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We tested differences in female pheromone production and male response in three species of the genus Adoxophyes in Korea. Females of all three species produced mixtures of (Z)-9-tetradecenyl acetate (Z9-14:OAc) and (Z)-11-tetradecenyl acetate (Z11-14:OAc) as major components but in quite different ratios. The ratio of Z9-14:OAc and Z11-14:OAc in pheromone gland extracts was estimated to be ca. 100:200 for Adoxophyes honmai, 100:25 for Adoxophyes orana, and 100:4,000 for Adoxophyes sp. Field tests showed that males of each species were preferentially attracted to the two-component blends of Z9-14:OAc and Z11-14:OAc mimicking the blends found in pheromone gland extracts of conspecific females. The effects of minor components identified in gland extracts on trap catches varied with species. Addition of 10-methyldodecyl acetate (10me-12:OAc) or (E)-11-tetradecenyl acetate (E11-14:OAc) to the binary blend of Z9-14:OAc and Z11-14:OAc significantly increased captures of A. honmai males, whereas E11-14:OAc exhibited a strongly antagonistic effect on catches of Adoxophyes sp. males. Moreover, (Z)-9-tetradecen-1-ol (Z9-14:OH) or (Z)-11-tetradecen-1-ol (Z11-14:OH) added to the binary blends increased attraction of male A. orana but not A. honmai and Adoxophyes sp. males, suggesting that these minor components, in addition to the relative ratios of the two major components, play an important role in reproductive isolation between Adoxophyes species in the southern and midwestern Korea where these species occur sympatrically.

To identify novel crystal proteins, Bacillus thuringiensis 2385-1 was isolated from Korean soil samples and characterized. The H-serotype of 2385-1 was identical to that of subsp. kenyae (H4a4c), and its crystal toxin was bipyramidal-shaped. However, 2385-1 showed a much higher toxicity towards Plutella xylostella and Spodoptera exigua larvae than subsp. kenyae. In addition, the crystal protein profile and plasmid DNA pattern of 2385-1 differed from those of subsp. kenyae. To verify the crystal protein gene types of 2385-1, a PCR-RFLP analysis was performed, and the results revealed that 2385-1 contained two novel cry1-type crystal protein genes, cry1-5 and cry1-12, in addition to the cry1Ja1 gene. The deduced amino acid sequences of cry1-5 and cry1-12 showed a 97.9% and 75.7% sequence similarity with the CrylAb and Cry1Ja crystal proteins, respectively. Among the novel crystal proteins, Cry1-5 showed a high toxicity towards P. xylostella and S. exigua larvae. In conclusion, B. thuringiensis 2385-1 is a new isolate in terms of its gene types, and should be a promising source for an insecticide to control lepidopteran larvae.

Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.

Acetylcholinesterase (AChE) has been known to be the target of organophosphorous and carbamate insecticides. Only a single AChE, however, existed in insects and was involved in insecticide resistance, recently another AChE is reported in mosquitoes and aphids. We have cloned cDNAs encoding two ace genes, designated as Ha-ace1 and Ha-ace2 by a combined degenerate PCR and RACE strategy from adult heads of the oriental tobacco budworm, Helicoverpa assulta. The Ha-ace1 and Ha-ace2 genes encode 664 and 647 amino acids, respectively and have conserved motifs including a catalytic triad, a choline-binding site and an acyl pocket. Both Ha-AChEs were determined to be secretory proteins based on the existence of a signal peptide. The Ha-ace1 gene, the first reported ace1 in lepidopterans, belongs to the ace1 subfamily whereas the Ha-ace2 gene showed high similarity to those in the ace2 subfamily. Phylogenetic analysis showed that the Ha-ace1 gene was completely diverged from the Ha-ace2, suggesting that the Ha-ace genes are duplicated. Quantitative real time-PCR revealed that expression level of the Ha-ace1 gene was much higher than that of the Ha-ace2 in all body parts examined. The biochemical properties of purified proteins by affinity chromatography showed substrate specificity for acetylthiocholine iodide, and inhibitor specificity for BW284C51 and eserine and their peptide sequences partially identified by a MALDI-TOF mass spectrometer demonstrated that two Ha-AChEs were expressed in vivo.

