Natural hyperthermia and expression of the heat shock protein Hsp70 affect developmental abnormalities in Drosophila melanogaster

We demonstrate that natural heat stress on wild larval Drosophila melanogaster results in severe developmental defects in >10% of eclosing adults, and that increased copy number of the gene encoding the major inducible heat shock protein of D. melanogaster, Hsp70, is sufficient to reduce the incidence of such abnormalities. Specifically, non-adult D. melanogaster inhabiting necrotic fruit experienced severe, often lethal heat stress in natural settings. Adult flies eclosing from wild larvae that had survived natural heat stress exhibited severe developmental anomalies of wing and abdominal morphology, which should dramatically affect fitness. The frequency of developmental abnormalities varied along two independent natural thermal gradients, exceeding 10% in adults eclosing from larvae developing in warm, sunlit fruit. When exposed to natural heat stress, D. melanogaster larvae with the wild-type number of hsp70 genes (n=10) developed abnormal wings significantly more frequently than a transgenic sister strain with 22 copies of the hsp70 gene. Received: 19 April 1999 / Accepted: 16 July 1999     

Influences of Domoic Acid Exposure on Cardiac Development and the Expression of Cardiovascular Relative Genes in Zebrafish (Daniorerio) Embryos

Domoic acid (DA) is a highly toxic phycotoxin that is generated from marine diatoms Pseudonitzschia spp. It has been found that bivalves or cephalopods can accumulate DA to a high level through their feeding activities and cause illness or death in consumers. Zebrafish have been used as a model to investigate and characterize the developmental toxicity of DA. However, there is no report about the relationship between DA and cardiac development in zebrafish. Here, zebrafish embryos were exposed to DA with at the dose of 1, 10, 100, and 1000 ng/L. High mortality and some developmental toxicity including pericardial and yolk sac edema, dorsal curvature, and cardiac defects were observed in the DA‐treated larvae. We found that DA exposure not only disrupted normal cardiac development but also altered the expression of some cardiac development correlated genes and calcium ion channels, such as Anf, Bnp, Atp2a2a, Atp2a2b, Ncx1h, Ryr2b, and Tbx5.     

Effects of Dual Targeting of Tumor Cells and Stroma in Human Glioblastoma Xenografts with a Tyrosine Kinase Inhibitor against c-MET and VEGFR2

Anti-angiogenic treatment of glioblastoma with Vascular Endothelial Growth Factor (VEGF)- or VEGF Receptor 2 (VEGFR2) inhibitors normalizes tumor vessels, resulting in a profound radiologic response and improved quality of life. This approach however does not halt tumor progression by diffuse infiltration, as this phenotype is less angiogenesis dependent. Combined inhibition of angiogenesis and diffuse infiltrative growth would therefore be a more effective treatment approach in these tumors. The HGF/c-MET axis is important in both angiogenesis and cell migration in several tumor types including glioma. We therefore analyzed the effects of the c-MET- and VEGFR2 tyrosine kinase inhibitor cabozantinib (XL184, Exelixis) on c-MET positive orthotopic E98 glioblastoma xenografts, which routinely present with angiogenesis-dependent areas of tumor growth, as well as diffuse infiltrative growth. In in vitro cultures of E98 cells, cabozantinib effectively inhibited c-MET phosphorylation, concomitant with inhibitory effects on AKT and ERK1/2 phosphorylation, and cell proliferation and migration. VEGFR2 activation in endothelial cells was also effectively inhibited in vitro. Treatment of BALB/c nu/nu mice carrying orthotopic E98 xenografts resulted in a significant increase in overall survival. Cabozantinib effectively inhibited angiogenesis, resulting in increased hypoxia in angiogenesis-dependent tumor areas, and induced vessel normalization. Yet, tumors ultimately escaped cabozantinib therapy by diffuse infiltrative outgrowth via vessel co-option. Of importance, in contrast to the results from in vitro experiments, in vivo blockade of c-MET activation was incomplete, possibly due to multiple factors including restoration of the blood-brain barrier resulting from cabozantinib-induced VEGFR2 inhibition. In conclusion, cabozantinib is a promising therapy for c-MET positive glioma, but improving delivery of the drug to the tumor and/or the surrounding tissue may be needed for full activity.     

