Identification and characterization of male specific serum proteins in the Mediterranean fruit fly Ceratitis capitata

Two major and three minor male specific serum proteins (MSSPs) have been identified in the Mediterranean fruit fly Ceratitis capitata. All five MSSPs accumulate in the haemolymph during the first 3 days of the adult development and represent more than 50% of the total haemolymph proteins in the adult males. All MSSPs are dimeric proteins consisting of two subunits with molecular weights between 13.5 and 14.5 kDa. MSSP-1, 3, 4 and 5 are homodimers while MSSP-2 is a heterodimer. The two major MSSPs (MSSP-1 and 5) have been isolated. Antisera against these two MSSPs cross-react partially with each other's antigens but did not give any reaction with haemolymph from adult females and third instar larvae.     

Adipokinetic hormone-induced lipid mobilization and lipophorin interconversions in fifth larval instar locusts

The response of fifth larval instar locusts to injected adipokinetic hormone (AKH) is only poor, as is reflected in both a very moderate elevation of the haemolymph lipid concentration and the slight occurrence of the haemolymph lipophorin interconversions characteristic for adult locusts, resulting in formation of only small quantities of the low density lipophorin (A⁺). However, an additional lipophorin fraction (A′) is induced, which is intermediate in density and size between high and low density lipophorin and which is not identified in adult haemolymph. As in adults, larval A⁺ formation includes association of the resting high density lipophorin with a non-lipid containing protein (C₂), the haemolymph concentration of which is only one-fifth relative to adults. However, the larval haemolymph protein composition is not the primary cause of the incomplete adipokinetic response, as elevation of the concentration of protein C₂ by injection of isolated adult C₂, whether or not in combination with adult high density lipophorin, did not increase lipophorin conversions nor haemolymph lipid elevation.In vitro incubation of larval fat bodies in adult haemolymph showed that competency to both the AKH-induced lipid release and the haemolymph lipophorin conversions of the larval fat body are reduced compared to equal amounts of adult tissue. Reciprocal incubation of adult fat body in larval haemolymph resulted in only a very moderate adipokinetic response, demonstrating that larval haemolymph protein composition is restrictive for full development of hormone action.Both immunoblotting experiments and enzyme-linked immunosorbent assays (ELISA), using monoclonal antibodies specific for the adult lipophorin apoproteins, indicated that the larval lipophorins closely resemble the adult forms. Apparently the structure of locust lipophorins is remarkably constant throughout development despite changes in metabolic functions.     

Hormone-induced rearrangement of locust haemolymph lipoproteins: The involvement of glycoprotein C2

Formation of lipoprotein A⁺ and elevation of lipoprotein fraction O in locust (Locusta migratoria migratorioides) haemolymph as induced by adipokinetic hormone (AKH) includes the participation of non-lipid carrying proteins (fraction C), which was examined in more detail. By using gel filtration chromatography, the rather heterogenous C-proteins were resolved into three protein fractions, only one of which (C₂) appeared to be actually involved in the lipoprotein reassociation. The changes in amino acid composition of the elevated lipoprotein fractions as compared with those from the lipoproteins in the resting situation are accounted for by the contribution of the rather specific amino acid composition of this C₂-fraction. Polyacrylamide gel electrophoresis (PAGE) indicates that the C₂-protein is migrating as only one band; SDS-PAGE revealed that the C₂-protein consists of one single polypeptide chain with an approximate molecular weight of 20,000. This chain is also recovered in the subunit structure of the lipoprotein fractions induced by AKH-injection (A⁺, O_AKH) in contrast with that of the lipoprotein fractions in resting haemolymph. Unlike the other C-proteins, protein C₂ displayed immunoreactivity with antiserum raised against lipoprotein A⁺. From carbohydrate analyses, C₂ appeared to be a glycoprotein containing approx. 12.5% carbohydrate. In vivo pilot studies on the dynamics of C₂-proteins using ³H-labelled glycoprotein C₂ gave evidence for the incorporation of radiolabel into both A⁺ and O_AKH. Possible functions of the involvement of the glycoprotein to A⁺ formation are discussed.     

