Annals of The Entomological Society of America

Volume 62 JANUARY 15, 1969 Number 1

The Pine Chrysomelid, Glyptoscelis pubescens,1 in Northwestern Wisconsin2 Downloaded from https://academic.oup.com/aesa/article/62/1/1/102404 by guest on 02 October 2021 MICHAEL G. KLEIN AND HARRY C. COPPEL3 Department of Entomology, University of Wisconsin, Madison 53706 ABSTRACT A study was made of Glyptoscelis pubescens (F.) rion similis (Hartig), and then sealed the opening with (Coleoptera: Chrysomelidae) in northwestern Wisconsin a mixture of needle-chips and a fast-drying liquid. Eggs between 1963 and 1966. Although the has been averaged 1.38x0.56 mm. Egg development was com- reported on several coniferous hosts throughout the east- plete in about 2 weeks, but larvae remained in cocoons ern half of the United States, in Wisconsin it could be up to 10 additional days before emerging. Newly emerged found on only eastern white pine, Pinus strobus L. Both larvae averaged 1.7x0.51 mm. Mortality of eggs and lst- males and females fed on new and old foliage, removing instar larvae in the cocoons was 27% for both 1963 and needles at their bases, as well as cutting notches in their 1964. First-instar larvae dropped to the ground and dis- entire length. Adults were obtained from May 15 to appeared beneath the soil surface. No late-instar larvae August 29, with females averaging slightly longer (8.66 or pupae were found. Two species of parasites were col- vs. 8.12 mm) and wider (4.48 vs. 4.04 mm) than the lected, with Eupelmus sp. in 25% of field collected co- males. Females deposited an average of 26 eggs in se- coons, and larvae of Microctonus, n. sp. taken from dying lected empty cocoons of the introduced pine sawfly, Dip- G. pubescens adults.

The pine chrysomelid, Glyptoscelis pubescens (F.), Eumolpus pini Say, 1826: 295. is a potential pest of pine in eastern North America. Glyptoscelis hirtus, Fitch 1858: 746. Although the 1st published record for the species in Glyptoscelis pubescens, Horn 1886: 143. Wisconsin was in 1960 (Coppel 1960), specimens MATERIALS AND METHODS in the collection of the Department of Entomology, University of Wisconsin, date back to 1940. During Field work was undertaken in central Polk County, investigations on the biological control of the intro- near Amery, Wis. Adult were collected on a duced pine sawfly, Diprion similis (Hartig), it was cloth mat by beating eastern white pine, Pinus strobus established that the pine chrysomelid utilized empty L., branches. Study areas, consisting of woodlots of sawfly cocoons for oviposition sites. Because of this pine and mixed hardwoods, provided trees of all age bizarre relationship, a study was undertaken to sup- classes and with considerable variation in ground plement the little-known bionomics of G. pubescens, cover. with particular reference to the adults, eggs, and lst- In 1963 specimens were reared in a basement lab- instar larvae. oratory at 20°C and 80% RH. In 1964 rearing was in a 2nd-story room where the temperature fluctuated NOMENCLATURE from 17 to 28°C and about 55% RH. Additional stud- Contrary to the synonomy of Clavareau (1914) and ies were undertaken in the laboratory of the Univer- Krauss (1937), the original description of G. pubes- sity of Wisconsin, Department of Entomology, at cens was in the genus Cryptoccphalus. The following Madison. synonomy is offered to give agreement between refer- Empty cocoons of the introduced pine sawfly were ences and the generic names used. glued to white pine stems and placed in 18x18x13- cm cages for observations of oviposition habits. Daily Cryptoccphalus pubescens Fabricius, 1776: 220. Cryptoccphalus hirsutus Gmelin, 1791: 1703. examinations established the period between oviposi- Eumolpus hirttts Olivier, 1808: 906. tion and the time larvae left the cocoons, and adult longevity. 1 Coleoptera: Chrysomelidae. 3 Approved for publication by the Director of the Wisconsin DISTRIBUTION AND HOSTS Agricultural Experiment Station. Supported in part by the University of Wisconsin Research Committee of the Graduate School with funds supplied by the Wisconsin Alumni Research Glyptoscelis pubescens is distributed primarily east Foundation, and in part by the Conservation Division, Wisconsin Department of Natural Resources. Accepted for publication March of the Mississippi River (Fig. 1), in 20 of the 26 18, 1968. States, and Washington D.C., and from Ontario and s Research Assistant and Professor of Entomology, respec- tively, University of Wisconsin, Madison. Quebec in Canada. It has been reported also from 1 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA [Vol.62. No. 1 Downloaded from https://academic.oup.com/aesa/article/62/1/1/102404 by guest on 02 October 2021

