The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents.     

Morphology and preliminary enzyme characterization of the salivary glands from the predatory bug Podisus nigrispinus (Heteroptera: Pentatomidae)

Podisus nigrispinus (Dallas) is a common predator in agricultural and natural systems in Neotropical America. Its feeding strategy involves extra-oral digestion and to better understand this process its salivary glands were extracted and subjected to morphological and preliminary enzyme characterization. The salivary glands of P. nigrispinus are formed by a pair of main and accessory gland complexes. The main salivary glands are further divided into an anterior and a posterior lobe. The compartmentalization of the salivary gland complex is likely to be important for the production, activation and release of the digestive enzymes used in the extra-oral digestion of prey items. Proteases and lipase, important digestive enzymes involved in zoophagy, were detected in the salivary glands of P. nigrispinus. The prevailing trypsin-like protease activity was characterized by using the serine-protease substrate N-alpha-benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and the trypsin inhibitors tosyl-L-lysine chloromethyl ketone (TLCK) and benzamidine. The KM value obtained for trypsin-like activity was 1.57 mm and the different peaks of optimum pH and temperature activity suggest the presence of multiple forms of this enzyme in P. nigrispinus. Detection of amylase activity in the salivary glands of this predator suggests its ability to digest starch and obtain nutrients from plants, which may have adaptative value under prey scarcity.     

Host hemolymph proteins and protein digestion in larval Habrobracon hebetor (Hymenoptera: braconidae)

Host plasma proteins and protein digestion in larval parasitoids were studied during trophic interactions of the ectoparasitoid Habrobracon hebetor Say (Hymenoptera: Braconidae), with a host, larvae of the Indianmeal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae). We could detect no apparent differences in host hemolymph protein patterns up to 72 h after paralysation and/or parasitization by H. hebetor. A 190 kDa putative apolipophorin I present in host hemolymph could not be detected in the midguts of feeding H. hebetor larvae indicating that it is rapidly digested. The major 60 kDa storage proteins (putative hexamerins) in host hemolymph were detected in the parasitoid midgut and were completely digested 24 h after cessation of feeding and the beginning of cocoon formation. Host hemolymph had a pH of about 6.4. The pH optima of the midgut proteinases in the larval parasitoid were in the alkaline region, but midgut fluid in feeding parasitoid larvae was about pH 6. 8. Based on enzyme activity against selected artificial proteinase substrates including azocasein, N-alpha-benzoyl-L-Arg p-nitroanilide (BApNA), succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA), succinyl-Ala-Ala-Pro-Leu p-nitroanilide (SAAPLpNA), and inhibition by selected proteinase inhibitors, serine proteinases appear to be the predominant class of enzymes involved in protein digestion in the midguts of H. hebetor. There is also an active aminopeptidase (LpNA) associated with the microsomal fraction of midgut preparations. There was no evidence for preoral digestion or ingestion of proteinases from host hemolymph by the parasitoid larva. There was a very active BApNAase in the soluble fraction of midgut extracts. This activity increased on a per midgut basis up to 24 h after the beginning of cocoon formation but decreased rapidly by 48 h. Two major (P1 and P3) and several minor proteinases were detected in midgut extracts of H. hebetor analysed with gelatin zymograms. The apparent molecular mass of P1 varied from 95 to 49 kDa depending on protein loading. P3 had an apparent molecular mass of 39 kDa that was independent of protein loading. In summary, electrophoretic evidence indicates that host hemolymph protein patterns do not change significantly for at least 72 h after paralysation by H. hebetor. The role, if any, of envenomization in preventing breakdown of hemolymph proteins during this time remains to be determined. Because the predominant host hemolymph proteins, a putative apolipophorin I and the putative hexamerins, are readily digested by the serine proteinases present in the midguts of this parasitoid larva, these or similar proteins would provide an easily digested source of dietary amino acids that could be used for development of artificial diets for this beneficial insect.     

