggs is tough and resilient, so that it requires softening or removal before sections can be cut. The method of De Coursey and Webster (1952) for the removal of the hard chorion of Aedes eggs with aqua regia was too violent and tended to distort blepharocerid eggs. A modification of the sodium hypochlorite method (Mortenson, 1950) was finally used and the fixed eggs were immersed for twenty-four hours in a 3% calcium hypochlorite solution at approximately 20°C. This treatment softened and sufficiently dissolved the chorion to enable sections to be cut, or for it to be removed with fine forceps.

Eggs for sectioning were orientated on small meat cubes with Mayer’s Albumen, the albumen then being hardened by a short immersion in absolute alcohol. The preparation was embedded using Peterfi’s Double Embedding Technique. Sections were cut at 4-6/x and stained with Delafield’s Haematoxylin and alcoholic Eosin.

Telford used Mayer’s Carmalum to obtain fine surface detail of Aedes egg whole mounts; however, excellent surface details of N. chiltoni embryos were obtained with Chlorazol Black E.

Observations

The Egg: The eggs of known New Zealand Blepharoceridae are roughly paraellipsoid in shape, with a slight flattening of the chorion on the ultimate ventral surface which is attached firmly to the substrate. The microsculpture of the dorsal or upper surface of the chorion is granulous, the raised portions being darker than the intervening portions of the chorion. The ventral surface of the chorion shows no microsculpturing at XIOO magnifications. According to Giudicelli the chorion of the eggs of A. elegans and L. bischoffi are completely smooth.

At X 1,200 magnifications the chorion in N. chiltoni and N. campbelli appears to consist of three distinct layers overlaid by a clear substance, 51—64// thick, which probably represents a cement layer similar to that described by Gambrell on Simulium pictipes eggs. The exochorion consists of a light brown layer 64—99//, thick, with the raised portions of the microsculpture protruding from the outer surface into the clear, cement-like layer; the endochorion is 26// thick and is heavily pigmented, accounting for the greater part of the colour of the chorion. The innermost layer is a strongly eosinophilic serosal cuticle, 32// thick.

The micropyle, at the anterior end of the dorsal surface of the chorion, usually consists of a rosette of dark spots arranged around a central dark region, the surrounding areas being lighter in colour than the rest of the chorion (fig. 2). The following descriptions of eggs are based on fresh material; the remarks concerning colour refer only to the dorsal or upper surface of the chorion.

Neocurupira campbelli Dumbleton (fig. 1) Size: 065 X 0.25 mm. Very heavily granulous, almost black in colour. Micropyle not in rosette form, but a dense black structure with a small clear central region. Neocurupira chiltoni (Campbell) (fig. 2) Size: 0.68 X 0.26 mm. Ends more rounded than other eggs. Lighter in colour than N. campbelli but darker than N. hudsoni. Without dark spots at ends of rosette arms as in N. hudsoni.

Neocurupira hudsoni Lamb (fig. 3) Size: 0.70 X 0.25 mm. Ends more tapered than in other species of Neocurupira and lighter in colour. Micropyle in rosette form with dark spots at ends of rosette arms.

Neocurupira tonnoiri Dumbleton (fig. 4) Size: 0.62 X 0.22 mm. Apart from smaller size, difficult to distinguish from eggs of N. chiltoni. Mature eggs of Peritheates spp. were not available but eggs dissected from adults and pharate adults of this genus are very similar in shape to those of Neocurupira though smaller in size:

Peritheates harrisi (Campbell) Size: 0.58 X 0.28 mm. Peritheates intermedins Tillyard Size: 0.52 X 0.16 mm. Peritheates turrifer Lamb Size: 0.59 X 0.19 mm.

The shape of New Zealand blepharocerid eggs is similar to that described and figured for Apistomyia elegans and Liponeura bischoffi by Giudicelli and for L. cinerascens Loew by Mannheims. The eggs of the primitive Australian blepharocerid Edwardsina australiensis are more flattened dorsoventrally and pointed apically than the eggs of New Zealand blepharocerids (fig. 5).