FIG. 1.—The reported distribution of G. pubesccns in the United States and Canada.

Iowa (Wickham 1911) and Oregon (Crotch and trees before new foliage was fully formed. In feeding Cantab 1873) west of the Mississippi. Both Horn tests beetles cut notches in needles in about equal (1892) and Krauss (1937) expressed doubt of the proportion to removing needles at their bases on validity of the Oregon record. Distribution records spruce, larch, and Austrian pine. Beetles cut a greater indicated that G. pubescens was found throughout proportion of notches in new and old foliage rather May, June, and early July in New York, April to than removing needles at their bases in red, jack, and June in North Carolina, and as early as mid-March in Scots pine. Severe infestation on Virginia and white Alabama. Adults were collected from white pine in pine in a North Carolina seed orchard caused loss of northwestern Wisconsin from May 15 to Aug. 29. growth (USDA 1965). As many as 100 beetles/ 18-in. However, Arery few beetles were collected after the branch were recorded. These beetles caused damage 1st week of July. Adults were found generally by feeding on the edges of needles which later turned throughout the study areas. Although most individual brown. white pine trees had only 1 or 2 beetles if any, a maximum of 32 adults was taken from a single tree. DESCRIPTIONS OF STAGES AND LIFE HISTORY The pine hosts of G. pubescens include eastern Viable eggs were found in the field from early June white pine; pitch pine, Pinus rigida Mill.; jack pine, to Aug. 29. Adults in the laboratory did not deposit P. banksiana Lamb.; and Virginia pine, P. virginiana viable eggs after the 1st week in September. Eggs Mill. Additional records were found for spruce, hem- were almost 3 times as long as wide, cylindrical, with lock, and southern pines as hosts. Although G. pubes- rounded ends, and were yellow (Fig. 5). Measure- ccns was observed only on eastern white pine under ments of 130 eggs removed from D. similis cocoons natural conditions in Wisconsin, it could be force fed showed an average length of 1.38 mm (range 1.0- on a wide range of coniferous plants without any ap- 1.65). Average width at the widest point was 0.56 parent ill effects. We found no evidence that G. mm (range 0.5-0.6). The eggs had a sticky surface pubescens could feed on wild grape or hickory as re- which caused them to adhere to one another as well ported by Wilcox (1954). as to the walls of the cocoons. There were no hairs or other projections on the surface of the egg;. A FEEDING DAMAGE group of up to 30 pores at the anterior end made up Damage to pine trees was caused by adult feeding. what appeared as a micropylar area. The cells in this Major damage resulted when new foliage was cut off area protruded slightly. just below the point where the needle left the sheath. Larvae of G. pubescens were present in /). similis All 5 needles in a cluster were removed, leaving only cocoons in the field from late June until late Sep- a small cup formed by the needle sheath (Fig. 2). A tember. First-instar larvae were subcylindrical, pair of beetles normally removed 4-8 needle clusters slightly curved and had 3 pairs of well-developed over a 2-day period. Adults of G. pubescens also cut legs (Fig. 4). Measurements of 40 lst-instar larvae notches in the entire length of either new or old from D. similis cocoons gave the following dimen- foliage (Fig. 3). Needles eaten in this manner some- sions: average length 1.7 mm (range 1.5-2.0) ; aver- times remained in place, but often broke off where a age depth 0.51 mm (range 0.50-0.54). General body deep notch occurred. Additional damage resulted in color of larvae was yellow with a large brown the spring when adults removed male flowers from sclerotized shield on the dorsum of prothorax and January 1969] KLEIN AND COPPEL : G. pubcsccns IN WISCONSIN small sclerotized areas on the pleura of thoracic and 90% of beetles collected after the first of July were abdominal segments. The head capsule was nearly females. Laboratory experiments confirmed the circular in outline when viewed frontally (Fig. 6). longevity of females. One $ was kept alive until Average length from the top of vertex to the tip Oct. 7, more than a month later than any male. of labrum was 0.39 mm (range 0.38-0.41). Average Adults of G. pubescens were robust, oblong-oval, width at the widest point was 0.38 mm (range brown with a brassy sheen, and covered with short 0.34-0.45). The capsule was light brown except pubescence. Measurements from 52 $ and 40 $ for the mandibles, which were dark brown at the (Table 1) were within the range of 7.5-9.5 mm tips. The mandibles were similar, short, and stout. reported by Krauss (1937). Body length alone Two distinct mandibular teeth could be seen in a could be used to separate 84% of the males from lateral view. A 3rd tooth could be seen in a frontal the females, with 8.4 mm long as a dividing point. view (Fig. 7). Three setae and 1 pit were present A combination of body length and antennal length