Connexin43 is associated with the progression of clear cell renal carcinoma and is regulated by tangeretin to sygergize with tyrosine kinase inhibitors

BackgroundThe roles of Connexin43 (Cx43) in clear cell renal cell carcinoma (ccRCC) microenviroment remains to be poorly defined.MethodsThe expression profile, prognosis and immune analysis of Cx43 in various cancers, particularly in ccRCC were performed using TCGA database, and various biological function assays were applied to explore the physiological role of Cx43 and tangeretin in ccRCC. Western blot were applied to examine the protein expression and Kunming mice were used to evaluate preliminary safety or anti-tumor activity of tangeretin and sunitinib.ResultsCompared with the normal group, higher expression levels of Cx43 in ccRCC, and distinct associations between Cx43 expression and ccRCC prognosis or immune infiltration, were found. Notably, the expression of Cx43 was found to be highly correlated with that of receptor tyrosine kinases (RTKs), particularly with VEGFR1, VEGFR2 and VEGFR3. The expression of Cx43 and EGFR was also found to be higher in ccRCC than that in the para-cancerous specimens. Knocking down Cx43 expression decreased RCC cell viability, cell migration, p-EGFR, MMP-9 and survivin expression. Using 14 Chinese medicine monomers, tangeretin was screened and found to inhibit tumor cell viability and Cx43 expression. Tangeretin also enhanced the sensitivity of RCC cells to tyrosine kinase inhibitors (TKIs) sunitinib and sorafenib. However, the same concentration of tangeretin exerted a less prominent effect on normal renal cell viability.ConclusionsCx43 is strongly associated with RTK expression and ccRCC progression, while tangeretin can inhibit RCC cell malignancy by inhibiting Cx43 expression and enhance the sensitivity of RCC cells to TKIs.     

Noninvasive Assessment of Tumor VEGF Receptors in Response to Treatment with Pazopanib: A Molecular Imaging Study

Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) drive angiogenesis, and several VEGFR inhibitors are already approved for use as single agents or in combination with chemotherapy. Although there is a clear benefit with these drugs in a variety of tumors, the clinical response varies markedly among individuals. Therefore, there is a need for an efficient method to identify patients who are likely to respond to antiangiogenic therapy and to monitor its effects over time. We have recently developed a molecular imaging tracer for imaging VEGFRs known as scVEGF/^99mTc; an engineered single-chain (sc) form of VEGF radiolabeled with technetium Tc 99m (^99mTc). After intravenous injection, scVEGF/^99mTc preferentially binds to and is internalized by VEGFRs expressed within tumor vasculature, providing information on prevalence of functionally active receptors. We now report that VEGFR imaging readily detects the effects of pazopanib, a small-molecule tyrosine kinase inhibitor under clinical development, which selectively targets VEGFR, PDGFR, and c-Kit in mice with HT29 tumor xenografts. Immunohistochemical analysis confirmed that the changes in VEGFR imaging reflect a dramatic pazopanib-induced decrease in the number of VEGFR-2⁺/CD31⁺ endothelial cells (ECs) within the tumor vasculature followed by a relative increase in the number of ECs at the tumor edges. We suggest that VEGFR imaging can be used for the identification of patients that are responding to VEGFR-targeted therapies and for guidance in rational design, dosing, and schedules for combination regimens of antiangiogenic treatment.     

Characteristics of patients with lung cancer in clinical practice and their potential eligibility for clinical trials evaluating tyrosine kinase inhibitors or immune checkpoint inhibitors