Pheromone biosynthesis activating neuropeptide (PBAN) produced in the subesophageal ganglion stimulates pheromone production in the pheromone gland. A cDNA isolated from female adult heads of the diamondback moth (Plutella xylostella (L.)) encodes 193 amino acids including PBAN, designated as Plx-PBAN, and four other neuropeptides (NPs): diapause hormone (DH) homologue, alpha-NP, beta-NP and gamma-NP. All of the peptides are amidated in their C-termini and shared a conserved motif, FXPR(or K)L structure, as reported from other PBAN cDNAs. Plx-PBAN consists of 30 amino acids, the shortest PBAN so far reported. Plx-PBAN exhibited below 50% homology, compared with other known PBANs. The Plx-DH homologue is structurally different from DH of Bombyx mori. The length of Plx-beta-NP (16 amino acids) was the shortest and showed relatively low similarity, whereas gamma-NP (10 amino acids in length) was the longest among examined gamma-NPs. When female adults were injected with synthetic Plx-PBAN, pheromone production showed a maximal increase 1h post-injection. RT-PCR screening revealed that Plx-PBAN cDNA was expressed in all examined body parts, with the highest expression level in the head of female adults. Analysis of RT-PCR products indicated the Plx-PBAN sequence was identical in all examined body parts of both sexes. Phylogenetic analysis revealed that the Plx-PBAN gene is distantly related to other PBANs, demonstrated by the relatively low similarity.

Molecular identification techniques are used where morphological characters are not useful for distinguishing species that resemble each other closely. The example studied here is the Adoxophyes species complex, in which A. orana (Fischer von Rösslerstamm) is officially the only known Korean species in the genus Adoxophyes (Lepidoptera: Tortricidae). However there have been suspicions that at least two types of A. orana exist in Korea based on the distribution and range of the host, with A. orana attacking apples and peaches, and another Adoxophyes sp. attacking tea and pears. The latter is presumed to be A. honmai (Yasuda), but the two have remained confused because of their extreme morphological similarity, despite several Asian studies of pheromonal and morphological characteristics. To confirm the occurrence of an Adoxophyes species other than A. orana in Korea, we compared 940 bp of the mitochondrial cytochrome oxidase I (COI) gene from 16 samples of Adoxophyes and found that there is a second Adoxophyes species different from A. orana. Comparison of the different sequences to that of Japanese A. honmai confirmed that they belong to the latter. From the sequence difference between the two Korean species, we were able to develop new PCR primer sets that distinguish them. This molecular identification technique with no enzyme digestion or sequencing step is a convenient and rapid way of differentiating between species that are hard to distinguish morphologically.

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis Cry(-)B strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the cry1Ac gene (pProAc-GFP). The B. thuringiensis Cry(-)B strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immunoblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single species, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis Cry(-)B strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuringiensis.

Production of sex pheromones, Z9-14:OAc and Z11-14:OAc, of the smaller tea tortrix, Adoxophyes sp. was stimulated by injection of the female or male head extracts as well as synthetic pheromone biosynthesis activating neuropeptide (PBAN) into decapitated females. The amount of pheromone produced reached a maximum level 3 h after injection of synthetic PBAN into females. A cDNA isolated from brain-suboesophageal ganglion complex (Br-SEG) of A. sp. females contained an ORF of 576 nucleotides encoding 192 amino acids. Based on endoproteolytic sites, it can be predicted to be cleaved into five putative peptide domains including PBAN and four other neuropeptides. Ado-PBAN consisting of 31-amino acids is the shortest PBAN so far reported. Four other putative PBAN-encoding gene neuropeptides (PGN) are predicted with PGN-24, PGN-7, PGN-20, and PGN-8 amino acids. All of the peptides are amidated in their C-termini with a FXPR(or I, K)L structure, except for PGN-8 (TVKLTPRLamide). PBAN-like immunoreactive material was observed in Br, SEG and ventral nerve cord (VNC) of the female adult. In the brain, 5-7 pairs of neurons containing PBAN-like immunoreactivity were found in each protocerebral hemisphere. Three groups of cell clusters found in the SEG corresponded to the mandibular, maxillary and labial neurons as in other moths. PBAN-like immunoreactive neurons in the VNC were found in thoracic (three pairs) and abdominal ganglia (two pairs). As compared to other moths, a relatively low similarity of peptide sequences deduced from Ado-PBAN gene and a different expression pattern of PBAN-like immunoreactivity could indicate phylogenetical distance from the other species., (Copyright 2004 Elsevier Ltd.)