Early Development and Germ Band Formation: As the living embryo is transparent and shows no surface detail until approximately the twenty-second day, the following description is based on fixed material. The ages quoted for embryos are for development at 12° C, and unless otherwise stated the embryos were examined prior to 10.00 hours on the days cited. When newly laid, the eggs of N. campbelli and N. chiltoni are creamy-white in colour, but turn bluish and then brown-black on the dorsal surface of the chorion within 3-4 hours. The dark central region of the micropyle appears immediately after oviposition and at the same time the oosome (Johannsen and Butt, 1941) appears at the posterior end of the egg.

Alexander and Mannheims each described similar changes for other blepharocerid eggs. Mannheims considered the darkening of the chorion to be due to chemicals in the water, however, Giudicelli states that the process of melanisation of the chorion is due to the action of a tyrosinase on breakdown products of protein. Attempts to detect early stages of development, such as maturation division and formation of the blastoderm, were not successful.

Sections of newly-laid eggs of N. chiltoni show the presence of an extremely thin vitelline membrane and sections of twenty-four-hour eggs of N. camphelli and N. chiltoni show the blastoderm to be spread evenly over the yolk. By the fourth day the embryonic membranes are formed and the germ band appears as a bilobed structure on the ventral surface of the yolk, showing the gastrular groove and the sero-amniotic opening (fig. 6). The amnion of N. camphelli and N. chiltoni is composed of extremely flattened cells with prominent nuclei while the serosa is made up of thicker cells (fig. 23).

The germ band, prior to blastokinesis, is directed away from the substrate, but nevertheless defines the ventral surface of'the yolk. As the germ band commences to elongate, on approximately the sixth day, yolk shrinkage occurs. This appears to be associated with the growth of the tail region of the germ band posteriorly and then dorsally over the yolk. By the end of the sixth day, the head lobe has curved over on to the anterior end of the yolk (figs. 7 and 16). Longitudinal shrinkage of the yolk in N. chiltoni continues until the ninth day, by which time the tail region of the embryo has progressed along the dorsal surface of the yolk and now almost

underlies the head lobe (fig. 17). In comparison the tail region and head lobe of N. campbelli are very closely applied during this stage.

Often in eight-day eggs of N. chiltoni the elongated germ band buckles laterally and describes a curved path across the yolk (fig. 7).

Segmentation : Sections of seven-day eggs of N. chiltoni show that the mesoderm of the germ band becomes segmented before the ectoderm. Segmentation on the surface of the germ band of N. chiltoni is visible at approximately the eighth day. Tonnoir (1923) states that the segmentation of Edwardsina australiensis was visible in fresh eggs after nineteen hours (temperature of incubation not given). A sample of E. australiensis eggs was available from Tonnoir’s collection, but sections unfortunately showed no signs of segmentation.

By early ninth day the antennal, mandibular, maxillary and labial segments are clearly defined. Lying between the antennal and mandibular segments are two triangular intercalary segments (Johannsen and Butt) (figs. 8 and 17). These intercalary segments are resorbed by the eleventh day. Segmentation progresses from the anterior to the posterior in a fashion similar to that described by De Coursey and Webster, and Rosay, so that by the tenth day a total of seventeen segments is apparent (fig. 18). The antennal and three gnathal segments are followed by three thoracic and ten abdominal segments.

There has been considerable disagreement concerning the number and arrangement of the segments of larval blepharocerids, due to the amount of fusion in the anterior and posterior segments. The main differences in interpretation centre around the number of abdominal segments fused into the cephalic and anal divisions, and the relation of the suckers to these segments. Some of the interpretations by various authors are presented in chronological order in Table I.

Further development of the embryos of N. campbelli and N. chiltoni shows that only the first abdominal segment fuses with the thorax and head to form the cephalic division and that there are four fused abdominal segments in the anal division. This is in complete agreement with Mannheims’ interpretation.

