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Morphological effects of insect growth regulating compounds on Aedes aegypti (Diptera: Culicidae) larvae
Life Sciences, Vol, 24, pp . 817-832 Printed in the U .S .A .
Pergamon Press
MORPHOLOGICAL EFFECTS OF INSECT GROWTH REGULATIN(; COMPOUNDS ON AEDES AE GYPT I (DIPTERA : CULICIDAE) LARVAE?/ J . Cocke,* A . C, Bridges,t R . T, Mayer,t and J, K . Olson° *Texas Ad~M Agricultural Research and Extension Center Weslaco, Texas tlleterinary Toxicology and Fntomology Research Laboratory Agricultural Research-Science and Education Administration U . S . Department of Agriculture College Station, Texas °°Department of Entomology, Texas AbM University College Station, Texas (Received in final form January 25, 1979) Surtmary Cuticular development of Aedes ae ti larvae was examined by electron microscopy and comparisons were made between larvae exposed to methoprene, isopropyl (E,E)-11-methoxy-3,7,11trimethyl-2,4-dodecadienoate, those treated with the fluorescent insect growth regulator, 5-[[[5-(dimeth lamino)-1-naphthalenyl]sulfor~yl]amino]-1,3-benzodioxole (DNSAB~, and untreated larvae . Larvae of all three groups were routinely fixed at 24, 48, and 72 hr posttreatment . Thin sections of the sixth-abdominal segment, anal papillae, midgut tissue, and Malpighian tubules were examined for morphological variations from controls . Methoprene and DNSAB appear to interfere with lysis and reabsorption of old endocuticle and prohibit the synthesis and deposition of new, well-structured procuticle by the epidermal cells . Disrupted mitochondria and numerous vesicles in other tissues examined are suggestive of possible changes in membrane selectivity and permeability . The ability of an insect growth regulating compound to penetrate the exoskeleton and be absorbed and/or accumulated in subcuticular tissues is of primary importance since it is one of the factors which determines the efficaciousness of the compound . Penetration of the growth regulating compound varies with the method of application, environment, target organism, and active lifetime of the compound . Mayer et al (1) reported the primary sites of absorption and/or accumulation of the ~Tuorescent insect growth regulator (FIGR), 5-[[[5-(dimethylApproved as Technical Article No . 13996 . Texas Agricultural Experiment Station . This research was conducted in cooperation with the Federal Agricultural-Science and Education Administration, U . S . Department of Agriculture . This paper reports the results of research only . Mention of a pesticide does not constitute a recommendation for use by the USDA nor does it imply registration under FIFRA as amended . Also, mention of a commercial or proprietary product does not constitute an endorsement by the USDA . 0024-3205/79/090817-1502 .00/0 Copyright (c) 1979 Pergamon Press Ltd
818
Insect Growth Regulation
Vol . 24, No . 9, 1979
amino)-1-naphthalenyl]sulfonyl]amino]-1,3-benzodioxole (DNSAB), in larvae of Aedes aegypti (L .) . The compound accumulated rapidly in the anal papillae acid in midgut cells . In addition, third-instar larval cuticle was incompletely shed from the sixth- and seventh-abdominal segments (2) at the time of ecdysis . Since the fluorescent probe characteristics of the compound indicated the sites of accumulation (1, 3), we decided to investigate the effects of the FIGR and a commercially available insect growth regulator (IGR), methoprene, iso propyl (E,E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate, at the ultrastructural level . Since only a limited number of investigations have been concerned with the effects of IGRs on epidermal cell activity and cuticle formation (4, 5, 6, 7), an ultrastructural study of the morphological effects of methoprene and DNSAB on mosquito cuticle and associated tissues is presented herein . Materials and Methods Third-instar larvae maintained in either deionized water, 7 .5 ppm DNSAB, or 0 .01 ppm methoprene were the primary subjects of this study . At 24, 48, and 72 hr posttreatment, five third-instar larvae from each group were washed five times with deionized water and dissected in a fixative containing 2X gluteraldehyde, 1X paraformaldehyde, and 0 .1 M sucrose in 0 .1 M phosphate buffer at pH 7 .3 . In addition, larvae that molted to the fourth instar were also prepared as above . The sixth- and seventh-abdominal segments, anal papillae, midguts, and Malpighian tubes were removed from each larva and fixed for 15 min at room temperature, then placed in ice for 1-3 hr . After rinsing and washing with cold 0 .1 M phosphate buffer (five times), the tissues were fixed in osmium tetroxide in 0 .1 M sucrose for 2 hr . Following five to ten distilled water rinses, the tissues were blockstained in 0 .5X aqueous uranyl acetate for 5 hr . After staining, the specimens were washed three to five times in distilled water and subsequently dehydrated through a 25, 50, 75, 95, and 100K ethyl alcohol series . Further dehydration was accomplished with three changes of acetone prior to incubation in an acetone/Spurn® resin (Polysciences, Inc ., Warrington, PA) (1 :1 ; v/v) mixture . After 2 hr, the 1 :1 mixture was replaced by a 1 :4 mixture and incubation allôwed to continue overnight . The following day, the tissues were placed in 100X Spurn and incubated at 70°C for 24 hr to allow complete polymerization .