on the outer face of each mandible. The lateral could be used for sexing adults with about 98% Downloaded from https://academic.oup.com/aesa/article/62/1/1/102404 by guest on 02 October 2021 ocelli were black. accuracy. Only 2 larvae beyond 1st instar were laboratory Female G. pubescens did not deposit viable eggs reared during our investigations. Both of these, unless they had been mated. As a result, eggs which died in the 2nd instar, differed little from deposited by the 1st females collected in May did lst-instar larvae, appearing only slightly larger. All not develop. In addition, the last few batches of attempts to rear or to collect larvae in the field eggs deposited in late summer often did not develop. failed. Krauss (1937) reported that larvae of this Females deposited viable eggs up to 46 clays after genus fed on roots at depths of 60-90 cm. Larvae last being mated. Mating was observed frequently of G. pubcsccns disappeared beneath the surface of in the laboratory from early June to mid-July. Males the soil within 15 sec after coming in contact with and females mated more than once and with differ- it. Larvae observed in plastic cages formed small ent partners. Six of 12 pairs under observation cells in the soil and remained apparently unchanged remained in the copulatory position after 10 hr, for a period of 8 months. with 18 hr being the maximum time recorded. A nearly equal sex ratio (34 9:30 $) was ob- Mating was observed at most hours of the day and tained from field collections made in early June. night under laboratory conditions. This observation This ratio gradually shifted to the point where about contrasted with field observations where single

FIG. 2, 3.—Feeding damage by G. pubescens to eastern white pine. 2 (above), Damage to new foliage (arrows) ; 3 (below), damage to needle. ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA [Vol. 62, No. 1 Downloaded from https://academic.oup.com/aesa/article/62/1/1/102404 by guest on 02 October 2021

