Crude Larval Extract Research Articles

Trichinella spiralis Larval Extract as a Biological Anti-Tumor Therapy in a Murine Model of Ehrlich SolidCarcinoma.

Trichinella spiralis (T. spiralis) is an immunomodulating parasite that can adversely affect tumor growth and extend host lifespan. The aim of this study was to elucidate the mechanisms by which T. spiralis larval antigens achieve this effect using Ehrlich solid carcinoma (ESC) murine model. Assessment was done by histopathological and immunohistochemical analysis of caspase-3, TNF-α, Ki-67 and CD31. Additionally, Bcl2 and Bcl2-associated protein X (Bax) relative gene expression was assessed by molecular analysis for studying the effect of T. spiralis crude larval extract (CLE) antigen on tumor necrosis, apoptosis, cell proliferation and angiogenesis. We found that both T. spiralis infection and CLE caused a decrease in the areas of necrosis in ESC. Moreover, they led to increased apoptosis through activation of caspase-3, up-regulation of pro-apoptotic gene, Bax and down-regulation of anti-apoptotic gene, Bcl2. Also, T. spiralis infection and CLE diminished ESC proliferation, as evidenced by decreasing Ki-67. T. spiralis infection and CLE were able to suppress the development of ESC by inhibiting tumor proliferation, inducing apoptosis and decreasing tumor necrosis, with subsequent decrease in tumor metastasis. T. spiralis CLE antigen may be considered as a promising complementary immunotherapeutic agent in the treatment of cancer.

Assesment of Hypoderma infestation in a wild population of Cervus elaphus from mountains Atlantic ecosystems in southwestern Europe (Spain)

Hypodermosis in Cervus elaphus was studied in the Riaño Regional Hunting Reserve, Province of León, north-western Spain. One hundred and ten red deer were examined for the presence of warble fly larvae. They were analyzed by PCR analysis of the COI region of mt-DNA and identified as Hypoderma actaeon. The prevalence of larvae was 42.7% with a mean intensity of 12.5 ± 18 (range 1–80) warbles/deer infested. The distribution of larvae in the infested animals showed an aggregated/overdispersed pattern (aggregation index = 25.84), where the larvae are not randomly or uniformly distributed, but strongly aggregated among their hosts. Larvae were found in all three states. First and second-instars were observed mainly in the autumn until the end of winter (November-March) and third-instars in late winter until mid-spring (March–May). The adult animals and the males had a higher prevalence than the young and the females, finding statistically significant differences only according to the sex of the animals. Seasonal variations were observed in the prevalence with the highest number of infested animals in winter and autumn, but not in terms of the mean intensity of parasites. Additionally, we assessed the presence of anti-Hypoderma antibodies in serum by means of indirect ELISA tests, using a crude larval extract (CLE) and a purified fraction the hypodermin C (HyC) obtained from first instars of Spanish isolates of Hypoderma lineatum (cattle). These findings confirm that H. actaeon is widely distributed in northern Spain, and provide new information about its chronobiology in mountainous Atlantic ecosystems from southwestern Europe.

Cytokine expression in bovine PBMC cultures stimulated with Hypoderma lineatum antigens

Hypoderma antigens are involved in host inflammation and immune response, conditioning larvae survival. In this study, peripheral blood mononuclear cell (PBMC) cultures from Hypoderma sensitized and unsensitized cattle were performed to determine the effect of H. lineatum antigens and incubation time (18, 24, 48 h) on IFN-γ, TNF-α, IL-10 and IL-4 mRNA gene expression determined by RT-qPCR. TNF-α and IL-4 gene expression were higher in Hypoderma previously sensitized PBMCs, suggesting that a mixed Th1/Th2 response may play a significant role in host defence reactions against Hypoderma exhibited by previously infested cattle. Incubation time had a significant effect on IL-10 and TNF-α gene expression, which decreased over time. Regarding to H. lineatum antigens, the crude larval extract and the purified fraction hypodermin B (HB) produced a significant reduction of the mRNA expression levels of the proinflammatory cytokine, IFN-γ; moreover, the HB had a stimulating effect on the mRNA gene expression of the anti-inflammatory cytokine IL-10, demonstrating that the parasite would modulate the host defence mechanisms by avoiding harmful immune responses that would limit its survival into the host tissues.