Properties of larval and imaginal membrane-bound digestive enzymes from Trichosia pubescens

Trichosia pubescens larval midgut ceca cells display in their plasma membranes α-glucosidases (M_r 95,000; pH_o 5.5; K_m 5.7 mM; K_i for TRIS 8.9 mM), trehalases (M_r 69,000; pH_o 5.3; K_m 0.92 mM; K_i for TRIS 57 mM), and aminopeptidases (M_r 95,000; pH_o 8.7; K_m 0.19 mM) which are solubilized by Triton X-100. The enzymes were purified by electrophoresis and used to raise antibodies in a rabbit. T. pubescens imaginal midgut cells display in their plasma membranes an α-glucosidase (M_r 156,000; pH_o 5.8; K_m 2.3 mM; K_i for TRIS 0.2 mM), a trehalase (M_r 93,000; pH_o 5.5; K_m 0.72 mM; K_i for TRIS 45.5 mM), and an aminopeptidase (M_r 210,000; pH_o 9.0; K_m 0.47 mM). Antiserum produced against the larval enzymes shows no precipitation arc when tested by double immunodiffusion or by immunoelectrophoresis with Triton X-100-solubilized membrane proteins from imaginal midguts. Otherwise, a similar test showed that larval midgut cecal enzymes and larval ventriculus enzymes display complete immunological identity. The data suggest that, despite the fact the larval and imaginal aminopeptidase, α-glucosidase, and trehalase probably have similar functions, the genes coding for them in larvae and imagoes must differ.     

Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.     

CARBOHYDRASES IN THE DIGESTIVE SYSTEM OF THE SPINED SOLDIER BUG,Podisus maculiventris (SAY) (HEMIPTERA: PENTATOMIDAE)

The spined soldier bug, Podisus maculiventris, is a generalist predator of insects and has been used in biological control. However, information on the digestion of food in this insect is lacking. Therefore, we have studied the digestive system in P. maculiventris, and further characterized carbohydrases in the digestive tract. The midgut of all developmental stages was composed of anterior, median, and posterior regions. The volumes of the anterior midgut decreased and the median midgut increased in older instars and adults, suggesting a more important role of the median midgut in food digestion. However, carbohydrase activities were predominant in the anterior midgut. In comparing the specific activity of carbohydrases, α‐amylase activity was more in the salivary glands (with two distinct activity bands in zymograms), and glucosidase and galactosidase activities were more in the midgut. Salivary α‐amylases were detected in the prey hemolymph, demonstrating the role of these enzymes in extra‐oral digestion. However, the catalytic efficiency of midgut α‐amylase activity was approximately twofold more than that of the salivary gland enzymes, and was more efficient in digesting soluble starch than glycogen. Midgut α‐amylases were developmentally regulated, as one isoform was found in first instar compared to three isoforms in fifth instar nymphs. Starvation significantly affected carbohydrase activities in the midgut, and acarbose inhibited α‐amylases from both the salivary glands and midgut in vitro and in vivo. The structural diversity and developmental regulation of carbohydrases in the digestive system of P. maculiventris demonstrate the importance of these enzymes in extra‐oral and intra‐tract digestion, and may explain the capability of the hemipteran to utilize diverse food sources.     

Consumption of food and spatial organization of digestion in the cassava hornworm, Erinnyis ello

Fifth-instar Erinnyis ello larvae eat 2.1 times their own weight per day of Euphorbia pulcherrima leaves, with a coefficient of digestibility of 45% and an efficiency of food conversion into tissue of 25%. The food takes about 150 min to go through the gut. Midgut contents have a pH of 9.3–9.8, depending on the region. Cellulase is absent from the gut in E. ello. Significant gut hydrolase activities are found only in midgut. Amylase and trypsin occur in the midgut tissue and contents and in regurgitated material, whereas aminopeptidase, α-glucosidase, β-glucosidase and trehalase are found in major amounts in the midgut tissue, in minor amounts in the midgut contents and are absent from regurgitated material. The results support the hypothesis that digestion starts in the endoperitrophic space under the action of amylase and trypsin and is largely completed in the ectoperitrophic space through the catalytic action of several oligomer and dimer hydrolases. Involvement of a membrane-bound aminopeptidase in the terminal digestion of oligopeptides cannot, at present, be excluded. The finding that less than 7% of the total amylase and trypsin are excreted, after a time identical to the passage time of the food bolus, leads to the proposal for the existence of some mechanism by which those enzymes are recovered from the undigested food before it is excreted.     

Nymphal Development and Reproduction of Podisus nigrispinus (Heteroptera: Pentatomidae) Fed With Combinations of Tenebrio molitor (Coleoptera: Tenebrionidae) Pupae and Musca domestica (Diptera: Muscidae) Larvae

The development and reproduction of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) fed on Tenebrio molitor L. (Coleoptera: Tenebrionidae) and Musca domestica L. (Diptera: Muscidae) were studied in four treatments. P. nigrispinus was permitted to feed on: (1) T. molitor pupae (T 1 ); (2) Musca domestica larvae (T 2 ); (3) both prey supplied simultaneously (T 3 ); (4) both prey supplied on alternate days (T 4 ). Duration of the nymphal period of P. nigrispinus was similar in all diets studied with nymphal viability of approximately 75%. Heavier females were obtained in T 1 and T 4 , but no correlation between this factor and the reproductive rate of the predator was found. Therefore, the use of body weight as a parameter to evaluate rearing quality should be approached with caution. However, females of this predator showed higher egg and nymph production when they received both prey. For this reason P. nigrispinus should be reared with T. molitor and M. domestica simultaneously or on alternate days.     