Aims: To investigate fusion expression between Bacillus thuringiensis crystal protein and a foreign protein, the expression of a fusion protein comprised of Cry1Ac, and enhanced green fluorescent protein (EGFP) in B. thuringiensis Cry(-)B strain was examined., Methods and Results: The N-terminal fusion expression of EGFP in Cry1Ac was attempted under the control of the native cry1Ac promoter. The EGFP gene was cloned into pProMu and named pProMu-EGFP. The transformant, ProMu-EGFP/CB produced parasporal inclusions that were of bipyramidal-shaped crystals in size ranging from 200 to 300 nm. The fusion protein was approximately 150 kDa and identified by the immunoblot analysis using a Cry1Ac antibody and also a GFP antibody. The LC(50) of the ProMu-EGFP/CB was twofold higher when compared with that by the ProAc/CB. However, the crystal protein produced by the ProMu-EGFP/CB was effective on Plutella xylostella larvae., Conclusions: The ProMu-EGFP/CB produced bipyramidal shaped and insecticidal crystals comprising fusion proteins., Significance and Impact of the Study: Through the N-terminal fusion expression of EGFP and Cry1Ac, expression and crystallization between the B. thuringiensis crystal protein and a foreign protein were validated.

Insect acetylcholinesterase (AChE) is known to be a primary target of organophosphorus and carbamate insecticides. However chronic exposure to these chemicals has led to resistance to applied insecticides, due usually to mutation of the AChE gene. Analysis of the AChE gene (hm) of Musca domestica (the housefly), which is cloned in this report, reveals the relationship between mutation and insecticide resistance. The 2,076 bp hm encodes a mature protein of 612 amino acids (67 kDa), and an 80 residue signal peptide. Unlike the enzyme of 'sensitive' strains, the AChE used in this study was resistant to the organophosphorus insecticide, trichlorphon. DNA sequencing showed that this AChE is identical to that of the sensitive strains with the exception of three amino acids Met-82, Ala-262, and Tyr-327. Site-directed mutagenesis of the Ala-262 and Tyr-327 residues largely restored sensitivity to the insecticide, suggesting that these two residues are the key structural elements controlling sensitivity. In addition to these residues, Glu-234 and Ala-236 in the conserved sequence FGESAG are thought to play a role in modulating sensitivity to organophosphorus insecticides.

The sex pheromone of the American population of Lyonetia prunifoliella (Lepidoptera: Lyonetiidae) was reported to be composed of three components, 10,14-dimethyloctadec-1-ene (10me14me-1-ene-18Hy), 5,9-dimethyloctadecane (5me9me-18Hy), and 5,9-dimethylheptadecane (5me9me-17Hy). Studies with a Korean population showed that the 10S, 14S isomer of the major compound was the major electrophysiologically by active stereoisomer. The 5S,9S isomers of the minar constituents also elicited electrophysiological responses. In field trappings, among the three components, the racemic mixture of 10me14me-1-ene-18Hy attracted L. prunifoliella males strongly, and the binary or tertiary combinations with racemic mixtures of the other two components did not increase trap catches. Among the four stereoisomers of 10me14me-1-ene-18Hy, only the 10S,14S isomer attracted L. prunifoliella males, as suggested by the EAG test, and attraction to the other three isomers was not observed. All stereoisomers of each minor component, as well as their racemic components, did not seem to have any additional effect on male attraction in Korea.

Sex pheromones of many Lepidopteran species have relatively simple structures consisting of a hydrocarbon chain with a functional group and usually one to several double bonds. The sex pheromones are usually derived from fatty acids through a specific biosynthetic pathway. We investigated the incorporation of deuterium-labeled palmitic and stearic acid precursors into pheromone components of Helicoverpa zea and Helicoverpa assulta. The major pheromone component for H. zea is (Z)11-hexadecenal (Z11-16:Ald) while H. assulta utilizes (Z)9-hexadecenal (Z9-16:Ald). We found that H. zea uses palmitic acid to form Z11-16:Ald via delta 11 desaturation and reduction, but also requires stearic acid to biosynthesize the minor pheromone components Z9-16:Ald and Z7-16:Ald. The Z9-16:Ald is produced by delta 11 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. The Z7-16:Ald is produced by delta 9 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. H. assulta uses palmitic acid as a substrate to form Z9-16:Ald, Z11-16:Ald and 16:Ald. The amount of labeling indicated that the delta 9 desaturase is the major desaturase present in the pheromone gland cells of H. assulta; whereas, the delta 11 desaturase is the major desaturase in pheromone glands of H. zea. It also appears that H. assulta lacks chain-shortening enzymes since stearic acid did not label any of the 16-carbon aldehydes.