(-0.23mmH |-Olmm-| I—005mm—| FIG. 4.—First-instar larva of G. pubescens. FIG. 5.—Egg of G. pubescens. FIG. 6.—Frontal view of head capsule of lst-instar G. pubescens larva. FIG. 7.—Frontal view of mandible of lst-instar G. pubescens larva. beetles rested at the base of the foliage both at night ditions in the laboratory. Eggs generally filled and in early daylight hours. about Yi the volume of the cocoons (Fig. 9) and Cocoons of D. similis adhere to eastern white usually rested on the sawfly remains. An average of pine twigs for several years. Females of G. pubescens 3 days (range 1-12) elapsed between 2 successive utilized cocoons opened by sawfly adults, rodents, ovipositions by a female. Female beetles required birds, and parasites as oviposition sites. The 35-60 min to deposit 20-25 eggs in a cocoon. opening was sealed with a mixture of needle chips The method for sealing cocoons after oviposition and fragments, and a fast-drying liquid after ovi- was described by Klein and Coppel (1965). About position. Field collections indicated that cocoons 40-75 min were required to complete a seal. Newly opened by sawfly adults were most numerous and formed seals were light green, and gradually turned were most frequently utilized for oviposition sites brown in the field. Degree of exposure to drying (75%). Sealed cocoons were taken from foliage, rather than length of time after formation appeared twigs, branches, and shrubs from ground level to to control the final color. Seals formed in the 2 m. In laboratory tests, no preference was shown laboratory remained green for several months. Seals toward cocoons opened by any one of the 4 agents from 3 cocoons removed from northern prickly previously mentioned. Females of G. pubescens ex- ash contained chips similar to those in seals of plored the cocoons both with the antennae and cocoons on white pine. with the styli at the tip of the tenth abdominal Laboratory studies showed that 12% of cocoons segment before initiating oviposition. The female utilized were sealed more than once. The only 2 positioned herself on the rim of the cocoon (Fig. 8) cocoons containing 2 batches of eggs were originally and deposited eggs through the flexible extended opened by sawfly adults. Remaining cocoons sealed ovipositor. The number of eggs deposited per twice contained 1 batch of eggs and had been cocoon (Table 2) was probably lower in the field opened originally by parasites. In these cocoons, than in the laboratory because of undisturbed con- the adult beetle had formed a barrier between January 1969] KLEIN AND COPPEL : G. pubescens IN WISCONSIN Table 1.—Body measurements (mm) of Glyptoscclis Table 2.—Number of eggs deposited per cocoon under pubcscens adults (52 9, 40 $) from Wisconsin. field and laboratory conditions. Polk County, Wis., 1963, 1964, and 1966. Male Female No. eggs per cocoon Measurement Max Min Avg Max Min Avg Source of cocoons Max Min Avg Body length 8.6 7.6 8.12 9.1 8.2 8.66 Width at 1963 laboratory 57 3 24 widest point 4.4 3.8 4.04 4.7 4.3 4.48 1964 laboratory 86 5 29 Body depth 3.4 2.9 3.18 3.6 3.3 3.50 1964 field 68 4 19 Antennal 1966 laboratory 53 12 28 length 4.4 3.8 4.12 4.1 3.6 3.88

where all 21 eggs were deposited in a crack. These Downloaded from https://academic.oup.com/aesa/article/62/1/1/102404 by guest on 02 October 2021 parasite and sawfly remains, and the eggs. The latter hatched and the larvae were recovered. Only flexibility of the terminal abdominal segments was 11% of eggs in the remainder of the cages hatched. shown by the ability of the female to form a seal Females placed in cages containing white pine inside the cocoon with access only through the bark utilized cracks and crevices in bark as oviposi- small hole. In addition to those cocoons sealed tion sites. Eggs were covered with the needle-chip twice, 3 of 202 cocoons contained a mixture of mixture in 3 of 4 cases. Additional observations eggs and the needle-chip material throughout the showed that beetles utilized empty tubes of the pine cocoon. These eggs failed to hatch. tube moth, Argyrotaenia pinatubana (Kearfott), as Since G. pubcscens had been reported from areas oviposition sites. Three tubes were collected con- where D. similis did not occur, studies were under- taining 8, 11, and 12 eggs and sealed at the end taken to establish oviposition habits of the beetle in with the needle-chip mixture. the absence of sawfly cocoons. When beetles were Laboratory studies in 1963 showed an average placed in cages with white pine foliage but no time of 27 days (range 20-30) was required between cocoons, eggs were found adhering to pine twigs the time of oviposition and the time larvae left the and needles, in the cup formed by the dropping cocoon. Eggs hatched after 18-20 days, and the of flowers or removal of new foliage, between larvae remained in the cocoons for as long as 10 needles and the sheath, in cracks in rearing cages, additional days. An average of 19 days (range and on the floors of cages. The cracks in cages 12-27) after oviposition was determined as the in which eggs were placed were sealed with needle- length of time required for larvae to leave cocoons chip mixture. In addition, 2 of the pockets formed in 1964. As in 1963, larvae spent up to 10 days by removal of the new foliage each contained an in the cocoons after emerging from the eggs. The egg surrounded by the needle-chip mixture. These shorter time required for egg development in 1964 eggs appeared desiccated after 48 hr and the embryos was probably the result of higher and fluctuating never developed. Kggs on cage floors made up temperatures during incubation. The 19-day average 60-80% of the total deposited, except in 1 cage in 1964 was near the 22-day average found for a limited number of field cocoons. The mortality of eggs and lst-instar larvae (Table