lx
0
Sections ca . 500 A thick were cut from the tissue blocks using a Dupont® diamond knife mounted on a Sorvall® MT-2B or Porter Blum ultramicrotome . These sections were stained with 0 .5X aqueous uranyl acetate and Reynolds' lead citrate (8) and subsequently examined using a Phillips 300 transmission electron microscope at 60 Kv . Results Control Larval Cuticle--Cuticle from the sixth-abdominal segment of both third- an ourt -instar arvae was examined at 24, 48, and 72 hr after the tests were begun . Figure 1 demonstrates the normal cuticle layers and epider mal cell structure, A dense exocuticle (EPC) (ca . 0 .05u) covering a rather thin (ca . 0 .25u), rugose exocuticle (EXC) was observed in both instars . The exocuticle appeared to have a coarse, granular texture and consisted of two to three lamellae, while the endocuticle (ENC) appeared to have a light, fine texture and consisted of four to five lamellae . The epidermal cells averaged approximately 1 .Ou thick and 8 .Ou in length . Each lateral epidermal cell membrane (CM) was interconnected to the adjacent lateral cell membrane via septate desmosomes .
Vol . 24, No . 9, 1979
Insect Growth Regulation
819
The outer cell membrane displayed typical microvilli . Each microvillus showed at its apical region a darkened granulose area which extended a short distance into the endocuticle . These cells displayed large numbers of rough endoplasmic reticula and dense granular materials in their cytoplasm . Numerous mitochondria (M) were present in the upper half of each epidermal cell in these larvae . In some third-instar specimens fixed at 48 hr, an ecdysial membrane was found above a retracted outer epidermal cell membrane (FIG . 1) . The space between the cell membrane and the ecdysial membrane (EM) was continuous along the upper epidermal surface and measured from ca . 0 .04u to ca . 0 .06u .
FIG . 1 . Micrograph of section of integument taken from the sixthabdominal segment of a third-instar A . ae ti untreated larva at 48 hr after the test was begun showing e le (EPC), exocuticle (EXC), endocuticle (ENC), ecdysial membrane (EM), cell membrane (CM), mitochondria (M), and nucleus (N) . Early fourth-instar larvae fixed at 48 hr showed an average cuticle thickness of ca . 0 .35 u (FIG . 2) . The epicuticle appeared smooth and darkly stained . Only one undifferentiated layer of procuticle (PrC) was observed in these specimens . The developing endocuticle (procuticle) was divided into two distinct undifferentiated zones : an upper granulose, darkly stained zone and a lower fine grained, lightly stained zone . The outer cell membrane displayed processes (CMP) (or plasma membrane plaques) (9) where cuticulin deposition occurs . The cuticulin granules appeared to be continuous from the tips of the processes with strands of granules extending into the light staining layer of the endocuticle . The cytoplasm of these cells was densely packed with ribosome , rough endoplasmic reticula, and glycogen . Numerous microtubules (ca . 100 ~ diameter) projected upward towards the cell membrane processes and appeared tö terminate along the upper epidermal cell membrane surface . Fourth-instar larvae fixed at 72 hr displayed a cuticle thickness averaging ca . 0 .70 u . The upper layers (cement, wax, lipid) of the epicuticle in most specimens examined showed disruption (FIG . 3) . The wax and lipid layers appeared to have been dissolved during the dehydration process from between the cement (C) and cuticulin layers leaving a space approximately 0 .06u between the two . The exocuticle showed more compacted lamellae and stained more intensely than did the endocuticle . The endocuticle was composed of four to five )emgllae . In some areas of the cuticle, pore canals (PC) measuring ca . 150-200 A
Pergamon Press
MORPHOLOGICAL EFFECTS OF INSECT GROWTH REGULATIN(; COMPOUNDS ON AEDES AE GYPT I (DIPTERA : CULICIDAE) LARVAE?