A strain of Bacillus thuringiensis that showed significantly high toxicity to Plutella xylostella and Spodoptera exigua was isolated from a Korean soil sample and characterized. The isolate, named B. thuringiensis K1, was determined to belong to ssp. kurstaki (H3a3b3c) type by an H antisera agglutination test and produced bipyramidal inclusions. Plasmid pattern of K1 was different from that of the reference strain, ssp. kurstaki HD-1, but the parasporal inclusion protein profile of K1 had two major bands that were similar in size to those of ssp. kurstaki HD-1. To verify the delta-endotoxin gene types of K1, PCR analysis with specific cry gene primers was performed to show that K1 contained a new cry gene in addition to cry1Aa, cry1Ab, cry1Ac, cry1E and cry2 genes. PCR-amplified region of the new cry gene, cryX, showed 79% similarity to cry1Fa1 gene (GenBank Accession No. M63897). In an insect toxicity assay, K1 had higher toxicity against Plutella xylostella and S. exigua than ssp. kurstaki HD-1.

The enantiomerically pure diastereoisomers (1R,4S,4aR,7S,7aR)- (1) and (1R,4R,4aR,7S,7aR)-dihydronepetalactol (2) were synthesized diastereoselectively from a renewable resource, (4aS,7S,7aR)-nepetalactone (3), isolated as the main constituent of the essential oil of the catmint plant Nepeta cataria. The stereochemistry of the compounds was determined by NMR spectroscopy and X-ray crystallography, and the compounds were identified, respectively, as neomatatabiol and isoneomatatabiol, natural products from Actinidia polygama, for which the lactol stereochemistry was previously incompletely defined. Compound 1 was found to catch significant numbers of three species of lacewing in the field: in Korea. Chrysopa cognata, and in the United Kingdom, Nineta vittata and most notably Peverimhoffina gracilis. All species caught in significant numbers were found more frequently in traps releasing 1 than 2, while more C. cognata, C. formosa, and C. phyllochroma were found in traps releasing (1R,4aS,7S,7aR)-nepetalactol (4). The catch of P. gracilis with 1 is of particular interest as this lacewing has only recently been recorded in the United Kingdom. Where sexed, the lacewings of all species trapped were found to be male, implying a possible pheromonal role for these or structurally related compounds.

A strain of Bacillus thuringiensis with dual toxicity was isolated from Korean soil samples and named K2. K2 was determined as ssp. kurstaki (H3a3b3c) by serological test and produced bipyramidal-shaped parasporal inclusions. The plasmid and protein profiles of B. thuringiensis K2 were different from those of the reference strain, ssp. kurstaki HD-1. To verify gene type of B. thuringiensis K2, PCR analysis with specific cry gene primers was performed. The result showed that B. thuringiensis K2 had cry1Aa, cry1Ab, cry1C, and cry1D type genes, whereas ssp. kurstaki HD-1 had cry1Aa, cry1Ab, cry1Ac, and cry2 type genes. In addition, B. thuringiensis K2 had high toxicity against Spodoptera exigua and Culex pipiens, whereas B. thuringiensis ssp. kurstaki HD-1 does not have high toxicity against these two insect species.

To study the structural features of genes for luciferase in Luciola lateralis, two different, functional luciferase genes, Luc1 and Luc2, were cloned and their nucleotide sequences were determined. The two genes were nearly identical, but a deletion of 128 bp was found in the 5' upstream region of Luc2, and minor deletions and additions were also found in the 3' downstream region. Seven base substitutions in the coding region produced two amino acid residue differences between the two genes. Southern blot analysis of genomic DNA isolated from 33 individual fireflies revealed that 26 were homozygous for the Luc1 gene and one was homozygous for Luc2. The others were heterozygotes with two, or even three, different alleles for the luciferase gene.