Resposta imunológica a antígenos de Hysterothylacium deardorffoverstreetorum de peixes teleósteos

RESUMO Anisaquidose é uma doença provocada por parasitos da família Anisakidae e se caracteriza por manifestações gastrointestinais e alérgicas. O Anisakis simplex é o parasito mais patogênico ao homem e altamente alergênico. Porém, outros anisaquídeos também são danosos aos humanos, mas é desconhecida a imunogenicidade dessas larvas. O objetivo deste trabalho foi avaliar o potencial imunogênico do parasito Hysterothylacium deardorffoverestreetorum (HD) em modelo murino. Camundongos da linhagem BALB/c foram divididos em três grupos experimentais e receberam as preparações antigênicas obtidas de larvas de HD: extrato bruto de larvas (EBH), extrato secretado/ excretado de larvas (ESH) e extrato bruto de larvas após excreção/secreção (EEH). Amostras séricas foram obtidas em diferentes dias após imunização para determinação dos níveis de anticorpos específicos pelo ensaio imunoenzimático (ELISA). Os resultados demonstram aumento na produção de imunoglobulina (Ig) G após a segunda imunização, com aumento progressivo após a terceira imunização. Já em relação à IgE, a reatividade foi mais tardia, demonstrando aumento progressivo após a terceira imunização. Foi avaliada a imunidade celular por meio da intradermorreação, como resultado estatisticamente significativo em relação ao controle utilizado. Este experimento é a primeira descrição da potencialidade patogênica desse parasito em mamíferos e representa um avanço no diagnóstico da anisaquidose humana.

Modulatory effects of interferon-γ and interleukin-4 on cellular immune responses against Hypoderma lineatum antigens.

This study investigates the in vitro modulatory effects of interferon-γ (IFN-γ) and interleukin-4 (IL-4) on both proliferative bovine T cell responses and IL-10 production induced by different antigens [crude larval extract and the purified fractions hypodermin A, B and C (HyA, HyB, HyC)] obtained from first instars of Hypoderma lineatum (Diptera: Oestridae), alone or in the presence of the mitogen concanavalin A. Incubation with the different parasitic antigens resulted in significant inhibition of T cell proliferation and IL-10 production, which, in general, did not revert after the addition of IFN-γ and IL-4. In the absence of antigens, IL-4 induced significant inhibition of mitogen-induced T cell responses. Exogenous IFN-γ exhibited an inhibitory effect on cell proliferation in the presence of the purified fractions HyB and HyC. These in vitro data suggest that far from neutralizing the effects of larval antigens, the addition of IFN-γ potentiates their anti-proliferative activity; by contrast, IL-4 had no consistent effects on proliferative responses to Hypoderma. IL-4 provoked an increment of IL-10 levels in supernatants of HyB-stimulated cells. In conclusion, exogenous IFN-γ and IL-4 were unable to counteract the suppressor effects of H. lineatum antigens.

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.

Use of a crude extract or purified antigen from first-instar cattle grubs,Hypoderma lineatum, for the detection of anti-Hypodermaantibodies in free-ranging cervids from southern Spain

During the 2003-2005 hunting seasons, a total of 120 Cervidae, including 39 red deer (Cervus elaphus hispanicus) and 81 fallow deer (Dama dama), were examined for subcutaneous myiasis. Animals were shot from January to June in southern Spain. Specific antibodies against Hypodermatinae (Diptera: Oestridae) were detected by indirect enzyme-linked immunosorbent assay (iELISA) using a crude larval extract (CLE) and a purified antigen [hypodermin C (HC)] obtained from first instars of Hypoderma lineatum (De Villers) (Diptera: Oestridae). Hypoderma actaeon Brauer was the only species detected in this study, which represents the first confirmation of this species in fallow deer from Spain. The overall prevalence of animals presenting subcutaneous larvae (14.2%) was considerably lower than the prevalences determined by iELISA with CLE (43.3%) and HC (40.0%). Red deer showed a higher prevalence of Hypoderma than fallow deer. The concordance between larval examination during the hunting season and iELISA using both antigens was low, whereas the concordance between the CLE and HC ELISAs was good. Larval antigens obtained from H. lineatum constitute a good tool for the diagnosis of H. actaeon in Cervidae, especially when the hunting season does not coincide with the maximum presence of larvae on the back.