IntroductionIn- and exclusion criteria of randomized clinical trials (RCTs) aim to include a homogeneous study-population. This study compared characteristics of lung cancer patients from phase III RCTs evaluating tyrosine kinase inhibitors (TKIs) or immune checkpoint inhibitors (ICIs) with characteristics of lung cancer patients in a real world setting in the United Kingdom.MethodsA retrospective study was conducted using the Clinical Practice Research Datalink GOLD. Patients (N = 9239) with a first ever lung cancer registration between 2014 and 2018 were identified. Eligibility for inclusion was assessed for twelve RCTs (evaluating TKIs or ICIs). Reasons for potential exclusion and the number of unmet criteria were assessed for each RCT independently. OS was assessed using Kaplan-Meier and Cox proportional hazards analyses.ResultsThe proportion of potentially eligible patients was 74.3% and 51.9% for TKI and ICI RCTs, respectively. History of another malignancy, renal insufficiency or concomitant drug-use were main reasons for exclusion. OS was considerably longer for potentially eligible patients. Hazards ratios varied from 1.17 (95% confidence interval, 1.11–1.24) to 1.35 (1.20–1.42) across the RCTs.ConclusionThis study showed that a considerable proportion of lung cancer patients in a real-world setting would have been ineligible for participation in phase III RCTs and that potentially ineligible patients experienced a shorter OS.     

Inhibitors of Eicosanoid Biosynthesis and Their Effect upon Bacillus thuringiensisδ-Endotoxin Response in Cultured Insect Cells and Developing Larvae

Twelve inhibitors of eicosanoid biosynthesis were examined for their ability to affect the response of insect cells in vitro and developing larvae to δ-endotoxin from Bacillus thuringiensis. The response of cultured insect cells from Manduca sexta, Choristoneura fumiferana, and Plodia interpunctella to CryIA(c) and CryIC protein from Bacillus thuringiensis was measured while exposed to various concentrations of specific cyclooxygenase and/or lipoxygenase inhibitors. Five of the inhibitors (curcumin, baicalein, nordihydroguaiaretic acid, indomethacin, and eicosatetraynoic acid) were toxic to the cells at high concentrations (>20 μM). Surprisingly, the same inhibitors had no significant effect upon normal larval development, except for nordihydroguaiaretic acid. No true, consistent difference was detected with either lipoxygenase or cyclooxygenase inhibitors for cells or larvae treated with δ-endotoxin. However, the δ-endotoxin response of insect cells in vitro and developing larvae in the presence of nordihydroguaiaretic acid was strong evidence of an involvement with P₄₅₀ cytochromes in the B. thuringiensis toxic response.     

Design,synthesis, in vitro inhibition and toxicological evaluation of human carbonic anhydrases I,II and IX inhibitors in 5-nitroimidazole series

Abstract

With the aim to obtain novel compounds possessing both strong affinity against human carbonic anhydrases and low toxicity, we synthesised novel thiourea and sulphonamide derivatives 3, 4 and 10, and studied their in vitro inhibitory properties against human CA I, CA II and CA IX. We also evaluated the toxicity of these compounds using zebrafish larvae. Among the three compounds, derivative 4 showed efficient inhibition against hCA II (KI = 58.6?nM). Compound 10 showed moderate inhibition against hCA II (KI = 199.2?nM) and hCA IX (KI = 147.3?nM), whereas it inhibited hCA I less weakly at micromolar concentrations (KI = 6428.4?nM). All other inhibition constants for these compounds were in the submicromolar range. The toxicity evaluation studies showed no adverse effects on the zebrafish larvae. Our study suggests that these compounds are suitable for further preclinical characterisation as potential inhibitors of hCA I, II and IX.     

Cabozantinib Inhibits Growth of Androgen-Sensitive and Castration-Resistant Prostate Cancer and Affects Bone Remodeling

Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. In vivo, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease.     

Physiological effects and bioconcentration of triclosan on amphibian larvae

We examined the acute effects of triclosan (TCS) exposure, a common antimicrobial found as a contaminant in the field, on survival and physiology of amphibian larvae. LC50 values were determined after 96 h for North American larval species: Acris crepitans blanchardii, Bufo woodhousii woodhousii, Rana sphenocephala, and for a developmental model: Xenopus laevis. Amphibian larvae were most sensitive to TCS exposure during early development based upon 96-h LC50 values. Heart rates for X. laevis and North American larvae exposed to TCS were variable throughout development. Metabolic rates of X. laevis and R. sphenocephala larvae exposed to TCS were significantly affected in larvae exposed to [50% LC50] and [LC50]. Tissue uptake and tissue bioconcentration factor (BCF) of TCS were investigated in X. laevis, B. woodhousii woodhousii, and R. sphenocephala. In general, a significant increase was observed as exposure concentration increased. Tissue BCF values were dependent upon stage and species. While TCS concentrations used here are higher than environmental concentrations, exposure to TCS was dependent upon species and developmental stage, with early developmental stages being most sensitive to TCS exposure.     