Fatty acid and lipid analysis of the house cricket,Acheta domesticus

The fatty acid content and composition of the house cricket Acheta domesticus have been investigated in entire insects at different developmental stages and in selected organs of male and female adults. We have also determined the fatty acid composition of the various lipid classes within extracts of the organs of adult female insects. Fatty acids were analysed by capillary gas chromatography or mass spectrometry as their methyl esters (FAMEs) after direct transesterification of insect material or separated lipid classes.The major esterified fatty acids in all extracts were palmitate (C_16:0), stearate (C_18:0), oleate (C_18:1) and linoleate (C_18:2). Levels of esterified fatty acid varied considerably between organs but the fatty acid compositions showed only small variations. The levels of polyunsaturated fatty acids of the C₁₈ series were considerably higher in phospholipid fractions than in other lipid classes. Triacylglycerols formed the major lipid class in ovaries, fat-body and newly-laid eggs, whereas diacylglycerols and phospholipid predominate in the haemolymph. Triacylglycerols, phospholipids, diacylglycerols and free fatty acids were all found in significant amounts in the gut tissue.     

Hydrocarbons,sterols and tocopherols in the seeds of six Adansonia species

The composition of the unsaponifiable matter of the lipids of six Adansonia species (A. grandidieri, A. za, A. fony, A. madagascariensis, A. digitata and A. suarezensis) was investigated. The total unsaponifiable content, its general composition and the identity of the components of the hydrocarbon, sterol and tocopherol fractions are presented. The unsaponifiable content in oil ranges from 0.4 to 1.1% (hexane method) and from 0.6 to 2.2% (diethyl ether method). In two species (A. grandidieri and A. suarezensis) the major components are 4-demethylsterols (23–42%) tocopherols (37-10%) and hydrocarbons (15–17%). In both species examined, eight 4-demethylsterols occur in the sterol fraction with sitosterol (81–88%) being predominant. Among the four tocopherols present, γ-tocopherol (68–98%) is the major compound. Each Adansonia species shows a characteristic gas liquid chromatography pattern for the hydrocarbon fraction. Squalene is the major component for five species (40–75%). Iso-, anteiso- and other branched hydrocarbons were not identified but were present in small amounts in comparison with n-alkanes. The dominance of odd- over even-carbon number chain length of n-alkanes was not observed in any species. The results show that C₂₂, C₂₅, C₂₆, C₂₇, C₂₈ and C₂₉ are the most frequent major constituents.     

Proteins of the fat body of non-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella: Effect of juvenile hormone

The proteins of the fat body of non-diapausing, pre-diapausing, and newly-diapaused larvae of the southwestern corn borer, Diatraea grandiosella, were examined. Since a low titre of juvenile hormone (JH) is present in the haemolymph throughout the final instar of non-diapausing larvae, the hormone does not appear to stimulate the pre-metamorphic synthesis of proteins. In contrast, the high titre of JH in the haemolymph during the final instar of pre-diapausing larvae appears to stimulate the synthesis of selected proteins. For example, pre-diapausing larvae store in their fat body a low molecular weight protein which has been named the ‘diapause-associated protein’. When non-diapausing larvae were treated topically with C₁₇-JH or a JH mimic, from 50 to 70% entered a diapause-like state as fully grown larvae. These hormone-treated larvae accumulated the diapause-associated protein and a high molecular weight protein in their fat bodies. Both of these proteins were shown to be released from the fat body of newly-diapaused larvae in vitro, and may function in the haemolymph during diapause. The high molecular weight protein, isolated from the haemolymph, was shown to contain neutral and polar lipids, including biochromes. Its storage in the fat body and release into the haemolymph may be essential for the transport of lipids during diapause. The fat body proteins of newly-diapaused larvae of the southern cornstalk borer, Diatraea crambidiodes, were also examined electrophoretically. They were found to contain a similar protein pattern to that of D. grandiosella, including the presence of a diapause-associated protein.     