Immunomodulatory effect of Hypoderma lineatum antigens: in vitro effect on bovine lymphocyte proliferation and cytokine production

This study examines the immunomodulatory effect of a crude larval extract (CLE), obtained from first stage larvae (L1) of H. lineatum, and the purified fractions hypodermin A (HyA), HyB and HyC. Proliferative responses of peripheral blood mononuclear cells (PBMC) from uninfested and previously infested cattle and the production of the cytokines IL-10, IL-4 and IFN-gamma, in response to concanavalin A (Con A), were determined. The stimulation index of peripheral blood mononuclear cells from uninfested cattle was significantly lower than that from infested animals with the different antigens assayed. The HyA was the antigen that most inhibited the proliferative response, followed by the HyB, the HyC and the CLE. This hypodermin provoked an increase of IFN-gamma and a suppression of IL-10 production that would support a Th1-like cytokine response. The HyB reduced the production of IL-10 stimulated by the Con A in cultures from infested animals. The HyC did not modulate the production of cytokines. Finally, the CLE induced a marked suppression in the production of the different cytokines in cultures from naïve and previously sensitized animals. Our results indicate that Hypoderma larval secretions are comprised of different components (hypodermins) that individually induce distinct but partially overlapping modulatory responses.

Protein expression profile of Gasterophilus intestinalis larvae causing horse gastric myiasis and characterization of horse immune reaction

BackgroundLittle information is available on the immunological aspect of parasitic Gasterophilus intestinalis (Diptera, Oestridae) larvae causing horse gastric myiasis. The objectives of this research were to analyze the protein content of larval crude extracts of the migrating second and third larvae (L2 and L3) of G. intestinalis in order to characterize the immune response of horses.ResultsThe proteomic profile of L2 and L3, investigated by using one and two dimensional approaches, revealed a migration pattern specific to each larval stage. Furthermore, Western blots were performed with horse sera and with sera of Balb/c mice immunised with the larval crude extracts of L2 or L3, revealing a different immune reaction in naturally infected horses vs. artificially induced immune reaction in mice. The comparisons of the immunoblot profiles demonstrate that the stage L2 is more immunogenic than the stage L3 most likely as an effect of the highest enzymatic production of L2 while migrating through the host tissues. Fifteen proteins were identified by mass spectrometry.ConclusionThis work provides further information into the understanding of the interaction between G. intestinalis and their host and by contributing a novel scheme of the proteomic profile of the main larval stages.

Allergenic activity of Molicola horridus (Cestoda, Trypanorhyncha), a cosmopolitan fish parasite, in a mouse model

The cestode Molicola horridus is a muscle parasite of teleost fish. The ability of molecules present in this parasite to induce allergic response is not known yet. Since fish-borne parasitic allergens can induce allergic manifestations even when the parasitized fish is well cooked, the knowledge of potential allergens present in food is important in order to provide a save products for consumers. The aim of the study was to determine the allergenic potential of the components present in the crude larval extract (CLE) of M. horridus. Two mouse models were exposed to the CLE: adult BALB/c mice that were intraperitoneally (i.p.) immunized and newborn BALB/c mice that were orally exposed. Specific antibody levels in serum and faeces were measured by ELISA. The cellular immune response was determined by proliferation assay of splenocytes from sensitized mice. The protein profile of CLE was analysed by SDS-PAGE and western blot. In adult mice, specific IgG and IgA were detected in sera and faeces, whereas specific IgE were detected in sera only. In newborn mice, specific IgG were detected in sera and a low level of IgA was detected in faeces. SDS-PAGE revealed the CLE protein profile, with most of the proteins running from 15 to 50 kDa. Specific IgG recognized mainly the 26 and 75 kDa proteins and a molecular complex below 100 kDa by immunoblot. Specific IgE recognized the same 26 kDa protein as IgG did, and, with less intensity, another protein at 30 kDa. Splenocytes from CLE-immunized mice proliferated when stimulated with CLE in a dose-dependent manner. The crude larval extract from M. horridus has potential allergenic molecules which can represent a risk for fish consumers.