/ J . Cocke,* A . C, Bridges,t R . T, Mayer,t and J, K . Olson° *Texas Ad~M Agricultural Research and Extension Center Weslaco, Texas tlleterinary Toxicology and Fntomology Research Laboratory Agricultural Research-Science and Education Administration U . S . Department of Agriculture College Station, Texas °°Department of Entomology, Texas AbM University College Station, Texas (Received in final form January 25, 1979) Surtmary Cuticular development of Aedes ae ti larvae was examined by electron microscopy and comparisons were made between larvae exposed to methoprene, isopropyl (E,E)-11-methoxy-3,7,11trimethyl-2,4-dodecadienoate, those treated with the fluorescent insect growth regulator, 5-[[[5-(dimeth lamino)-1-naphthalenyl]sulfor~yl]amino]-1,3-benzodioxole (DNSAB~, and untreated larvae . Larvae of all three groups were routinely fixed at 24, 48, and 72 hr posttreatment . Thin sections of the sixth-abdominal segment, anal papillae, midgut tissue, and Malpighian tubules were examined for morphological variations from controls . Methoprene and DNSAB appear to interfere with lysis and reabsorption of old endocuticle and prohibit the synthesis and deposition of new, well-structured procuticle by the epidermal cells . Disrupted mitochondria and numerous vesicles in other tissues examined are suggestive of possible changes in membrane selectivity and permeability . The ability of an insect growth regulating compound to penetrate the exoskeleton and be absorbed and/or accumulated in subcuticular tissues is of primary importance since it is one of the factors which determines the efficaciousness of the compound . Penetration of the growth regulating compound varies with the method of application, environment, target organism, and active lifetime of the compound . Mayer et al (1) reported the primary sites of absorption and/or accumulation of the ~Tuorescent insect growth regulator (FIGR), 5-[[[5-(dimethylApproved as Technical Article No . 13996 . Texas Agricultural Experiment Station . This research was conducted in cooperation with the Federal Agricultural-Science and Education Administration, U . S . Department of Agriculture . This paper reports the results of research only . Mention of a pesticide does not constitute a recommendation for use by the USDA nor does it imply registration under FIFRA as amended . Also, mention of a commercial or proprietary product does not constitute an endorsement by the USDA . 0024-3205/79/090817-1502 .00/0 Copyright (c) 1979 Pergamon Press Ltd
818
Insect Growth Regulation
Vol . 24, No . 9, 1979
amino)-1-naphthalenyl]sulfonyl]amino]-1,3-benzodioxole (DNSAB), in larvae of Aedes aegypti (L .) . The compound accumulated rapidly in the anal papillae acid in midgut cells . In addition, third-instar larval cuticle was incompletely shed from the sixth- and seventh-abdominal segments (2) at the time of ecdysis . Since the fluorescent probe characteristics of the compound indicated the sites of accumulation (1, 3), we decided to investigate the effects of the FIGR and a commercially available insect growth regulator (IGR), methoprene, iso propyl (E,E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate, at the ultrastructural level . Since only a limited number of investigations have been concerned with the effects of IGRs on epidermal cell activity and cuticle formation (4, 5, 6, 7), an ultrastructural study of the morphological effects of methoprene and DNSAB on mosquito cuticle and associated tissues is presented herein . Materials and Methods Third-instar larvae maintained in either deionized water, 7 .5 ppm DNSAB, or 0 .01 ppm methoprene were the primary subjects of this study . At 24, 48, and 72 hr posttreatment, five third-instar larvae from each group were washed five times with deionized water and dissected in a fixative containing 2X gluteraldehyde, 1X paraformaldehyde, and 0 .1 M sucrose in 0 .1 M phosphate buffer at pH 7 .3 . In addition, larvae that molted to the fourth instar were also prepared as above . The sixth- and seventh-abdominal segments, anal papillae, midguts, and Malpighian tubes were removed from each larva and fixed for 15 min at room temperature, then placed in ice for 1-3 hr . After rinsing and washing with cold 0 .1 M phosphate buffer (five times), the tissues were fixed in osmium tetroxide in 0 .1 M sucrose for 2 hr . Following five to ten distilled water rinses, the tissues were blockstained in 0 .5X aqueous uranyl acetate for 5 hr . After staining, the specimens were washed three to five times in distilled water and subsequently dehydrated through a 25, 50, 75, 95, and 100K ethyl alcohol series . Further dehydration was accomplished with three changes of acetone prior to incubation in an acetone/Spurn® resin (Polysciences, Inc ., Warrington, PA) (1 :1 ; v/v) mixture . After 2 hr, the 1 :1 mixture was replaced by a 1 :4 mixture and incubation allôwed to continue overnight . The following day, the tissues were placed in 100X Spurn and incubated at 70°C for 24 hr to allow complete polymerization .