Inorganic ions in spermathecal fluid and their transport across the spermathecal membrane of the queen bee, Apis mellifera

Micropuncture and microanalytical methods were employed to investigate the rôle of the spermathecal epithelium of the honey queen-bee in providing the appropriate conditions for the prolonged storage of spermatozoa. It was found that the epithelium maintains large concentration gradients of inorganic ions, generates an electrical potential difference of 21 mV, lumen positive, and produces a pH difference of up to 2.4 pH units between spermathecal fluid (SF) and haemolymph (H). The $\text{SF}\text{H}$ concentration ratios for K⁺, Na⁺, Ca⁺⁺, Cl^?1, HPO₄^??, H₂PO₄^? and amino acids were 7.7; 0.5; 0.8; 0.4; 1.03; 0.004; 0.3, respectively. While the pH value of haemolymph was constant at 6.17, the pH of SF increased with age from 7.3 to 8.6 over the first 3 days. The calculated electrochemical potential differences suggest that the epithelium of the spermathecal wall secretes K⁺ (and possibly HCO₃^? or OH^?) actively into the lumen, but handles Na⁺ passively. This pattern conforms with the organization of ion transport in other insects.     

Differential synthesis of bacteria-induced proteins of Manduca sexta larvae and pupae

The range of inducible antibacterial and other associated haemolymph proteins in Manduca sexta larvae and pupae was examined by high resolution two-dimensional (2D) isoelectric focusing-polyacrylamide gel electrophoresis. Twenty-two major inducible proteins were consistently resolved on gels of haemolymph from bacteria-injected larvae. Haemolymph from bacteria-injected pupae showed a different pattern of induced proteins. The proteins of the two stages include those which (i) are induced in both stages, (ii) those which are exclusively induced in either larvae or pupae, (iii) those which are inducible in larvae, but consititutively present in pupae, and, (iv) those which are induced in larvae, and which are present at intermediate levels but may be induced to higher levels in pupae.The antibacterial activity of the haemolymph from larvae and pupae was compared on acidpolyacrylamide gels, and the apparent M_r and pI of the inducible proteins determined. Certain of the inducible proteins appear to resemble the cecropin and attacin proteins of Hylophora cecropia.     

The composition of external lipids from adult whiteflies,Bemisia tabaci and Trialeurodes vaporariorum

The total surface lipids, including the wax particles, of the adult whiteflies of Bemisia tabaci and Trialeurodes vaporariorum were characterized. At eclosion, there were similar amounts of long-chain hydrocarbons, aldehydes, alcohols and wax esters. Within a few hours post-eclosion, long-chain aldehydes and long-chain alcohols were the dominant surface lipid components, C₃₄ on B. tabaci and C₃₂ on T. vaporariorum. Hydrocarbons, mainly n-alkanes, were minor components of the surface lipids. The major wax esters were C₄₆ on B. tabaci and C₄₂ on T. vaporariorum. The major acid and alcohol moieties in the wax esters of B. tabaci were C₂₀ and C₂₆, respectively, and of T. vaporariorum were C₂₀ and C₂₂, respectively. Both B. tabaci and T. vaporariorum had a minor wax ester composed of the fatty acid C_18:1 esterified to the major alcohols, C₃₄ and C₃₂, respectively. Bemisia were readily distinguished from Trialeurodes based on the composition of their wax particles and/or their wax esters; however, no differentiating surface lipid components were detected between biotypes A and B of B. tabaci.     

Regulation of oxaloacetate, aspartate, and malate formation in mesophyll protoplast extracts of three types of c(4) plants

The use of mesophyll protoplast extracts from various C₄ species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C₄ photosynthesis. Conditions regulating the formation of the C₄ acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C₄ species, Eleusineindica, an NAD-malic enzyme C₄ species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C₄ species. Light-dependent induction of CO₂ fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO₂ fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C₄ mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO₂ fixation was not.     