Formation of the Nervous System : Before the intercalary segment is resorbed on the eleventh day, a deep neural groove is formed (fig. 8). This extends from the stomodaeum to the last abdominal segment. External evidence of neurulation is obliterated by the thirteenth day (fig. 9) and sections at this stage show that each thoracic and abdominal segment has already a definite ganglion.

As the thoracic segments regress, the individual thoracic ganglia are observable through the thin ectoderm (figs. 11, 13, 24 and 27). As external evidence of the thorax becomes obliterated by the anterior movement of the developing first abdominal sucker, the sub-oesophageal and the first thoracic ganglia become closely applied but retain their individual nature. However, the second and third thoracic ganglia plus the first abdominal ganglion become fused into a single structure. The original constituents of the fused ganglion can be clearly identified in longitudinal sections. The identity of the first abdominal ganglion is further suggested by the first abdominal sucker apparently retaining its innervation from this ganglion (fig. 28).

The regression and fusion of the seventh to tenth abdominal segments is such that by the eighteenth day the ganglia protrude beyond the remainder of the abdominal extoderm (figs. 11, 21 and 25). The ganglia of the seventh to tenth segments then completely fuse into a single posterior ganglion and there is then no longer any indication of the individual ganglia in longitudinal sections.

Mannheims investigated the structure of the ganglion in the cephalic division of larval blepharocerids but failed to determine the relationship of the fused thoracic and first abdominal ganglia. He pointed out that such an investigation would be better carried out on embryonic blepharocerids.

C: segment incorporated into the cephalic division; s: sucker; A: segment incorporated into the anal division; —: segment not present; Blank spaces: information not given.

Mouthpart Formation: The mandibular rudiment, though large initially, regresses and becomes comparatively small as the development of the maxilla proceeds (figs. 9, 13 and 14). The maxillary rudiment constricts into two portions, distal and basal (figs. 9 and 19). The distal portion eventually forms the maxillary palp, which grows dorsally then posteriorly and later covers the lateral margin of the labial rudiment and a small part of the first thoracic segment (figs. 19 and 20). The posterior part of the maxillary palp grows medially at the same time as the two labial rudiments fuse into a single median structure (figs. 9, 10 and 11). The basal portion of the maxillary rudiment grows anteriorly forming a curved plate which covers the mandibles laterally and medially (figs. 13, 14, 15, 21 and 22). This plate forms the galea and the lacinia. The labrum arises as a single lobe anterior to the stomodaeum and by the eleventh day is bilobed, becoming single lobed again by the thirteenth day.

Bischoff (1928) interpreted the structure formed from the distal lobe-like portion of the maxillary rudiment as the “ Mentallappen (Bolster) ” and the median sensory structure on the maxilla as the maxillary palp. Craig (1967) on the basis of comparative embryology with other insect orders, and with evidence from the musculature of the larval maxilla, shows that the distal lobe is probably the true maxillary palp and the median sensory structure the lacinia.

Blastokinesis : Blastokinesis or the rotation of the yolk and embryo through 180°, occurs in N. chiltoni on approximately the eleventh day, normally taking only a few hours but in some cases requiring up to two days to complete. The ventral surface of the embryo is then directed towards the substrate. (Figures 19, 20, 21 and 22 are presented in the pre-blastokinesis orientation for clarity.)

Associated with blastokinesis of N. chiltoni is the shrinkage of the tail region which until now has lain along the dorsal surface of the yolk. By the fourteenth day the tail region lies almost completely on the ventral surface (fig. 19).

At the same time as blastokinesis occurs in N. chiltoni the embryo begins to swell, completely filling the chorion by the twenty-second day. Considerable pressure appears to be exerted on the chorion as the egg changes shape slightly and the initial hatching movement is violent.