Population growth rate of the depredating Podisus nigrispinus (Heteroptera: Pentatomidae) and of the Tuta absoluta (Leptoptera: gelechiidae) in wintering place

The fertility life table of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) preying either on Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) caterpillars or on alternative prey Tenebrio molitor L. (Coleoptera: Tenebrionidae) under greenhouse conditions (30 +/- 5 degrees C, 61 +/- 23% RH) were studied. The life table was also determined for the pest T. absoluta under the same conditions. The net reproductive rate (Ro) and the intrinsic rate of natural increase (rm) were higher 14.13 and 46.32 times for predators fed on T. molitor prey, however, the generation time (T) was similar between prey. The pest T. absoluta showed Ro and rm higher 2.15 and 32.10 times than those achieved for predators fed on this pest. However, females fed on a suitable prey T. molitor showed higher Ro and rm than those yielded for the pest. The survival curves were similar for P. nigrispinus females fed on both prey and classified as being type II by Weibull analysis. The results suggest that P. nigrispinus is able to maintain its population preying only on T. absoluta caterpillars; however, the life table parameters determined individually for both showed that the pest produces more generations per year and faster population natural growth than the predator.     

Functional response of the predators Podisus maculiventris (Say) and Podisus nigrispinus (Dallas) (Het., Pentatomidae) to the beet armyworm, Spodoptera exigua (Hübner) (Lep., Noctuidae): ef fect of temperature

Predation abilities of Podisus maculiventris and Podisus nigrispinus on caterpillars of the beet armyworm, Spodoptera exigua (Hübner), were compared at three different temperatures (18, 23 and 27°C) by performing functional response tests. In both species, predation capacity was a function of temperature and prey density: more prey were captured as temperature and number of prey offered increased. Results indicated that type II and III functional responses provided the best fit to the data obtained for P. nigrispinus at 18 and 23°C, and at 27°C, respectively. However, the data for P. maculiventris showed a better fit to type II at 18°C and to type III at higher temperatures. In both pentatomids the handling time decreased with increasing temperature. At higher temperatures, P. nigrispinus demonstrated greater predation rates than P. maculiventris . The implications of these findings for the control of caterpillar pests in glasshouses are discussed.     

Effects of Metarhizium anisopliae (Metsch.) Sorok. and Beauveria bassiana (Bals.) Vuill. on the predatory stinkbug Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae)

Entomopathogenic fungi and the predatory stinkbug Podisus nigrispinus (Dallas) can target simultaneously the same or different pests in agroecosystems. Topical contact during fungal dispersion or spraying, walking on plant surfaces and ingestion of contaminated prey are some of possible ways of interactions between fungi and the predatory stinkbug. The impact of three isolates of Metarhizium anisopliae (Metsch.) Sorok. (866, 1022 and 1189) and Beauveria bassiana (Bals.) Vuill. (604, 634 and 561) against nymphs and adults of P. nigrispinus was investigated under laboratory conditions. M. anisopliae caused higher mortality compared to B. bassiana either by topical application or by dry residue on cotton leaves. Topic contact with both fungi caused higher mortality to the predator. No mortality confirmation was reported for the adults. Nymphs fed with cotton leafworm larvae Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) contaminated by the fungi had their reproduction affected, but this was not observed when adult predators fed on contaminated larvae. These findings suggest that isolates of M. anisopliae can cause mortality of P. nigrispinus nymphs by topical contact, while isolates of B. bassiana were less harmful by all ways of infection as compared to M. anisopliae.     

Bloodmeal digestion in the midgut of Phlebotomus papatasi and Phlebotomus langeroni

Abstract. Bloodmeal digestion in midguts of the sandflies Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) was investigated in optimized assays to detect general protease, trypsin and aminopeptidase activities using synthetic substrates. Optimal activity occurred at pH 8-9 for all enzymes examined in both species. Protease activity peaked at 24-34h post human bloodmeal in midguts of P.papatasi and 34-48h in P'.langeroni; all endo- and exoprotease activities were completed by 50 h in P.papatasi compared to 72 h in P. langeroni. Hydrolysis of two chymotrypsin substrates was <2% of trypsin activity in both species. Aminopeptidase activity was associated mainly with the midgut wall, whereas trypsin activity was confined to the midgut lumen. A feature of digestion in P.langeroni was the high level of aminopeptidase recorded within 10h of the bloodmeal.     