Different course of proteolytic inhibitory activity and proteolytic activity in Galleria mellonella larvae infected by Nosema algerae and Vairimorpha heterosporum

The activity of protease inhibitors and proteases was studied in the hemolymph, gut, and fat body of 7th-instar larvae of Galleria mellonella infected by two microsporidia, Nosema algerae and Vairimorpha heterosporum. The increase in inhibitory activity in the hemolymph was substantial, and coincided with the development of the disease. The increase in inhibitory activity in the gut was almost doubled by N. algerae as compared with V. heterosporum, whereas the increase in inhibitory activity in fat body was found only in V. heterosporum-infected larvae. The course of proteolytic activity followed an inverse pattern to the elevated activity of inhibitors in the gut and the fat body, and rose only in moribund larvae at the end of the course of V. heterosporum infection. The differences in the pattern of proteases and inhibitors reflect the organ specificity of each of the microsporidia.     

Impact of Metarhizium brunneum (Hypocreales: Clavicipitaceae) on pre-imaginal Rhagoletis indifferens (Diptera: Tephritidae) within and on the surface of orchard soil

When last instar laboratory-reared Rhagoletis indifferens were allowed to pupate within non-sterile orchard soil containing incorporated Metarhizium brunneum isolate F52 conidia, a dose-related proportion died from developmental abnormalities and mycosis. When larvae entered soil superficially treated with M. brunneum, over 80% of the pupae died of developmental abnormalities.     

Effects of potato plants expressing a barley cystatin on the predatory bug Podisus maculiventris via herbivorous prey feeding on the plant

The aim of this study was to assess the effects of potato plants expressing a barley cystatin on a potentially cystatin-susceptible natural enemy by predation on susceptible and non-susceptible preys feeding on the plant. We have focussed on the impact of the variant HvCPI-1 C⁶⁸ → G, in which the only cysteine residue was changed by a glycine, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the Egyptian cotton leafworm (ECW), Spodoptera littoralis. Moreover, we have studied the prey-mediated effects of the barley cystatin at the third trophic level, using the predatory spined soldier bug (SSB), Podisus maculiventris, as a model. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing the C⁶⁸ → G variant resulted in significantly lower weight gains compared to those fed on non-transformed (NT) plants. On the contrary, larger weight gains were obtained when ECW larvae, that lack digestive cysteine proteases, were reared on transgenic potato expressing the cystatin, as compared to larvae fed on NT plants. No negative effects on survival and growth were observed when SSB nymphs were exposed to HvCPI-1 C⁶⁸ → G by predation on either CPB or ECW larvae reared on transgenic potato plants expressing the barley cystatin, despite the fact that the inhibitor suppressed in vitro gut proteolysis of the predatory bug. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays. Fernando álvarez-Alfageme and Manuel Martínez are contributed equally to this work.     

Effect of neem on the growth and development of the obliquebanded leafroller, Choristoneura rosaceana

Neem, Azadirachta indica A. Juss. (Meliaceae), seed oil (NSO) added to meridic diet at concentrations as low as 0.016% reduced pupation and prevented adult eclosion of obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae). At a rate of 0.0016%, NSO reduced the fitness of C. rosaceana, resulting in longer developmental times, lower adult eclosion rates, and reduced egg production compared with controls. The neem insecticide Margosan-O ^TM produced comparable results based on concentrations of the most biologically active constituent, azadirachtin. Pupation was completely inhibited at concentrations of 0.25% and 1.0% for larvae exposed at 5th or 6th instar, respectively; rates as low as 0.016% reduced pupal weights and adult eclosion rates. For larvae transferred to treated diet at 5th instar, physical abnormalities in the wings of adults occurred at a rate of 0.004% NSO and increased with increasing treatment rates. NSO at concentrations as high as 2.0% was not antifeedant to neonate larvae, based on 24 and 48 h choice test bioassays, when incorporated into a meridic diet.     