Effect of RH-2485 on development, metamorphosis and synthesis of major proteins in female silkworm Bombyx mori

Toxicological data on silkworm Bombyx mori are quite comparable to those of other lepidopteran pest insects, therefore, it is considered as a suitable model for exploring effects of any new synthetic formulations. In this study, female V instar larvae of silk moth B. mori were chosen to evaluate the lethal and sublethal toxicity effects of RH-2485 (methoxyfenozide), a non-steroidal ecdysteroid agonist and to substantiate the ecdysteroid mimicking action of RH-2485 on ovary development, vitellogenin incorporation and egg production in isolated pupal abdomen (IPA). Probit analysis was carried out to find the median lethal dose (LD₅₀) from 96 h cumulative mortality percent. Protein profile of haemolymph, fat body, ovary and eggs were separated in SDS-PAGE. Western blot analysis was carried out to confirm vitellogenin in the ovary. Sublethal effects on feeding, cocoon spinning, pupation, adult emergence and egg production were studied at doses of 1/5^th, 1/10^th and 1/20^th of LD₅₀. Significant changes were observed in all these parameters at all three sublethal doses. The morphological effects were related to underlying biochemical changes by finding the changes in haemolymph, fat body, ovary and egg protein profile. Marked changes were observed in storage proteins (80 kDa) and 30 kDa proteins in the haemolymph at all three sublethal doses. The larvae that escaped the sublethal effects at a dose of 1/20 of LD₅₀ and emerged as adults with malformed wings produced significantly lower number of eggs. The isolated pupal abdomen (IPA) treated with RH-2485 did not metamorphose into adult but the oocyte development and vitellogenesis were normal but the egg precursor processing was incomplete leading to failure in choriogenesis.     

Dehydrodinosterol,dinosterone and related sterols of a non-photosynthetic dinoflagellate, Crypthecodinium cohnii

The heterotrophic dinoflagellate Crypthecodinium cohnii contained the 4α-methyl sterols, dinosterol, dehydrodinosterol (4α,23,24-trimethylcholesta-5,22-dien-3β-ol) and the tentatively identified 4α,24-dimethyl-cholestan-3β-ol and 4α,24-dimethylcholest-5-en-3β-ol. The major 4-demethyl sterol was cholesta-5,7-dien-3β-ol which was accompanied by a smaller amount of cholesterol and traces of several other C₂₇,C₂₈ and C₂₉ sterols. In addition, a 3-oxo-steroid fraction was isolated and the major component identified as dinosterone (4α,23,24-trimethylcholest-22-en-3-one). The possible biosynthetic relationships of these compounds are discussed.     

Fatty acid esters of testosterone in rat brain: identification, distribution, and some properties of enzymes which synthesize and hydrolyze the esters

[4-¹⁴C]Testosterone was converted to an unknown compound with a much higher R_f on thin layer chromatogram than the substrate when it was incubated with a rat brain microsomal preparation. Evidence from its mass, infrared, and ultraviolet spectra indicated that the enzymic product is a mixture of fatty acid esters of testosterone. Saponification of the product yielded testosterone and a mixture of C_12:0, C_14:0, C_16:0, C_18:0, and C_18:1 fatty acids. The enzymic product was identical to testosterone laurate and testosterone stearate which were synthesized chemically. The enzyme system had a pH optimum at 4.9 with acetate buffer. The apparent K_m was 8.3 × 10^?5m for testosterone and 5.0 × 10^?5m for palmityl CoA. An enzyme which hydrolyzes testosterone[1-¹⁴C]oleate was also detected in rat brain. Most of this activity was in the nuclear and mitochondrial fractions. This enzyme had an optimum pH at 6.5 with phosphate buffer and its apparent K_m was 2.1 × 10^?4m. A low level of synthetic activity was found in fetal brain tissue which increased and reached a maximum at 3 weeks of age. The synthetic activity rapidly decreased with further increase in age. Hydrolytic activity was nearly undetectable in fetal rat brain, increased gradually until the animal reaches 3 weeks old, and remained at this level. Both synthetic and hydrolytic enzyme activities were higher in the brain than in other tissues examined.     

