Lycorine induces cell death in the amitochondriate parasite, Trichomonas vaginalis, via an alternative non-apoptotic death pathway

In this study, the mechanism of action of the pro-apoptotic alkaloid lycorine on an amitochondriate cell, the parasite Trichomonas vaginalis, was investigated. The cytotoxicity of lycorine against T. vaginalis was studied from 2.5 to 1000 μM and several important ultrastructural alterations were observed by electron microscopy. Lycorine arrested the T. vaginalis cell cycle, although no hallmarks of apoptosis, such as apoptotic bodies, were observed. Consequently, the underlying mechanism of action fails to completely fulfill the criteria for apoptosis. However, some similarities to paraptotic cell death were observed.     

Morphological,anatomical, and ultrastructural changes (visualized through scanning electron microscopy) inducedin Triticum aestivum by Pb2+ treatment

Lead (Pb) causes severe damage to crops, ecosystems, and humans, and alters the physiology and biochemistry of various plant species. It is hypothesized that Pb-induced metabolic alterations could manifest as structural variations in the roots of plants. In light of this, the morphological, anatomical, and ultrastructural variations (through scanning electron microscopy, SEM) were studied in 4-day-old seedlings of Triticum aestivum grown under Pb stress (0, 8, 16, 40, and 80 mg Pb²⁺ l^?1; mild to highly toxic). The toxic effect was more pronounced in radicle growth than on the plumule growth. The SEM of the root of T. aestivum depicted morphological alterations and surface ultrastructural changes. Compared to intact and uniform surface cells in the control roots, cells were irregular and desiccated in Pb²⁺-treated roots. In Pb²⁺-treated roots, the number of root hairs increased manifold, showing dense growth, and these were apparently longer. Apart from the deformity in surface morphology and anatomy of the roots in response to Pb²⁺ toxicity, considerable anatomical alterations were also observed. Pb²⁺-treated root exhibited signs of injury in the form of cell distortion, particularly in the cortical cells. The endodermis and pericycle region showed loss of uniformity post Pb²⁺ exposure (at 80 mg l^?1 Pb²⁺). The cells appeared to be squeezed with greater depositions observed all over the tissue. The study concludes that Pb²⁺ treatment caused structural anomalies and induced anatomical and surface ultrastructural changes in T. aestivum.     

20-Epiheyneanine,an iboga alkaloid from Peschiera affinis

20-Epiheyneanine, an epimer of the known iboga alkaloid heyneanine, is the major base of the root bark of Peschiera affinis. It is accompanied by lesser quantities of coronatidine, coronaridine pseudoindoxyl, affinisine and olivacine, as well as sitosterol, β-amyrin, lupeol and 3-O-acetyllupeol.     

A Quinoxaline Derivative as a Potent Chemotherapeutic Agent,Alone or in Combination with Benznidazole,against Trypanosoma cruzi

Background

Chagas’ disease is a condition caused by the protozoan Trypanosoma cruzi that affects millions of people, mainly in Latin America where it is considered endemic. The chemotherapy for Chagas disease remains a problem; the standard treatment currently relies on a single drug, benznidazole, which unfortunately induces several side effects and it is not successful in the cure of most of the chronic patients. In order to improve the drug armamentarium against Chagas’ disease, in the present study we describe the synthesis of the compound 3-chloro-7-methoxy-2-(methylsulfonyl) quinoxaline (quinoxaline 4) and its activity, alone or in combination with benznidazole, against Trypanosoma cruzi in vitro.

Methodology/Principal Findings

Quinoxaline 4 was found to be strongly active against Trypanosoma cruzi Y strain and more effective against the proliferative forms. The cytotoxicity against LLCMK₂ cells provided selective indices above one for all of the parasite forms. The drug induced very low hemolysis, but its anti-protozoan activity was partially inhibited when mouse blood was added in the experiment against trypomastigotes, an effect that was specifically related to blood cells. A synergistic effect between quinoxaline 4 and benznidazole was observed against epimastigotes and trypomastigotes, accompanied by an antagonistic interaction against LLCMK₂ cells. Quinoxaline 4 induced several ultrastructural alterations, including formations of vesicular bodies, profiles of reticulum endoplasmic surrounding organelles and disorganization of Golgi complex. These alterations were also companied by cell volume reduction and maintenance of cell membrane integrity of treated-parasites.