Specific IgG antibody responses in Oestrus ovis L. (Diptera: Oestridae) infected sheep: Associations with intensity of infection and larval development

Larvae of Oestrus ovis (Diptera: Oestridae) are ubiquitous parasites of nasal and sinusal cavities of sheep and goats. According to the chronobiology of O. ovis infections in Sardinia and the seasonal pattern of the IgG response, the optimal period to investigate the relationships between O. ovis larval populations and intensity of local and systemic IgG antibody responses was mid-July in the summer season. Sarda × Lacaune ewes ( n = 186), divided into three ram-families were used in the study. Systemic and local IgG responses were measured by ELISA tests using second stage larval crude extracts (L2CE) and L2 (L2SGC) and L3 (L3SGC) salivary gland contents as coating antigens. The number of larval instars, larval length of L1, L2 and L3 larvae, and larval weight of L2 and L3 larvae were individually recorded after ewe necropsy. Negative correlations among larval establishment and/or larval development on the one hand and intensity of local or systemic IgG responses on the other hand were found in two out of three studied ram-families.

Expression and purification of horseradish peroxidase in insect larvae

A strategy for obtaining a high-level expression of horseradish peroxidase isozyme C (HRPC) in insect larvae is herein described. The HRPC-6xHis coding sequence was inserted into the AcNPV genome via the pAcGP67HRPC-6xHis transfer vector to originate an AcHRPC-6xHis recombinant baculovirus of phenotype occ −. Rachiplusia nu larvae were injected with a viral stock derived from culture supernatants of Sf9 cells infected with AcHRPC-6xHis. Enzyme concentration at day 3 post-infection was 230 ± 10 mg/kg haemolymph and 100 ± 14 mg/kg larvae, an expression level never attained by other expression systems. HRPC-6xHis was purified from the crude larval extract or haemolymph by immobilised metal ion affinity chromatography with yields of 88.8% and 89.0% and purification factors of 18.9 and 14.0, respectively, in a single step. The purity of the final product was 90%.

Vaccination of mice against intestinal Trichinella spiralis infections by oral administration of antigens microencapsulated in methacrilic acid copolymers

BALB/c and NIH mice have been successfully vaccinated against the intestinal nematode Trichinella spiralis by oral administration of crude larval extracts (CLE) and excretory–secretory (ES) products derived from first stage T. spiralis larvae (L1) encapsulated in microcapsules made of copolymers of the metacrylic acid (Eudragit L100). Oral vaccination stimulated the secretion of IFN-γ and inhibited the secretion of IL-4 in spleen and mesenteric lymph nodes (MLN) of BALB/c mice. In vaccinated mice the proportion of CD4+ cells increased ( p < 0.05) in Peyer's patches (PP) and decreased ( p < 0.05) in spleen whereas the proportion of CD19+ cells decreased ( p < 0.05) in both PP and spleen, with regard to unvaccinated controls. No variation was evident for the proportion of CD8+ cells. Oral vaccination elevated the antigen-specific serum IgG1 and IgA ( p < 0.05) as well as the antigen-specific IgA response in MLN ( p < 0.05). It is concluded that this way of vaccination induced a concurrent Th1/ Th2 local and systemic responses that are protective and at the same time they may help balancing the strong Th2 response triggered by helminth infections.

Comparative analysis of Trichinella spiralis and Trichinella nativa proteins by two-dimensional gel electrophoresis

Trichinella spiralis and Trichinella nativa are both common wildlife parasites in Finland. However, they differ substantially in their resistance to below 0 degrees C temperatures in their natural hosts. T. nativa can live in frozen fox meat for years, whereas T. spiralis dies when frozen. In mouse muscle, the difference is not as evident; even T. nativa cannot maintain infectivity when kept at -20 degrees C for 1 week. Crude larval protein extracts of these two parasite species were analyzed by two-dimensional gel electrophoresis (2DE). The protein patterns showed clear differences, but matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) peptide mass fingerprint followed by database searches failed to identify these proteins, suggesting that they may still be uncharacterized. The patterns compared after freezing treatment at -20 degrees C revealed changes in the intensity of some protein spots. The antigenic differences of the species were analyzed with two-dimensional Western blots, which showed T. spiralis-specific proteins.