Identification of a Gene Encoding a Transporter Essential for Utilization of C4 Dicarboxylates in Corynebacterium glutamicum

The Corynebacterium glutamicum R genome contains a total of eight genes encoding proteins with sequence similarity to C₄-dicarboxylate transporters identified from other bacteria. Three of the genes encode proteins within the dicarboxylate/amino acid:cation symporter (DAACS) family, another three encode proteins within the tripartite ATP-independent periplasmic transporter family, and two encode proteins within the divalent anion:Na⁺ symporter (DASS) family. We observed that a mutant strain deficient in one of these genes, designated dcsT, of the DASS family did not aerobically grow on the C₄ dicarboxylates succinate, fumarate, and malate as the sole carbon sources. Mutant strains deficient in each of the other seven genes grew as well as the wild-type strain under the same conditions, although one of these genes is a homologue of dctA of the DAACS family, involved in aerobic growth on C₄ dicarboxylates in various bacteria. The utilization of C₄ dicarboxylates was markedly enhanced by overexpression of the dcsT gene. We confirmed that the uptake of [¹³C]labeled succinate observed for the wild-type cells was hardly detected in the dcsT-deficient mutant but was markedly enhanced in a dcsT-overexpressing strain. These results suggested that in C. glutamicum, the uptake of C₄ dicarboxylates for aerobic growth was mainly mediated by the DASS transporter encoded by dcsT. The expression level of the dcsT gene transiently increased in the early exponential phase during growth on nutrient-rich medium. This expression was enhanced by the addition of succinate in the mid-exponential phase and was repressed by the addition of glucose in the early exponential phase.     

Climate change,nutrition and immunity: Effects of elevated CO2 and temperature on the immune function of an insect herbivore

Balanced nutrition is fundamental to health and immunity. For herbivorous insects, nutrient-compositional shifts in host plants due to elevated atmospheric CO₂ concentrations and temperature may compromise this balance. Therefore, understanding their immune responses to such shifts is vital if we are to predict the outcomes of climate change for plant–herbivore–parasitoid and pathogen interactions. We tested the immune response of Paropsis atomaria Olivier (Coleoptera: Chrysomelidae) feeding on Eucalyptus tereticornis Sm. seedlings exposed to elevated CO₂ (640 μmol mol⁻¹; C_E) and temperature (ambient plus 4 °C; T_E). Larvae were immune-challenged with a nylon monofilament in order to simulate parasitoid or pathogen attack without other effects of actual parasitism or pathology. The cellular (in vivo melanisation) and humoral (in vitro phenoloxidase PO activity) immune responses were assessed, and linked to changes in leaf chemistry. C_E reduced foliar nitrogen (N) concentrations and increased C:N ratios and concentrations of total phenolics. The humoral response was reduced at C_E. PO activity and haemolymph protein concentrations decreased at C_E, while haemolymph protein concentrations were positively correlated with foliar N concentrations. However, the cellular response increased at C_E and this was not correlated with any foliar traits. Immune parameters were not impacted by T_E. Our study revealed that opposite cellular and humoral immune responses occurred as a result of plant-mediated effects at C_E. In contrast, elevated temperatures within the tested range had minimal impact on immune responses. These complex interactions may alter the outcomes of parasitoid and pathogen attack in future climates.     

Molecular cloning and characterization of neutral ceramidase homologue from the red flour beetle, Tribolium castaneum

Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C₆, C₁₂, C₁₆, C_18:1, and C_24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe²⁺. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.     

In vitro and in vivo effects of copper on haemocyanin-o2 binding in the shore crab,Carcinus maenas

1. When added in vitro to crab haemolymph at concentrations of 50 or 100 mg.1⁻¹, copper decreased both haemocyanin-O₂ affinity and the cooperativity of O₂ binding.2. In crabs contaminated by a lethal dose of waterborne copper (2mg.l⁻¹), haemolymph total concentration of the metal never reached levels that could affect O₂ binding properties directly.3. Exogenous copper added in vitro or entering the animal in contaminated water was found for the most part non filterable and thus probably bound to haemolymph proteins.     

A basidiomycetous yeast, Pseudozyma crassa, produces novel diastereomers of conventional mannosylerythritol lipids as glycolipid biosurfactants