Conclusion/Significance

Our results demonstrated that quinoxaline 4, alone or in combination with benznidazole, has promising effects against all the main forms of T. cruzi. The compound at low concentrations induced several ultrastructural alterations and led the parasite to an autophagic-like cell death. Taken together these results may support the further development of a combination therapy as an alternative more effective in Chagas’ disease treatment.     

The effect of fasting and refeeding on the composition and synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in rat liver

Refeeding a high-sucrose, fat-free diet to fasted rats caused drastic alterations in the fatty acid composition of hepatic diacylglycerols, triacylglycerols, and phosphatidylcholines. However, the fatty acid profile of phosphatidylethanolamines did not change significantly. These results suggest that the fatty acid composition of diacylglycerols may influence the distribution of diacylglycerols among triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. Fasting and refeeding also affected the activities in vitro of a number of enzymes responsible for the formation of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. The activity of hepatic phosphatidate phosphatase increased fourfold upon refeeding. However, fasting the rats did not affect the activity of this enzyme despite the reduced triacylglycerol synthesis in the fasted liver in vivo. Fasting and refeeding induced alterations in the activities of diacylglycerol acyltransferase, cholinephosphotransferase, and ethanolaminephosphotransferase which correlated reasonably well with the changes observed in the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in vivo, although the changes in diacylglycerol acyltransferase were too moderate. The changes in the activity of cholinephosphate cytidylyltransferase, which is suggested to catalyze the rate-limiting step in the formation of CDP-choline, ran parallel with the alterations in the synthesis of phosphatidylcholines in vivo. No such correlation was found between the activity of ethanolaminephosphate cytidylyltransferase and the rate of phosphatidylethanolamine synthesis. The present results indicate that the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines is controlled by the availability of the various substrates as well as by the activities of several enzymes involved in these processes.     

Vitellogenesis of the digenean Plagiorchis elegans (Rudolphi, 1802) (Plagiorchioidea,Plagiorchiidae)

The ultrastructural organization of vitellogenesis of Plagiorchis elegans (Rudolphi, 1802), experimentally obtained from the golden hamster Mesocricetus auratus (Linnaeus, 1758), is described using transmission electron microscopy. This study is the first ultrastructural study of vitellogenesis in a member of the superfamily Plagiorchioidea. The four stages usually observed during vitellogenesis are described: stage I, cytoplasm of the vitellocytes mainly filled with ribosomes and few mitochondria; stage II, beginning of the synthetic activity; stage III, active synthesis of the shell globule clusters; stage IV, vitellocytes are filled with shell globule clusters and contain several lipid droplets, and glycogen granules are grouped around clusters and droplets. Vitellogenesis in P. elegans is compared with that of other Digenea. The differences among P. elegans and previously studied digeneans include, but are not limited to the occurrence of dense coiled endoplasmic reticulum saccules and the concentration of glycogen in the mesenchyme, which may be considered as a fifth stage of maturation of the vitelline glands. This peculiarity was not observed in all trematodes, which clearly indicates differences in the vitellogenesis in various digenean lineages at different stages of maturation of their vitelline cells.     