Isolation, partial purification, and characterization of a novel petromyzonol sulfotransferase from Petromyzon marinus (lamprey) larval liver

We have isolated, partially purified, and characterized the 5 alpha-petromyzonol (5 alpha-PZ), (5 alpha-cholan- 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-) sulfotransferase (PZ-SULT) from larval lamprey liver. Crude liver extracts exhibited a PZ-SULT activity of 0.9120 pmol/min/mg in juvenile and 12.62 pmol/min/mg in larvae. Using crude larval liver extracts and various 5 beta-cholan substrates and allocholic acid there was negligible activity, however, with 5 alpha-PZ and 3-keto-5 alpha-PZ the SULT activity was 231.5 pmol/min/mg and 180.8 pmol/min/mg respectively. This established that the sulfotransferase of lamprey larval liver extracts prefers (5 alpha) substrates and it is selective for hydroxyl at C-24. PZ-SULT was purified through various chromatography procedures. Partially purified PZ-SULT exhibited a pH optimum of 8.0, a temperature optimum of 22 degrees C, and activity was linear for 1h. PZ-SULT exhibited a K(m) of 2.5 microM for PAPS and a K(m) of 8 microM for PZ. The affinity purified peak PZ-SULT exhibited a specific activity of 2,038 pmol/min/mg. The peak protein upon SDS-PAGE, correlated to an Mw 47 kDa. Photoaffinity labeling with PAP(35)S, specifically crosslinked the 47 kDa protein, further confirming the identity of PZ-SULT. Partial amino acid sequencing of the putative 47 kDa PZ-SULT protein yielded a peptide sequence (M)SISQAVDAAFXEI, which possessed an overall (approximately 35-40%) homology with mammalian SULT2B1a.

Comparison of IgG3 responses to carbohydrates following mouse infection or immunization with six species ofTrichinella

The IgG3 antibody responses to carbohydrate epitopes were compared in BALB/c mice infected or immunized with six species of Trichinella: T. spiralis (T1), T. nativa (T2), T. britovi (T3), T6, T. nelsoni (T7), and T8. The dynamics of IgG3 responses and antigen recognition following infection or immunization were measured by ELISA and Western blot respectively, using glycosylated and deglycosylated larval crude extracts (LCE) prepared from homologous isolates. A high degree of protein glycosylation was found in all species and with similar profiles. Deglycosylation was completely achieved only in LCE from T1 and T6 isolates. The dynamics of IgG3 responses following infection or immunization significantly differed whereas the antigen recognition profiles appeared similar. Variations in the levels and antigen recognition patterns of IgG3 among the different species were apparent. The highest IgG3 levels were recorded in infections by the T8 isolate and the lowest in infections by the T6 isolate, whereas for immunization the highest IgG3 response was induced by T7 and the lowest by T8. Following antigen deglycosylation, the IgG3 responses were significantly reduced or abrogated and the recognition patterns markedly modified or suppressed in the different species of Trichinella.

Comparison of IgG3 responses to carbohydrates following mouse infection or immunization with six species of Trichinella

AbstractThe IgG3 antibody responses to carbohydrate epitopes were compared in BALB/c mice infected or immunized with six species of Trichinella: T. spiralis (T1), T. nativa (T2), T. britovi (T3), T6, T. nelsoni (T7), and T8. The dynamics of IgG3 responses and antigen recognition following infection or immunization were measured by ELISA and Western blot respectively, using glycosylated and deglycosylated larval crude extracts (LCE) prepared from homologous isolates. A high degree of protein glycosylation was found in all species and with similar profiles. Deglycosylation was completely achieved only in LCE from T1 and T6 isolates. The dynamics of IgG3 responses following infection or immunization significantly differed whereas the antigen recognition profiles appeared similar. Variations in the levels and antigen recognition patterns of IgG3 among the different species were apparent. The highest IgG3 levels were recorded in infections by the T8 isolate and the lowest in infections by the T6 isolate, whereas for immunization the highest IgG3 response was induced by T7 and the lowest by T8. Following antigen deglycosylation, the IgG3 responses were significantly reduced or abrogated and the recognition patterns markedly modified or suppressed in the different species of Trichinella.