Gut Formation: Stomodaeum formation takes place with the advent of segmentation and first appears as an anterior invagination of the germ band on the seventh day. Development of the proctodaeum does not begin until the tail region of the embryo shortens on approximately the fourteenth day. However, development of the proctodaeum is rapid and by the twentieth day it has assumed the simple coiled shape of the hind gut of larval blepharocerids described by Muller (1879). By the twentieth day the stomodaeum and the proctodaeum intrude into the yolk, but there is little evidence yet of midgut formation, although by this stage the yolk is becoming enclosed within the body wall (fig. 22). By the twenty-eighth day the midgut epithelial rudiment has enclosed the yolk completely (fig. 28).

Body Appendages: The thoracic appendages develop on the fourteenth day as medially directed lobes on the posterior margin of the thoracic segments. The line of the thoracic prolegs is continued on to the abdominal segments as a faintly visible abdominal ridge (fig. 9). By the sixteenth day the thoracic prolegs have become peg-like (fig. 10), and begin to move anteriorly as the embryo elongates and as the thoracic segments are compressed by the enlarging abdominal segments (fig. 11). By the twenty-second day the prolegs have completely regressed. Fusion of the thoracic segments is completed by the twenty-second day, but their regression continues until the twenty-eighth day by which time the developing first abdominal sucker has moved anteriorly (figs. 13, 14 and 15) and lies ventral to the first and second thoracic ganglia (fig. 28),

Development of the suckers begins on approximately the twentieth day, as the ventral ectoderm of the first to sixth abdominal segments becomes thickened and raised into median blocks (fig. 12). By the twenty-fourth day the pistons of the suckers show as distinct central regions on the raised blocks of ectoderm (fig. 13) . Sections at this stage show that the piston already has muscle attachments and is surrounded by a cylinder of ectoderm (fig. 26), which develops into the fleshy rim of the sucker (fig. 28). Bischoff, Hora (1930) and Komareck (1914) give detailed accounts of the fully developed blepharocerid sucker.

The abdominal prolegs (pseudopods, Johannsen) of the first six abdominal segments begin development on the twentieth day (fig. 12) as low projections from the abdominal ridge which lies in series with the now almost regressed thoracic prolegs. The method of development and the histological similarities (fig. 24 and 26) between the thoracic and the abdominal prolegs suggest that they are homologous structures. The large lobes of ectoderm flanking the protruding ganglia of the seventh to the tenth abdominal segments (figs. 11 and 21) show serial and histological similarities (figs. 11, 12, 24, 25 and 26) to the thoracic prolegs and to the prolegs of the first six abdominal segments. This suggests that they may represent the rudimentary prolegs of the seventh to tenth abdominal segments.

The prolegs of the first six abdominal segments become cone-shaped and by the thirty-second day (fig. 15) have developed the extensile tip described by Tonnoir (1924) as typical of first instar larvae of blepharocerids. The ectodermal lobes of the seventh to tenth abdominal segments regress and fuse into the anal division.

Late Embryonic Development: The anal blood gills begin development on approximately the twenty-fifth day. The two pairs are initially the same size, but the more lateral pair grow larger by the twenty-sixth day (fig. 14) and by the thirty-second day have moved posteriorly from the smaller anterior blood gills (fig. 15). By the twenty-eighth day the ocelli are visible as red structures on the anterio-lateral surface of the cephalic division. The suckers become pigmented on the thirty-second day and at approximately this time the embryo becomes capable of movement. Movements have been observed of mouth parts, suckers and abdominal prolegs. During the thirty-fourth day the sclerite of the egg-burster becomes heavily pigmented as do the latero-dorsal body spines, and by the thirtyseventh day all body spines are clearly visible through the chorion.