Mitochondrial biogenesis in Neurospora crassa. I. An ultrastructural and biochemical investigation of the effects of anaerobiosis and chloramphenicol inhibition

The isolation of a new class of mutants permitting facultative anaerobiosis in Neurospora crassa is described. Backcross analyses to the obligate aerobe prototroph (An ^-) indicate single nuclear gene inheritance (An ^-/An ⁺). An ⁺ and An ^- are indistinguishable in morphology and growth rates under aerobic conditions. Anaerobic growth requires nutritional supplements that are dispensable for aerobic growth. Conidiogenesis, conidial germination, and vegetative growth rate are suppressed by anaerobiosis. An ⁺ mutants produce substantial quantities of ethanol under anaerobic conditions. Anaerobiosis and chloramphenicol both affect mitochondrial enzyme activity and morphology. Chloramphenicol inhibition leads to reduction in cytochrome oxidase and swollen mitochondria with few cristae. Anaerobiosis leads to reduction in both cytochrome oxidase and malate dehydrogenase activities, enlarged mitochondria with fewer cristae, enlarged nuclei, and other alterations in cellular morphology. The fine structure of anaerobically grown cells changes with the time of anaerobic growth. We conclude that either inhibition of mitochondrial membrane synthesis or inhibition of respiration might lead to the observed alterations in mitochondria.     

Ligia italica (Isopoda,Oniscidea) as Bioindicator of Mercury Pollution of Marine Rocky Coasts

In this study, we evaluated the possible role of Ligia italica as a bioindicator for the monitoring of heavy metals pollution in the suppralittoral zone of marine rocky coasts. Between 2004 and 2011 specimens of L. italica were collected along the Eastern Sicilian coasts from sites known for their high pollution levels as they are near to an area where in September 2001 a refinery plant discharged into the sea some waste containing Hg. Other specimens were collected from the Vendicari Natural Reserve located about 30 miles from the polluted sites and used as control area. On a consistent number of animals, the concentration in toto of As, Cd, Cr, Hg, Ni, Pb, V, was determined by Atomic Absorption Spectrometry. On other animals, investigations were carried out in order to check for ultrastructural alterations of the hepatopancreas, that is the main metals storage organ in isopods. Results revealed the presence, in the animals collected in 2004 from the polluted sites, of considerable concentrations of Hg and of lower concentrations of other metals such as As, Pb and V. The Hg bioaccumulation resulted in remarkable ultrastructural alterations of the two cellular types (B and S cells) in the epithelium of the hepatopancreas. Surprisingly, a moderate amount of Hg was also found in specimens collected in 2004 from the Vendicari Natural Reserve, proving that the Hg pollution can also spread many miles away. Animals collected from the polluted sites in the following years showed a progressively decreasing Hg content, reaching very low levels in those from the last sampling. Also, the ultrastructural alterations found in the hepatopancreas of the animals from the last sample were quite irrelevant. In conclusion, Ligia italica can represent a good bioindicator and the ultrastructure of the hepatopancreas could be used as ultrastructural biomarker of heavy metals pollution in the supralittoral zones.     

Kidney mitochondrial metabolism of 25-hydroxyvitamin D3,: Evaluation of in vitro cation modulation

Oxidative phosphorylation and 1 α,25-dihydroxyvitamin D₃ [lα,25-(OH)₂D₃]synthesis in isolated mitochondria were decreased by the addition of strontium. Calcium effected a similar inhibition of 1α,25-(OH)₂D₃ synthesis which correlated with cation-induced mitochondrial swelling. The ultrastructural changes were found to be a consequence of experimental conditions and not a prerequisite for suppressed 1α,25-(OH)₂D₃ synthesis. Dietary administration of strontium or calcium also resulted in a decreased rate of 1α,25-(OH)₂D₃ synthesis; however, the decrease in 1-hydroxylase activity was accompanied by an induction of mitochondrial 25-hydroxyvitamin D₃ 24-hydroxylase activity. Such an in vivo-prompted mitochondrial response occurred in the absenee of morphological changes or extensive loss of oxidative phosphorylation activity. In contrast, no induction of 24-hydroxylase activity could be observed in acute studies using isolated mitochondria. Therefore, the in vitro action of calcium and strontium does not appear to reflect the in vivo mechanism whereby the cations act to change renal 25-hydroxyvitamin D₃ (25-OHD₃) hydroxylation. Results from in vitro studies corcerning the action of calcium to alter renal 25-OHD₃ metabolism should be interpreted in light of the cation's capacity to decrease oxidative phosphorylation and the subsequent intramitochondrial generation of NADPH.     