Acute toxicity and sub-lethal effects of a pyrethroid (cypermethrin) on survival,development and fitness of Helicoverpa armigera

The chickpea pod borer, Helicoverpa armigera (Hübner) is one of the most destructive pests of India and most of the countries. In this experiment, sub-lethal effects of cypermethrin were evaluated against sixth instar larvae of this pest that had ingested insecticide-treated chickpea pods. The LC₅₀, LC₃₀ and LC₁₀ values of cypermethrin were 80.38, 26.65 and 5.41?mg a.i.?L^?1, respectively, against sixth instar. The sub-lethal effects on fitness, eggs, larvae, pupae, adult longevity and reproduction were observed in H. armigera population that survived exposure to the sub-lethal doses of LC₅₀, LC₃₀ and LC₁₀ of cypermethrin. Survivorship was reduced to 36?days if H. armigera exposed to the sub-lethal dose of LC_50, whereas it was 42?days in unexposed groups. Larvae treated with LC₅₀ dose exhibit lowest intrinsic rate of increase (r_m) (0.0413 females/female/day) and highest (0.0517) with the unexposed group. Time taken by H. armigera to complete one generation (T_c) was 40.09?days, while this time significantly lowered to 32.85?days when exposed to the sub-lethal dose of LC₃₀. Hatching eggs were greatly reduced in the larvae exposed to cypermethrin at all the sub-lethal doses. About 430 eggs/female/generation were recorded after treatment with 80.38?mg/L as compared with untreated females (1390). Larvae that were exposed to sub-lethal concentrations of cypermethrin exhibited lower pupal weight and prolonged pupal developmental times compared control larvae. The overall fitness performance was better in the unexposed population as compared to the population exposed to sub-lethal doses.     

The combined effects of temperature and food consumption on body weight,egg production and developmental time in Chaoborus crystallinus De Geer (Diptera: Chaoboridae)

Summary Chaoborus crystallinus fourth-instar larvae were reared individually at 14°, 17° and 20° C under different food conditions. Daphnia magna of 1.25 mm average length served as prey. The following were measured: amount of prey ingested, larval weight gain, duration of fourth instar, body weight of the adults, and egg number per female. At a given temperature, the body weight, egg-number and developmental rate increased with food consumption. At a given food consumption, higher temperatures caused a decrease in body weight and egg number, and an increase in developmental rate. Gross production efficiencies for fourth-instar larvae were highest at temperatures around 17° C. The results clearly indicate that from an energetic point of view higher temperatures are disadvantageous. In C. crystallinus vertical migration is evidently a way of lowering the temperature to which the animals are exposed and hence optimizing food conversion into biomass and offspring production, especially if prey densities are below the saturation level.     

The effects of some selected bacteria on embryos and larvae of the American oyster,Crassostrea virginica

An investigation was conducted to determine the effects of selected bacteria on healthy embryos and larvae of the American oyster, Crassostrea virginica. A total of 156 bacterial types were isolated from moribund larvae and particulate matter collected with a 36 μm Nitex screen. Most of the isolates were nonpigmented motile, gram-negative rods sensitive to chloramphenicol and neomycin, and were classified as Pseudomonas and Vibrio.Three oyster developmental stages were exposed to selected bacteria: embryos in the first 48 hr of development, 2-day-old larvae, and presetting, 10-day-old larvae. Twenty of the bacterial strains tested caused gross morphological abnormalities, decreased growth, and/or increased mortality for one or more of the three developmental stages. Neither culture filtrate nor heat-killed bacteria produced these effects.     