Detection and micro-scale isolation of a low molecular mass paralysis toxin from the tick, Argas (Persicargas) walkerae.

This study describes the isolation of a 11 kDa paralysis toxin from crude larval extracts of Argas (Persicargas) walkerae by exploiting the cross-reactivity of a monoclonal antibody (4B12), directed against the paralysis toxin of Rhipicephalus evertsi evertsi. This low molecular mass is in contrast to previous findings of a 60-70 kDa toxin for A. (P.) walkerae, but is similar to neurotoxins isolated from venomous forms of the class Arachnida, which comprise the orders Araneae (spiders). Scorpionida (Scorpions) and Acari (ticks and mites). Since numerous antigenic bands, ranging between 11 and 115 kDa, were detected by the monoclonal antibody 4B12, the possibility of toxin-complex formation and the effect of pH on the latter were investigated by means of HPLC and ammonium sulphate precipitation. The results suggest that physiological conditions, with respect to pH and ionic strength, promote the formation of heterogeneous toxin-complexes while an acidic pH favours the formation of a more homogeneous toxin-containing complex. Furthermore, the effect of partially purified toxin on neurotransmitter release from crude rat brain synaptosomes was investigated, since tick paralysis toxins are hypothesised to inhibit neurotransmitter release from the presynaptic terminal. Both calcium-dependent, as well as calcium-independent release was inhibited by the toxin-containing sample.

Optimization of the protein fraction of microcapsules used in feeding of marine fish larvae using in vitro digestibility techniques

The development of artificial feeds for marine fish larvae is demanding and, taking into account the difficulties and costs associated with the rearing of the larvae of some fish, preliminary evaluation of feed ingredients using in vitro techniques may be an alternative to in vivo assays. Some proteins used in microfeeds for marine fish were tested in vitro for examination of their effect on the proteases of seabream larvae. Casein and cuttlefish meal did not affect protease activity of crude larval extracts, whereas ovalbumin produced a 60% inhibition. The use of a pH-stat for the assessment of the degree of hydrolysis (DH) of proteins confirmed the low nutritive value of ovalbumin, since a much lower value was obtained for microcapsules prepared using this protein source when compared with those prepared using casein or cuttlefish meal (3.2 vs. 7.3 and 7.6, respectively). Products resulting from such hydrolysis were analysed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Image analysis of such gels led to the formulation of an index named the coefficient of protein degradation (CPD) which expressed the extent to which the main protein fractions were hydrolysed by larval proteases in a given time. A significant correlation (r2 = 0.98, P < 0.05) was obtained between DH and CPD values measured for either protein sources or microcapsules. The combination of protease inhibition assays with measurements of DH and CPD is proposed as a preliminary evaluation of protein ingredients used in the formulation of artificial feeds for larval fish.

The α-amylase from the yellow meal worm: complete primary structure, crystallization and preliminary X-ray analysis

The α-amylase from Tenebrio molitor larvae (TMA) was purified from a crude larval extract. After removal of the N-terminal pyroglutamate residue and identification of the following 17 residues by Edman sequencing, the cDNA of mature TMA was cloned from larval mRNA. The encoded enzyme consists of 471 amino acid residues and has 57–79% sequence identity to other insect α-amylases and also shows high homology to the mammalian enzymes. TMA was crystallized in form of well-ordered orthorhombic crystals of space group P2 12 12 1 diffracting beyond 1.6 Å resolution with unit cell dimensions of a=51.24 Å, b=93.46 Å, c=96.95 Å. TMA may serve as model system for the future analysis of interactions between insect α-amylase and proteinaceous plant inhibitors on the molecular level.