Effect of T4 infection on initiation of protein synthesis and messenger specificity of initiation factor 3

No alteration in the messenger specificity of initiation factor 3 (IF-3) is observed upon T4 phage infection of several strains of Escherichia coli. IF-3 present in the 1.0 m NH₄Cl washes of ribosomes from T4-infected cells supports the translation of f2 RNA and T4 late mRNA with the same degree of efficiency as the IF-3 in the ribosomal washes obtained from uninfected cells. At high concentrations the ribosomal washes obtained from T4-infected cells are more inhibitory for both f2 RNA- and T4 late mRNA-directed protein synthesis than the ribosomal washes from uninfected cells. Furthermore, this increased inhibition is also observed in the poly(U)-directed synthesis of polyphenylalanine. These data suggest that translational controls exerted at the level of IF-3 probably do not account for the alterations in protein synthesis observed upon T4 infection.     

Ultrastructure of Cosmarium strains (Zygnematophyceae,Streptophyta) collected from various geographic locations shows species-specific differences both at optimal and stress temperatures

Plant species collected from various climatic zones and stressed in vitro at various temperatures reveal changes in cellular ultrastructure which are in accordance with the climate at their sampling sites. This observation initiated the investigation to establish if stress at different temperatures may cause diverse extents of changes in the ultrastructure of microalgal strains originating from different geographic zones. The study revealed that the six Cosmarium strains demonstrated ultrastructural characteristics that were consistent with their source location under optimal, low and high temperature conditions, pointing to their preference to specific climatic niches. Interestingly, chloroplasts of all of the Cosmarium strains correspond to a sun-adapted type, which is concomitant with earlier statements that these strains are rendered as high-light adapted algae. The Cosmarium strains developed multiple ultrastructural responses which enabled them to cope with excessive temperatures, occasionally occurring in desmid natural habitats. The appearance of cubic membranes and increased number of plastoglobules may represent the first line in protection against high-temperature stress, which is accompanied by the alteration of protein synthesis and the appearance of stress granules in order to preserve cell homeostasis. However, the prolonged warm- or cold-temperature stress obviously initiated the programmed cell death, as concluded from the appearance of several ultrastructural features observed in all of the Cosmarium strains. The fair acclimation possibilities and the ability to undergo programmed cell death in order to save the population, certainly favor the cosmopolitan distribution of the genus Cosmarium.     

The action of α-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

α-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of α-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of α-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10–20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by α-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

Cytoskeleton structure,pattern of mitochondrial activity and ultrastructure of frozen or vitrified sheep embryos

Even though sheep embryo cryopreservation is a commonly used procedure the survival and pregnancy outcomes can vary greatly. This study investigated whether cryopreservation was causing subtle changes in ultrastructure, mitochondrial activity or cytoskeletal integrity. Sheep embryos were either slow cooled in 1.5 M EG (n = 22), or vitrified in 20% EG + 20% DMSO with 0.5 M sucrose in Open Pulled Straws (OPS) (n = 24). One hour after warming the cryopreserved embryos differed from control embryos in that they had no mitochondrial activity combined with cytoskeletal disorganization and large vesicles. Vitrified embryos also showed many points of cytoskeleton disruption. Ultrastructural alterations resulting from actin filaments disorganization were observed in both cryopreserved groups. This includes areas presenting no cytoplasmic organelles, Golgi complex located far from the nucleus and a decrease of specialized intercellular junctions. Additionally, large vesicles were observed in vitrified morulae and early blastocysts. The alterations after cryopreservation were proportional to embryo quality as assessed using the stereomicroscope. Even in the absence of mitochondrial activity, grade I and II cryopreserved embryos contained mitochondria with normal ultrastructure. Embryos classified as grade I or II in the stereomicroscope revealed mild ultrastructural alterations, meaning that this tool is efficient to evaluate embryos after cryopreservation.     