Mating behavior,spawning, parental care,and embryonic development of some marine pseudocerotid flatworms (Platyhelminthes: Rhabditophora: Polycladida) in Singapore

The reproductive biology of tropical marine cotylean polyclads is presently poorly known. Reproductive aspects of 16 polyclad species from the family Pseudocerotidae in the genera Acanthozoon, Nymphozoon, Phrikoceros, Pseudobiceros, Pseudoceros, Thysanozoon, and Tytthosoceros from Singapore were documented for the first time. Penis fencing was observed to be just a mating ritual and not necessary for insemination, not always aggressive, and could also result in eventual reciprocal insemination. Results showed that all species underwent similar embryonic developmental stages and hatched as Müller's larvae. Only Pseudoceros concinnus and Pseudoceros laingensis, with mean developmental times of >20 days and mean brood sizes of <1,000 eggs, displayed long‐term parental care. Polyclads producing larger brood sizes had shorter developmental times and only covered their egg masses for about one day. Phrikoceros baibaiye and Pseudobiceros spp. produced egg capsules with pointed opercula, whereas all other species possessed smooth, rounded opercula. All genera hatched with brownish‐orange larvae, except for Pseudoceros spp., which hatched with reddish‐purple larvae regardless of the initial embryo color (either grayish‐yellow or reddish‐purple). These could potentially complement current taxonomic characters in distinguishing polyclad genera and species.     

Effects of juvenile hormone I and precocene I & II on Microplitis rufiventris when administered via its host, Spodoptera littoralis

We investigated the effect of juvenile hormoneI (JHI) and precocene I & II (PI & PII) onthe endo-developmental stages, reproductiveperformance and longevity of Microplitisrufiventris Kok. after topical treatment ofhost larvae of Spodoptera littoralis(Boisd.). Host larvae were treated with each ofthe test compounds by a single dose 2–4 h afterparasitization or 1 day prior to parasitoidemergence, or by repeated daily applications 2–4 h after parasitization to parasitoid emergence.JHI-treatments increased parasitoiddevelopmental time, induced developmentalabnormalities and inhibition of parasitoidegression. Interestingly, one of the causal factorsthat inhibited the parasitoid egression was themoulting of some parasitoid larvae into asupernumerary instar. Applications of PI or PIIto parasitized S. littoralis larvae didnot affect parasitoid developmental time, butsome developmental abnormalities were noted.Melanin formation in host's blood was greaterin PI-treatments than in PII-treatments.PII-treatments arrested development of somefirst instar parasitoid larvae typical of JHIexcess. Adult female wasps that emerged fromJHI-treated hosts lived for significantlylonger periods than control females, but didnot produce more offspring. The longevity andreproductive capacity of females whichdeveloped in hosts treated by precocenes werereduced compared with the females emerged fromcontrol hosts. Even the small number ofadult wasps from PII-treated hosts which livedfor the same period as control wasps, showed asignificant reduction in their reproductiveperformance. Repeated daily applications of thetest compounds reduced wasp fecundity andlongevity to a significantly greater degreethan the single applications, except for a lowdose of JHI (1 µg). The study demonstratesthat growth disrupters, such as JHs andprecocenes have a delayed developmental effect onM. rufiventris and points out the need tobe aware of these effects in implementingcontrol strategies of S. littoralis innatural habitats.     

Characterisation of digestive protease in the rose sawfly,Arge rosae Linnaeus (Hymenoptera: Argidae)

Insect midgut proteases are excellent targets for insecticidal agents such as protease inhibitors. These inhibitors are used for producing transgenic plants, resistant to pests. For achieving this goal, it is necessary to find the nature of specific proteases and their properties for adopting possible pest management procedure. Therefore, characterisation of the enzymes in the gut of the rose sawfly, Arge rosae (Hymenoptera: Argidae), responsible for proteolysis, was performed using a range of synthetic substrates and specific inhibitors. The optimum conditions for general proteases and trypsin were achieved at pH 10. The highest activity for general proteases was obtained at a temperature of 45°C. The use of specific inhibitors and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) provided evidence to suggest that most of the proteases belonged to the serine group because of high inhibitory effect of phenyl methane sulfonyl fluoride on total proteolytic activity. Also, inhibition assays and zymogram analysis showed that metalloproteases are present in A. rosae digestive system. These results indicated that A. rosae larvae mainly used serine proteases for protein digestion, with chymotrypsin as the dominant form. The kinetic parameters of trypsin-like proteases using N-benzoyl-dl-arg-p-nitroanilide as substrate indicated that the K _m and V _max values of trypsin in the gut of the fifth instar larvae were 730 ± 17.3 μM and 456 ± 13.85 nmol min^?1 mg^?1 protein, respectively.