Archives of insect biochemistry and physiology guide for authors

Several carbohydrases and glycosidases from the alimentary cancal and/or salivary glands of feeding larvae of mayetiola destructor have been identified. Pectinase activity was identified in the midgut and may be present in the salivary glands. No endocellulase activity was found in larvae; however, hemicellulase activity was detected in extract of larvae. Amylase activity was present in midguts from feeding larvae and at a low level in extract of salivary glands. Amylases detected in the midgut showed mobilities during polyacrylamide gel electrophoresis similar to the two major amylases in tissues of the insect's host plant. The possibility exists that Hessian fly larvae utilize amylases obtained from their host plant in the digestion of starch. The major glycosidases detected in the midgut lumen of larve were: α-D-glucosidase and α-D-and β-D-galactosidase. The role of these enzymes in the feeding process of Hessian fly larvae is discussed as well as their potential role in feeding damage to wheat.     

Protein digestion in larvae of the red oak borer Enaphalodes rufulus

Abstract In the Ozark Mountains of the U.S.A., the red oak borer Enaphalodes rufulus contributes to the destruction of red oaks. To understand nutrient digestion in E. rufulus larvae, digestive proteinases are compared in both larvae fed heartwood phloem and those transferred to artificial diet. The pH of gut extracts is approximately 6.3 in the midgut and foregut and decreases to 5.5 in the hindgut region. The hydrolysis of casein by midgut extracts from E. rufulus larvae fed either artificial diet or phloem from tree sections increases in buffers greater than pH 6.19, with maximum hydrolysis being observed at pH 10.1. Casein zymogram analysis reveals two major proteinase activities in larval midgut extracts of diet‐fed larvae, with molecular masses of approximately 25 and 40–60 kDa, whereas phloem‐fed larvae have proteinase activities corresponding to 40, 45, 60, 80 and >100 kDa. Substrate analysis indicates at least one major trypsin‐like activity in both gut extracts with a molecular mass of >100 kDa, but two chymotrypsin‐like activities of approximately 25 and >200 kDa are found only in diet‐fed larvae. Inhibitors of serine proteinases are most effective in reducing the general proteolytic activity of midgut extracts from larvae fed either food source. The data indicate that serine proteinase inhibitors have the potential to reduce E. rufulus larval damage to oaks. In particular, transgenic technologies incoporating trypsin inhibitors may be effective in reducing protein digestion in phloem‐feeding larvae.     

The cathepsin L-like proteinases from the midgut of Tenebrio molitor larvae: sequence, properties, immunocytochemical localization and function

CDNAs coding for five procathepsin L-like proteinases (pCALs) were cloned and sequenced from a cDNA library prepared from Tenebrio molitor larval midguts: pCAL1a (with the isoforms pCAL1b and pCAL1c), pCAL2, and pCAL3. All the pCALs have the active residues Cys 25, His 169, Asn 175, and Gln 19 (papain numbering), the ERFNIN motif of papain-like enzymes and their sequences are homologous to cathepsin L enzymes. pCAL1a was expressed in bacterial systems. It is auto-catalytically activated at low pH, has kinetic properties and N-terminal sequence identical to hemocyte cathepsin L-like proteinase (CAL) and was used to raise antibodies. Semi-quantitative RT-PCR data showed that mRNAs for pCAL2 and pCAL3 were transcribed in midgut and in lesser amounts in hemolymph, whereas that for pCAL1a was transcribed in these tissues and also in fat body, Malpighian tubules, and carcass. Imunochemical detection recognized pCAL1a translation in all tissue homogenates, except anterior midgut. At this region, the presence of pCAL2 is suggested on the grounds of electrophoretical migration and high recovery of CAL2 activity from anterior midgut cells and from isolated midgut contents. Immunocytochemical localization data revealed that pCAL1a occurs in lysosome-like vesicles in all tissues, except anterior midgut, where a labelling considered to correspond to pCAL2 is found in large acidic granules being released by apocrine secretion. Putative pCAL2 was also detected in midgut contents, probably in the form of CAL2, the major luminal CAL, which was purified to homogeneity. A cladogram of insect CALs result in a monophyletic branch with lysosomal T. molitor enzymes and enzymes from five insect orders and in a polyphyletic array of coleopteran sequences, including digestive CALs from T. molitor. The data suggest that only Coleoptera have digestive CALs that may originate by gene duplication and independent evolution relative to the gene encoding the lysosomal enzyme.     