Methionine metabolism and ultrastructural changes with D-galactosamine in isolated rat hepatocytes

The biochemical and morphological effects of 2, 10 and 100 mM of D-galactosamine (GalN) were studied in isolated rat hepatocytes during 2 h of incubation. Lactate dehydrogenase (LDH), alanine aminotransferase (ALAT) and cell viability did not change, whatever the concentration used. The variations observed, which were dose dependent, included a large drop in ATP levels and inhibition of RNA and protein synthesis. A very high concentration of GalN was necessary, however, to induce a significant decline in methionine adenosyltransferase activity compared to control cells.The use of L-[methyl-¹⁴C]methionine during cell incubation with GalN demonstrated a decrease of S-adenosyl-L-methionine (SAMe) and an accumulation of L-methionine content related to the GalN concentration. These results suggested that an hepatotoxic agent such as GalN was able to induce disturbances of methionine metabolism.Some of the ultrastructural changes observed were different from those previously found in vivo, in rats given GalN intraperitoneally, underlining the marked difference between in vivo and in vitro intoxication.     

Enzyme regulation during the synchronous nuclear cycle of Physarum polycephalum: Effects of methotrexate and hydroxyurea on thymidine kinase and deoxycytidine kinase

Specific enzyme activities of thymidine kinase (TK) and deoxy-cytidine kinase (dCK) increase sharply at the onset of synchronous mitosis in macroplasmodia of Physarum polycephalum. They reach a maximum in early S-phase (Physarum lacks a G1 period) and decline to a minimum in late G2. Partial inhibition of DNA synthesis with methotrexate (MTX) or hydroxyurea (HU) retards the onset of the next mitosis and provokes a superinduction of both enzymes, with dCK responding stronger than TK. The temporal pattern observed suggests that the drugs interfere with the postmitotic down-regulation of enzyme expression, possibly due to alterations of the chromatin structure. Moderate inhibition of DNA synthesis still permits the appearance of (delayed) mitoses associated with peaks of enzyme activity at elevated levels. On the other hand, stronger inhibition completely suppresses the onset of mitosis and keeps the enzyme activities at an elevated level without further oscillations. The timing mechanism of periodic enzyme induction therefore appears to be functionally linked to the mitotic signal and does not persist under a stringent DNA block.     

Fat body cells of female reproductive castes of Attini ants (Hymenoptera: Formicidae): An ultrastructural and chemical analysis

The ultrastructural study on the fat body of gynes (virgin queens) of the basal ant species Cyphomyrmex rimosus and Mycetarotes parallelus and the derived Acromyrmex disciger and Atta laevigata queens showed vesicular rough endoplasmic reticulum, Golgi complex, and mitochondria in trophocytes, suggesting the involvement of these cells in protein synthesis, in addition to digestive vacuoles associated with the digestion of endocytosed compounds or rejected cell organelles. Oenocytes, another cell type present in the fat body of these species exhibit mitochondria, digestive vacuoles, and vesicles, indicating a mobilization of compounds by these cells. In A. laevigata, oenocytes also exhibited large storage sites of glycogen, in addition to a well-developed vesicular rough endoplasmic reticulum, suggesting an intensive participation of these cells in protein synthesis. The ultrastructural cytochemistry study also revealed electrodense granules of basic proteins present throughout the cytoplasm of trophocytes. The same was observed in oenocytes, although with smaller amounts of proteins. In the cytoplasm of trophocytes and oenocytes were also found droplets or electrodense granules of lipids. In oenocytes of A. disciger and in trophocytes of A. laevigata, lipids were observed in mitochondria, suggesting that this organelle might be a site of synthesis of these compounds. The chemical analysis of lipids revealed that in gynes, the main compounds present in fat body cells were saturated fatty acids, while in queens, saturated as well as unsaturated fatty acids were found. In conclusion, the present study showed that the fat body cells of gynes and queens, in general, maintained the same compounds and original features through the evolution process of the Attini tribe.     