The egg-burster (egg-tooth, Wigglesworth 1964; hatching-spine, Clements 1963) of N. campbelli and N. chiltoni is a relatively long blade-like structure supported by a strong median sclerite lying between the ocelli. The supporting sclerite is sunken into the surrounding cuticle so that the sharp edge of the egg-burster is just below the lip of the groove. The blade-like egg-burster of the blepharocerids is probably forced against the chorion of the egg by pressure of the body fluids as no protractor muscles have been detected in sections. The mouth part movements prior to eclosion may indicate manipulation of the egg-burster or perhaps active gnawing at the chorion. Egg-bursters have been reported for simuliids (Puri, 1925) and the culicids (Clements, and Wigglesworth).

Eclosion : Eclosion takes place on the thirty-ninth to fortieth day. the chorion splits dorsally, commencing at the micropyle and progressing posteriorly, the split almost without exception curving away to the right. Giudicelli reports similar dehiscence of the chorion in eggs of Apistomyia and Liponeura. The larva is partially ejected in the violent hatching movement, suggesting a hatching mechanism of a hydraulic nature. The larva then attaches the anterior suckers to the substrate and pulls the remainder of its body free. The newly hatched larvae of N. chiltoni

range from o.Bmm to 1.1. mm in length. The colour of the newly hatched larva is initially creamy-white but darkens to black-brown within two to three hours.

Rates of Embryonic Development: Eggs of N. chiltoni laid in the laboratory and of known age were incubated at a series of constant temperatures ranging from O°C to 27°G (fig. 29). The temperature of any one experiment never varied more than ± 1.5° C from the mean temperature.

Development and hatching were normal only at temperatures between 4°G and 18°G. Eggs reared at 20°G developed to full term embryos, but few hatched. Eggs incubated at O°C failed to develop and it was found that exposure to this temperature for as little as twenty minutes was lethal. A constant temperature of 25°G was found to be lethal, however, eggs could withstand exposure to 27°C for up to five hours before failing to develop at suitable lower temperatures.

Discussion

The structure of the chorion of N. campbelli and N. chiltoni is similar to that of the culicid eggs as described by Clements, who also pointed out that the exochorion and endochorion are soluble in hypochlorite solutions. Gambrell describes only two membranes on the eggs of Simulium pictipes besides the cement layer, a thin chorion and vitelline membrane. Early gastrulation and formation of the embryonic membranes similar to that occurring in the embryos of N. campbelli and N. chiltoni, is described for S. pictipes by Gambrell. However, the embryonic membranes of the culicids (Clements) and of the chironomids (Miall and Hammond 1900) are formed at a much later stage than gastrulation. These membranes of N. campbelli and N. chiltoni, in contrast to those of simuliids and culicids, have a serosa composed of thick cells and an amnion of very thin cells with prominent nuclei (fig. 23).

The germ bands on the yolk of N. campbelli and N. chiltoni at the eight-day stage are relatively narrow structures and are at the most only slightly embedded in the yolk. The germ bands of some simuliids (Gambrell, and Mecznikow 1866) are also narrow at a comparable stage, but the tail regions are deeply embedded in the yolk. The germ bands of the culicids (Clements, Rosay 1959) and of the chironomids (Miall and Hammond) are considerably wider, the tail region of the chironomids being deeply embedded in the yolk.

At a stage comparable to that of an eight-day N. chiltoni egg, where the germ band curves laterally over the yolk, Rosay describes vertical folding in the germ band of Culex tarsalis. The faint indentations visible on the germ band of the eight-day N. chiltoni egg (fig. 7) are early indications of segmentation and not merely folding as in C. tarsalis.

The initiation of segmentation in the mesoderm of the germ band of N. chiltoni follows the pattern common to many arthropods (Johannsen and Butt, Manton 1960), However, Clements states that in the culicids the mesoderm differentiates later than the ectoderm. The association of blastokinesis and tail region shrinkage as in N. chiltoni also occurs in Culex tarsalis, but they do not appear to be associated in Simulium pictipes. The amount of rotation during blastokinesis of C. tarsalis and S. pictipes embryos is similar to that of N. chiltoni, however, Miall and Hammond state that blastokinesis in Chironomus eggs takes place in two stages through a total of 360°.