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These compounds show not only excellent surface-active properties, but also versatile biochemical actions. During a survey of new MEL producers, we found that a basidiomycetous yeast, Pseudozyma crassa, extracellularly produces three glycolipids. When glucose and oleic acid were used as the carbon source, the total amount of glycolipids reached approximately 4.6 g/L in the culture medium. The structures of these glycolipids were similar to those of well-known MEL-A, -B, and -C, respectively. Very interestingly, in all the present glycolipids, the configuration of the erythritol moiety was entirely opposite to that of conventional MELs. The present glycolipids were identified to have the carbohydrate structure of 4-O-β-d-mannopyranosyl-(2R,3S)-erythritol, stereochemically different from 4-O-β-d-mannopyranosyl-(2S,3R)-erythritol of conventional MELs. Furthermore, these new glycolipids possessed both short-chain acids (C₂ or C₄) and long-chain acids (C₁₄, C₁₆, or C₁₈) on the mannose moiety. The major component of the present glycolipids clearly showed different interfacial and biological properties, compared to conventional MELs comprising two medium-chain acids on the mannose moiety. Accordingly, the novel MEL diastereomers produced by P. crassa should provide us with different glycolipid functions, and facilitate a broad range of applications of MELs.     

Determination of structure and composition of suberin from the roots of carrot, parsnip, rutabaga, turnip, red beet, and sweet potato by combined gas-liquid chromatography and mass spectrometry

Suberin from the roots of carrots (Daucus carota), parsnip (Pastinaca sativa), rutabaga (Brassica napobrassica), turnip (Brassica rapa), red beet (Beta vulgaris), and sweet potato (Ipomoea batatas) was isolated by a combination of chemical and enzymatic techniques. Finely powdered suberin was depolymerized with 14% BF₃ in methanol, and soluble monomers (20-50% of suberin) were fractionated into phenolic (<10%) and aliphatic (13-35%) fractions. The aliphatic fractions consisted mainly of ω-hydroxyacids (29-43%), dicarboxylic acids (16-27%), fatty acids (4-18%), and fatty alcohols (3-6%). Each fraction was subjected to combined gas-liquid chromatography and mass spectrometry. Among the fatty acids very long chain acids (>C₂₀) were the dominant components in all six plants. In the alcohol fraction C₁₈, C₂₀, C₂₂, and C₂₄ saturated primary alcohols were the major components. C₁₆ and C₁₈ dicarboxylic acids were the major dicarboxylic acids of the suberin of all six plants and in all cases octadec-9-ene-1, 18-dioic acid was the major component except in rutabaga where hexadecane-1, 16-dioic acid was the major dicarboxylic acid. The composition of the ω-hydroxyacid fraction was quite similar to that of the dicarboxylic acids; 18-hydroxy-octadec-9-enoic acid was the major component in all plants except rutabaga, where equal quantities of 16-hydroxyhexadecanoic acid and 18-hydroxyoctadec-9-enoic acid (42% each) were found. Compounds which would be derived from 18-hydroxyoctadec-9-enoic acid and octadec-9-ene-1, 18-dioic acid by epoxidation, and epoxidation followed by hydration of the epoxide, were also detected in most of the suberin samples. The monomer composition of the six plants showed general similarities but quite clear taxonomic differences.     

Regulation of extracellular acid phosphatase biosynthesis by culture conditions in entomopathogenic fungusMetarhizium anisopliae strain CQMa102

Metarhizium anisopliae is an imperfect entomopathogenic fungus. Once invading into its host,M. anisopliae needs to absorb basic nutrients such as phosphorus from the host haemolymph. A large number of phosphorylated compounds in haemolymph cannot be directly utilised by the fungal cell and must be hydrolysed into available form by phosphatase before ingested. Aims of this paper were to investigate optimum fermentation conditions for production of acid phosphatase and phosphatase isoenzymes byMetarhizium anisopliae. The optimum fermentation conditions were: glucose, 20 g/l; (NH₄)₂SO₄, 2 g/l; casein, 4 g/l; MgSO₄, 0.5 g; KCl, 0.5 g; microelement salt solution, 10 ml; inoculum size, 1×10⁷ spores per 100 ml medium; initial medium pH, 6.0. Under these conditions, the highest total acid phosphatase activity was 3.05 U/ml in 4 days at 27 °C and 160 rpm. Synthesis of the acid phosphatase was repressed by 0.01% inorganic phosphate in culture medium. The spectrum of isoenzymes produced byM. anisopliae varied depending on the phosphorus source employed in the culture. A specific isoform with pI 9.45 was induced by casein, and another isoform of pI 8.21 was induced by phytic acid and disodium phenyl phosphate.