Growth and ultrastructure of the marine unicellular alga <Emphasis Type="Italic">Dunaliella salina</Emphasis> (Chlorophyta) after chronic selenium intoxication

The effects of selenium (0.01, 0.5, 1, 5 and 10 mg/liter) on the growth and ultrastructure of the microalga Dunaliella salina were investigated following its transfer into clean water. Selenium concentrations of 5 and 10 mg/liter were toxic to D. salina, and reinoculation of microalga into clean water did not prevent it from total mortality. When reinoculated from medium with 0.01 mg Se/liter, the cell population density of D. salina was restored in 14 days. The number of ultrastructural alterations in cells was the same as in the control, while the excretory activity of microalga between days 4 and 10 of this experiment was higher. Cell population growth of D. salina transferred from 0.5 and 1 mg Se/liter was lower than in the control. No ultrastructural defects were observed in microalga reinoculated from medium with a selenium concentration of 0.5 mg/liter and the excretion level corresponded to that at 0.01 mg/liter. Various types of ultrastructural damage were found in microalga from medium with 1 mg Se/liter, which was previously reported to be threshold for D. salina; however, the number of cell injuries decreased with increasing time in clean medium. Excretory activity was decreased at the beginning of experiment; but after 7 days, it was restored to the control level. Though there were no ultrastructural alterations in microalgal cells from medium with 0.5 mg Se/liter, we assume that they had molecular defects that could inhibit the cell population growth. The study of microalgae following their reinoculation from medium containing toxicants into clean medium can be a useful method for evaluating algal survival after toxic exposure.     

Half-lives of messenger RNA species during growth and differentiation of Dictyostelium discoideum

The half-lives of functional messenger RNAs were determined by a method employing the drugs actinomycin D and daunomycin for the inhibition of mRNA synthesis; the activity of extracted mRNAs was determined by an in vitro translation assay. Several controls indicated that this method yielded reliable values for mRNA half-lives; in particular, the declining rate of protein synthesis in the presence of the drugs is due predominantly to the decay of translatable mRNA. This method was used to determine the half-lives of two specific mRNAs—encoding actin and a protein of MW 51,000—as well as that of total cytoplasmic mRNA activity during growth and at several times in differentiation. The half-lives of at least these two mRNAs were shown to be distinctly different from that of the total mRNA population—about 4 hr. However, no significant change in any of these half-lives was observed between growing and developing cells. Therefore wholesale alterations in the degradation rates of total and at least specific messages do not appear to play a role in the regulation of gene expression during Dictyostelium development.     

Histopathology and ultrastructure of midgut of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) fed Bt-cotton

The interaction of Cry toxins from Bacillus thuringiensis in the midgut of some insect larvae determines their efficacies as insecticides, due to the expression and availability of sites of action of the toxin in the midgut. Researches point out cases of resistance to Cry toxin due to alterations in the binding sites in columnar cell membrane. We analyzed the effects of Cry1Ac toxin expressed by Bt-cotton plants on Alabama argillacea midgut morphophysiology clarifying in levels of morphological and ultrastructural. Larvae in the 4th instar of A. argillacea after 20 min from ingesting Bt-cotton leaves expressing 0.183 ng of Cry1Ac exhibited ultrastructural and morphological modifications in the columnar cells with significant changes in the mitochondrial polymorphism, cytoplasmic vacuolization, microvillus and basal labyrinth. Expressive morphological alterations were also observed in the goblet cells indicating that the columnar cells are not the only target of the Cry1Ac toxin. The regenerative cells did not modify their structures and exhibited decrease in regeneration capacity. In conclusion, the ingestion of 0.183 ± 0.077 ng of Cry1Ac was enough to promote alterations in the columnar and goblet cells, besides reducing significantly the number of regenerative cells, which may have contributed to larval death. Nevertheless, further studies are necessary to determine the true cause of death.