The development of the larval labrum of N. chiltoni, from a single lobe which becomes bilobed for a short period, agrees with the general labral development as put forward by Crampton (1921) and Manton. Butt (1960), however, states that in many insects the labrum arises as a double structure and that the labral lobes represent the appendages of the intercalary segments, the intercalary appendages migrating around the mouth preorally and fusing to form the labrum. The observations presented in this paper show clearly that the labrum is not formed by the fusion of preoral appendages and is definitely not formed from the intercalary segments. This agrees in principle with Crampton and with Manton.

Although the thoracic and abdominal prolegs of N. chiltoni show positional and histological similarities suggesting that they are homologous (page 202), Hinton (1955) believes that the abdominal prolegs of dipterous larvae have been secondarily evolved and he presents considerable evidence to show that they are not homologous with the thoracic prolegs. The origin of the suckers from raised ectodermal blocks on the ventral surface of the abdominal segments indicates that contrary to Hora’s (1930 and 1933) theory, they did not evolve from fused paired abdominal prolegs, but rather by modification of the ventral surface of the body as suggested by Tonnoir (1933).

Conclusion

Apart from the differences in the chorion, embryonic membranes and in the invagination of the tail region of the germ band, the initial emryonic development of N. campbelli and N. chiltoni shows greater similarities to the published accounts of simuliid embryology than to those of the embryology of other Nematocera. This may indicate that the Blepharoceridae have closer phylogenetic affinities to the Simuliidae than to other Nematocera.

Acknowledgments

I wish to thank Dr R. L. C. Pilgrim, Dr V. M. Stout and R. Craig for helpful criticism during the preparation of this paper, the Entomology Division, D.5.1.R., Nelson, for the loan of Edvuardsina australiensis eggs, and those who helped with techniques and the preparation of material.

LIST OF ABBREVIATIONS

Ab. 10, Abdominal segment 10; abg., Abdominal ganglion; abg. 1, Abdominal ganglion 1; abp., Abdominal proleg; abr., Abdominal ridge; ag., Anal gills; am., Amnion; ant., Antenna; eg., Cerebral ganglion; ect., Ectoderm; gb. Germ band; gg., Gastrular groove; hi., Head lobe; int., Intercalary segment; lb., Labium; lr., Labrum; mge., Midgut epithelium; md., Mandible; mi., Micropyle; mx., Maxilla; mxp., Maxillary palp; ng., Neural groove; p., Piston; pr., Proctodeum; sa., Sero-amniotic opening; se., Serosa; sk., Sucker; st, Stomodeum; subg., Sub-oesophageal ganglion; th 1., Thoracic segment 1; th 3., Thoracic segment 3; thg., Thoracic ganglion; thgl., Thoracic ganglion 1; thp., Thoracic proleg; Vent., Ventral surface; y., Yolk.

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Whitten, J. M., 1963. The Tracheal Pattern and Body Segmentation in the Blepharocerid larva. Proc. R. ent. Soc. Land. (A). 38(1-3): 39-44. Wiggles worth, V. 8., 1964. The Life of Insects. Weidenfeld and Nicolson, London, 351 pp.

D. A. Craig, Department of Entomology, The University of Alberta, Edmonton, Canada,

(1923) (1934) (1935) et (1957) (1957) (1963) Head G G G C G G G Segments 1 C G C G G G G Cephalic Thoracic C G c G C G C division 3 G G G C G G G 1 G Cs Cs G Cs C Gs 2 Cs s s Cs s Cs s 3 s s s s s s s Free 4 s s s s s s s Abdominal 5 s s s s s s s segments 6 s s s s s s s 7 s A A s A s A 8 A A A A A Anal 9 A A A A A? division 10 A — A — —

Table I.—lnterpretations of the Arrangement of the Segments of Larval Blepharoceridae.