Effect of diet on male reproductive tract of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae).

The morphology and histology of the reproductive tract of males of the predator Podisus nigrispinus (Dallas) fed on different diets were studied. P. nigrispinus was fed on diets of: larvae of Alabama argillacea (Hübner), Tenebrio molitor L., Musca domestica L., and an artificial diet. The male reproductive tract, independent of diet, showed testes with intense red coloration in a compact, circular, or slightly oval structure. The vasa deferentia were similar in color to the testes and formed long filaments, which joined with the yellow-cream colored ejaculatory duct. The morphological characteristics of the male reproductive tract were similar under all diets, except for the artificial one. The histological studies demonstrated that independent of the diet the testes of P. nigrispinus were composed of four to six follicles. The testes with six follicles generally had four developed and two atrophied follicles. The morphological and histological differences of the testes of P. nigrispinus when fed with different prey are presented and discussed.     

Differential expression of proteins in the midgut of Anopheles albimanus infected with Plasmodium berghei

The main vector for transmission of malaria in Mexico is the Anopheles albimanus mosquito. The midgut of disease-transmitting mosquitoes carries out a variety of functions that are related to blood feeding. We analyzed the midgut of A. albimanus infected with Plasmodium berghei (resistant mosquito) using a proteomic approach to identify putative short peptides that are enriched in the midgut after blood feeding. Mosquito midguts were analyzed by two-dimensional electrophoresis to determine the changes in protein profiles. We identified 21 spot proteins that are differentially expressed in the blood of mosquitoes during the immune challenge. Molecular weight of the spots varied from 13 to 36 kDa, with a broad isoelectric point range of 3.92–8.90. We identified the differentially expressed proteins using mass spectrometry and constructed a proteomic data base of the A. albimanus midgut with diverse functions, some of them proteins with digestive and immunologic functions. Identification of these proteins may have important implications for understanding the blood meal digestion process, as well as developing novel vector control strategies and understanding parasite vector interactions.     

Ultrastructural and biochemical aspects of digestion in the imagoes of the flyRhynchosciara americana

The midgut of adultRhynchosciara americana Wiedemann (Diptera: Sciaridae) displays, in contrast to the midguts of other adult Diptera, two caeca connected to a ventriculus. All midgut cells exhibit long apical microvilli, and narrow and ramified basal channels with openings to the underlying space. These morphological features are thought to be involved in the absorption of nutrients from food. Enzymatic assays inR. americana adults revealed that amylase occurs in salivary glands and midgut, whereas aminopeptidase, α-glucosidases and trypsin occur only in the midgut, mainly in the ventriculus. There is a soluble (Mr 105000) and a membrane-bound aminopeptidase (solubilized form, Mr 110000). Soluble α-glucosidase inactivates easily and could not be characterized, whereas membrane-bound α-glucosidases were resolved after solubilization into three molecular species (Mr 186000, 105000 and 84000) with different substrate specificities. The activities of trypsin (pH optimum 9.0), which was inhibited completely by soybean trypsin inhibitor, and of amylase (pH optimum 5.5), were not sufficiently high to be further characterized. The data support the assertion thatR. americana adults are able, to a limited extent, to digest and absorb starch and proteins, in addition to nectar sugars. The results, supported by published data, suggest that there is an inverse correlation between the digestive enzyme activities and midgut absorptive surface in insects which has nectar as a major food.     

Pharmacological Study of Intracellular Second Messengers that Affect Active Ion Transport in the Midgut of Tobacco Hornworm, Manduca sexta

ABSTRACT Midgut active ion transport changes during the final larval stage of the tobacco hornworm. The short-circuit current ( I _sc) of the posterior midguts dissected from feeding fifth instars (day 2) is higher than that of midguts from wandering larvae (day 5). The I _sc of midguts from day 2 larvae is inhibited by 1 mM cAMP and 0.1 mM cGMP, whereas the midguts from day 5 larvae are stimulated by cAMP but unaffected by cGMP. A similar pattern is observed if the midguts are exposed to the cyclic nucleotide derivative, 8-bromo-cGMP. Exposure to the calcium ionophore, A23187, or the endoplasmic calcium ATPase inhibitor, thapsigargin, slightly inhibited the I _sc of day 2 larval midguts, but this inhibition was not significant. Pharmacological agents known to modulate the activities of protein kinase A, protein kinase C, or protein phosphatases did not change the I _sc. These results indicate that midgut active ion transport is modulated by cyclic nucleotides, but the manner of the response depends on the developmental status of the insect.