lx
0
Sections ca . 500 A thick were cut from the tissue blocks using a Dupont® diamond knife mounted on a Sorvall® MT-2B or Porter Blum ultramicrotome . These sections were stained with 0 .5X aqueous uranyl acetate and Reynolds' lead citrate (8) and subsequently examined using a Phillips 300 transmission electron microscope at 60 Kv . Results Control Larval Cuticle--Cuticle from the sixth-abdominal segment of both third- an ourt -instar arvae was examined at 24, 48, and 72 hr after the tests were begun . Figure 1 demonstrates the normal cuticle layers and epider mal cell structure, A dense exocuticle (EPC) (ca . 0 .05u) covering a rather thin (ca . 0 .25u), rugose exocuticle (EXC) was observed in both instars . The exocuticle appeared to have a coarse, granular texture and consisted of two to three lamellae, while the endocuticle (ENC) appeared to have a light, fine texture and consisted of four to five lamellae . The epidermal cells averaged approximately 1 .Ou thick and 8 .Ou in length . Each lateral epidermal cell membrane (CM) was interconnected to the adjacent lateral cell membrane via septate desmosomes .
Vol . 24, No . 9, 1979
Insect Growth Regulation
819
The outer cell membrane displayed typical microvilli . Each microvillus showed at its apical region a darkened granulose area which extended a short distance into the endocuticle . These cells displayed large numbers of rough endoplasmic reticula and dense granular materials in their cytoplasm . Numerous mitochondria (M) were present in the upper half of each epidermal cell in these larvae . In some third-instar specimens fixed at 48 hr, an ecdysial membrane was found above a retracted outer epidermal cell membrane (FIG . 1) . The space between the cell membrane and the ecdysial membrane (EM) was continuous along the upper epidermal surface and measured from ca . 0 .04u to ca . 0 .06u .
FIG . 1 . Micrograph of section of integument taken from the sixthabdominal segment of a third-instar A . ae ti untreated larva at 48 hr after the test was begun showing e le (EPC), exocuticle (EXC), endocuticle (ENC), ecdysial membrane (EM), cell membrane (CM), mitochondria (M), and nucleus (N) . Early fourth-instar larvae fixed at 48 hr showed an average cuticle thickness of ca . 0 .35 u (FIG . 2) . The epicuticle appeared smooth and darkly stained . Only one undifferentiated layer of procuticle (PrC) was observed in these specimens . The developing endocuticle (procuticle) was divided into two distinct undifferentiated zones : an upper granulose, darkly stained zone and a lower fine grained, lightly stained zone . The outer cell membrane displayed processes (CMP) (or plasma membrane plaques) (9) where cuticulin deposition occurs . The cuticulin granules appeared to be continuous from the tips of the processes with strands of granules extending into the light staining layer of the endocuticle . The cytoplasm of these cells was densely packed with ribosome , rough endoplasmic reticula, and glycogen . Numerous microtubules (ca . 100 ~ diameter) projected upward towards the cell membrane processes and appeared tö terminate along the upper epidermal cell membrane surface . Fourth-instar larvae fixed at 72 hr displayed a cuticle thickness averaging ca . 0 .70 u . The upper layers (cement, wax, lipid) of the epicuticle in most specimens examined showed disruption (FIG . 3) . The wax and lipid layers appeared to have been dissolved during the dehydration process from between the cement (C) and cuticulin layers leaving a space approximately 0 .06u between the two . The exocuticle showed more compacted lamellae and stained more intensely than did the endocuticle . The endocuticle was composed of four to five )emgllae . In some areas of the cuticle, pore canals (PC) measuring ca . 150-200 A