FIG. 8.—Female of G. pubcscens depositing eggs in D. FIG. 9.—Eggs of G. pubcscens in cut-open cocoon of D. similis cocoon. similis. ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA [Vol. 62, No. 1

Table 3.—Mortality of eggs and lst-instar larvae, in parasitized cocoons and larvae in the remainder, cocoons of D. similis, expressed as a percent of the total but the number of eggs destroyed per cocoon did eggs. Polk County, Wis., 1963-1964. not show a direct correlation to the number of % % mortality parasites present. Sawfly cocoons parasitized by No. mortality of lst-instar Eupelmus sp. were characterized by the presence of a Source of eggs eggs of eggs larvae large, tough, brown, parasite cocoon usually situated in the bottom. When more than 1 parasite larva was 1963 laboratory 1385 6 21 present, the cocoons formed were held together 1964 laboratory 2611 18 9 1964 field 853 13 20 loosely, but each larva was in a distinct cell. Adult parasites emerged in January from cocoons held at room temperature (23°C) since the previous sum- 3) was 27% in both 1§>63 and 1964. The higher mer. No emergence hole was cut by an adult para- percent of eggs which failed to hatch in 1964 was site. Emergence apparently took place through the Downloaded from https://academic.oup.com/aesa/article/62/1/1/102404 by guest on 02 October 2021 probably caused by low humidity and female age. hole formed either by a G. pubescens larva or by Many eggs deposited in late summer did not hatch, the drying and shrinking of the seal. Three cocoons probably because they were infertile. Of lst-instar which had the seals intact contained dead parpsite larval mortality in 1964, 68% resulted from larvae adults, apparently incapable of escaping. which were unable to break through the seal on the One parasite not previously reported was ob- cocoon. Although 26% of cocoons utilized by tained. It was determined as Microctonns, n. sp. G. pubescens as oviposition sites in 1963 were (Hymenoptera: Braconidae). Parasite larvae which opened by sawfly adults, all larval mortality oc- emerged from dying beetle adults were reared. curred in cocoons opened by 1 of the other agents. No predators were observed feeding on G. pubes- Larvae were unable to cut through seals which cens. However, 1 dead adult was removed from were more than a few chips thick. In 28% of a spider web. Of the cocoons sealed by the beetle 84 cocoons sealed in the laboratory in 1964, the in the field, 10% contained dead eggm or larvae, seal had either broken or had pulled away from and evidence of some additional . Cast the side of the cocoon and provided a hole through skins and silk from mites were most often asso- which larvae could escape. The presence of a ready- ciated with cocoons containing G. pubescens. Cast made hole in the seal did not stimulate larvae to skins from neuropterous larvae (Chrysopidae) were leave the cocoon earlier than usual. found in association with dead larvae in 2 cocoons. One larva cuts an emergence hole in completely The role of these was impossible to sealed cocoons. All larvae in a cocoon used the determine because they occurred also in empty same exit hole in 84% of the cocoons, with 3 cocoons of D. similis (Coppel 1960). holes being maximum. Exit holes were cut at any point in the cocoon seal. A hole was made by re- REFERENCES CITED moval of needle chips from an area of the seal. A larva would cut around a large chip with its Clavareau, H. 1914. Chrysomelidae: . Co- mandibles and then remove some of the surrounding leop. Cat, pars 59. 242: 1-215. material. The hole was thus enlarged until the Coppel, H. C. 1960. Empty cocoons of the introduced head capsule could be forced through. The mouth pine sawfly Diprion similis, as habitat niches for parts were used to supplement body movements to arthropods. Ann. Entomol. Soc. Amer. 53: 847-8. get the legs through the hole. Larvae left the Crotch, G. R., and M. A. Cantab. 1873. Materials for cocoon 1 at a time, a second appearing at the hole the study of the phytophaga of the United States. within a minute after the 1st larva left. Proc. Acad. Natur. Sci. Philadelphia 25: 19-83. Larvae of G. pubescens did not feed on their own Fabricius, J. C. 1776. Genera insectorum mantissa, Chi- larvae or eggs in the cocoon, even when left until lonii. Litteris M. F. Bartschii. 310 p. dead. In addition, no evidence of feeding on Fitch, A. 1858. Fourth report of the noxious and other chorions was found. of the state of New York. Trans. N. Y. State Agr. Soc. 1857. 17: 687-814. PARASITES AND PREDATORS Gmelin, J. F. 1791. Systema naturae (Linnaeus) 13th The only information on parasites of G. pubescens ed. Vol. 1, pt 4, p. 1517-2224. was given by Coppel (1960) in a study of empty Horn, G. H. 1886. A review of the species described by Olivier in the "Entomologie." Trans. Amer. cocoons of D. similis as habitat niches. He reported Entomol. Soc. 13: 135-44. 4 hymenopterous parasites from cocoons sealed by 1892. The Eumolpinae of boreal America. Ibid. 19: the beetle. The only parasite determined during our 195-234. study from cocoons containing eggs and lst-instar Klein, M. G., and H. C. Coppel. 1965. Oviposition larvae of G. pubescetis was Eupelmus sp. Of 60 habits of a white pine chrysomelid Glyptoscelis field-collected cocoons, 25% contained parasites. Only pubescens (Fabr.). Proc. N. Cent. Br. Entomol. Soc. Amer. 20: 140-1. 45% of the cocoons containing parasites had been Krauss, N. L. H. 1937. A study of the genus Glypto- opened originally by sawfly adults. The average scelis Le Conte in America north of Mexico (Coleop- number of parasites per cocoon was 2 (range 1-6). tera, Chrysomelidae). Univ. Calif. Publ. Entomol. Eggs of G. pubescens were fed upon in 73% of 7: 21-32. Olivier, A. G. 1808. Entomologie, ou Histoire Naturelle des Insects. Vol. 6. Paris. 956 p. January 1969] KLEIN AXD COPPEL : G. pubesccns IN WISCONSIN Say, T. 1826. Descriptions of new species of coleopter- Wickham, H. F. 1911. A list of the Coleoptera of ous insects inhabiting the United States. J. Acad. Iowa. State Univ. Iowa, Bull. Lab. Natur. Hist. Natur. Sci. Philadelphia 5: 293-304. 6(2) : 1-40. U.S. Department of Agriculture. 1965. Southern For- Wilcox, J. A. 1954. Leaf beetles of Ohio (Chrysomel- est Pest Reporter, Forest Service, no. 2. 8 p. idae: Coleoptera). Ohio Biol. Surv. 8: 351-506.

Chorionic Melanization in the Eggs of Aedes aegypti1,2

WILLIAM F. WALKER AND R. E. MENZER Department of Entomology, University of Maryland, College Park 20742 Downloaded from https://academic.oup.com/aesa/article/62/1/1/102404 by guest on 02 October 2021 ABSTRACT Completely reversible inhibition of melanization was at 45°C for 10 minutes. Chorionic melanization induced achieved by incubation of freshly deposited eggs of the in freshly deposited eggs by brief immersion in various yellow-fever mosquito, Acdcs aegypti (L.), at 4°C for 3 organic solvents was inhibited by copper chelating agents days or less, in a nitrogen atmosphere for not longer than and by incubation at 45-52°C for 10 minutes, the precise several hours, or in hypertonic XaCl solutions for up to temperature depending on the time of immersion. Despite 90 minutes. Irreversible inhibition was produced by sev- several positive correlations, concurrent egg swelling ap- eral chemical treatments, incubation of intact egg batches parently does not instigate melanization. dissected from the ovary, and incubation of individual eggs

Insect egg chorions are generally said to consist eggs placed in a saturated solution of the phenolase of 2 layers, a thin outer exochorion and an inner inhibitor phenylthiourea blackened normally. Chris- endochorion. A series of papers (Harwood and tophers (1960) reported the observation by several Horsfall 1957, 1959; Harwood 1958) described the researchers that A. aegypti eggs are white when de- synthesis and microstructure of mosquito egg shells posited and darken after about 1-2 hr, going through and the role of the various sublayers in control of egg an intermediate blue stage. Macfie (1915) noted that hydration. Several observers (Beament 1949, Beckel incubation of freshly deposited A. aegypti eggs in 1958, MeFarlane I960) have reported that the endo- 2.0% NaCl prevented melanization. Christophers chorion undergoes melanization and tanning. Har- (1960) cited the work of Gander (1951) showing wood (1958) defined the melanized portion of egg that melanization of A. aegypti eggs occurs under shells of the yellow-fever mosquito, Acdes aegypti paraffin oil. This fact was interpreted as evidence for (L.) as the chorion, distinct from the thin endo- tanning of the chorion. Christophers (1960) found chorion, which is not formed until after melanization. phenolase activity in freshly deposited A. aegypti Presumably these processes are controlled by the re- eggs, since incubation in an aqueous catechol solution actions of phenolase-produced quinones, as is true of resulted in a pink chorion. cuticles of insects and many other organisms. How- In view of the small amount of information avail- ever, little definitive work has been directed toward able concerning the control of chorionic melanization, an understanding of the control of chorionic melani- we decided to investigate further the mechanism of zation. chorionic melanization of A. aegypti eggs. This mos- Beament (1949) found that the chorion from eggs quito offered several advantages in studying this of 1-day-old Rhodnius prolixits Stal turns from color- problem. Eggs of known uniform age can be obtained less to dark brown when incubated with p-benzo- at relatively low cost. Oviposition can be induced quinone. A strong positive reaction with Tollen's readily, so that precise time periods after oviposition reagent suggested the presence of polyphenols in the can be determined. The chorion undergoes a striking endochorion. McFarlane (1960) found that incuba- change in color from white to black within about 30 tion of Aclicta domesticus (L.) eggs in tyrosine min, beginning 40 min after oviposition. This uni- caused the endochorion to melanize. This change form and rapid process was a favorable circumstance was inhibited by prior incubation in saturated phe- for rapid and reliable determination of the effect of nylthiourea, 0.1 M KCX, or boiling water. Beckel experimental variables. (1958") found that the endochorion of Acdcs hexodo- iiotits Dyar eggs undergoes a striking melanization MATERIALS AND METHODS within 2 hr after deposition. Melanization was in- All experiments were performed with the Bangkok hibited by heating the eggs at 90°C for 1 min, dehy- strain no. 2 of A. aegypti obtained from Walter Reed dration, or incubation in 0.1 M KCN. However, white Army Institute of Research in June 1964. Batches of 1000 larvae were raised at 26°C in 18x9-in. porcelain 1 Dipteral Culicidae. " Scifiitific article no. A1420, contribution no. 4038, of the pans. Two-hundred-fifty nig of finely ground Purina® Maryland Agricultural Experiment Station, Department of Laboratory Chow were sprinkled on the water sur- Entomology. Part of a thesis presented to the Graduate School of the University of Maryland by the first author in partial face daily until larvae pupated. Adults were caged at fulfillment of the requirements for the M.S. degree. Accepted for publication February 20, 1968. 70% RH at 26°C with a 12-hr day length. They were