The present study is concerned with cloning and characterizing Has-PBAN cDNA which is 756 nucleotides long, isolated from the brain and suboesophageal ganglion complex (Br-Sg) of Helicoverpa assulta adults. The 194-amino acid sequence deduced from this cDNA possessed the proteolytic endocleavage sites to generate multiple peptides. From the processing of the prepro-hormone, it can be predicted that the cDNA has a PBAN domain with 33 amino acids and four additional peptide domains: 24 amino acid-, 7 amino acid-, 18 amino acid- and 8 amino acid-long sequences, with FXPR (or K) L (X = G, T or S) amidated at their C-termini. The amino acid sequence of all five predicted peptides, including the PBAN, are identical to that of Helicoverpa zea (Raina, A.K., Jaffe, H., Kempe, T.G., Keim, P., Blacher, R.W., Fales, H.M., Riley, C.T., Klun, J.A., Ridgway, R.L., Hayes, D.K., 1989. Identification of a neuropeptide hormone that regulates sex pheromone production in female moths. Science 244, 796-798 and Ma, P.W.K., Knipple, D.C., Roelofs, W.L., 1994. Structural organization of the Helicoverpa zea gene encoding the precursor protein for pheromone biosynthesis-activating neuropeptide and other neuropeptides. Proc. Natl. Acad. Sci., U.S.A. 91, 506-510). A single mRNA species corresponding to the size of Has-PBAN cDNA was detected from the Br-Sg of 1-3-day old female and male adults, and their expression was also at a similar level. Pheromone production was induced upon injection of female or male Br-Sg extracts or synthetic PBAN into the haemocoel of decapitated 1-3-day old female adults during the photophase when they are not supposed to produce pheromone. From these results, H. assulta adult females seem to use their own PBAN for regulating sex pheromone biosynthesis. Functions of the four other peptides ending with FXPR (or K) L in the Has-PBAN cDNA and of the male PBAN remain to be elucidated.

A proteinous antimicrobial substance was purified from the bacteria-challenged larvae of the fall webworm, Hyphantria cunea. It is a cecropin-like antibacterial peptide which exhibits antibacterial activity against Gram-negative and Gram-positive bacteria, and known as Hyphantria cecropin A. The cDNA clones corresponding to this peptide were isolated from a cDNA library constructed from the bacteria-challenged larvae and obtained complete nucleotide sequences. In addition to the Hyphantria cecropin A sequence, we obtained three other cDNAs exhibiting high sequence similarity with Hyphantria cecropin A. We synthesized the C-terminally amidated peptide of 35 residues based on the deduced sequence of the isolated cDNA of Hyphantria cecropin A. The synthetic peptide exhibited strong antibacterial activity against several microbes including medically important bacteria such as Salmonella, Shigella, and fungus such as Candida. A Southern blot experiment using these cloned cDNAs as probes predicted the existence of multiple forms of Hyphantria cecropin genes.

Solvent extracts of individual pheromone glands were prepared from femaleHelicoverpa assulta (Guenée) at 2-hr intervals throughout the scotophase. The amounts of female sex pheromone components, (Z)-9-hexadecenal, (Z)-11-hexadecenal, (Z)-9-hexadecenyl acetate, and (Z)-11-hexadecenyl acetate, in the extracts were determined by gas chromatographic analysis. Although females called from early scotophase (2 hr) until late scotophase (6 hr) the quantity of extracted pheromone remained high at 8 hr, the end of the scotophase. More than 70% of the pheromone gland extracts contained sex pheromone components regardless of whether the donor females had been called or resting. Pheromone components were absent from gland extracts prepared at the onset of the scotophase. The quantity of (Z)-9-hexadecenal and (Z)-11-hexadecenal increased rapidly to reach a maximum of approximately 260 and 30 ng/female, respectively, that was maintained for up to 8 hr, the duration of the scotophase. The quantity of (Z)-9-hexadecenyl acetate and (Z)-11-hexadecenyl acetate increased continuously during the scotophase to peak at 600 and 30 ng/female, respectively, 8 hr into the scotophase. At the end of scotophase the quantity of all pheromone components decreased significantly.

Analysis of ovipositor washings from virgin femaleHelicoverpa assulta (Guenée) (Lepidoptere: Noctuidae) from Korea by gas chromatography (GC) linked to electroantennography and GC linked to mass spectrometry resulted in the identification of nine compounds, hexadecanal, (Z)-9-hexadecenal, (Z)-11-hexadecenal, hexadecyl acetate, (Z)-9-hexadecenyl acetate, (Z)-11-hexadecenyl acetate, hexadecan-l-ol, (Z)-9-hexadecen-l-ol, and (Z)-11-hexadecen-1-ol. However, ovipositor washings from females from Thailand contained mainly the 16-carbon aldehydes with very small amounts of (Z)-9-hexadecenyl acetate. Field tests conducted in Korea, China, and Thailand indicated that a binary blend of (Z)-9-hexadecenal and (Z)-11-hexadecenal was sufficient for attraction, although the most attractive ratio of compounds varied with location. In Korea a 20∶1 blend of compounds was the most attractive, while in Thailand a 7.5∶1 blend was most attractive. In China both blends of hexadecenal isomers were equally attractive. Addition of the hexadecenyl acetates to the 20∶1 blend of hexadecenals in the ratio of 1∶3.3 increased the trap catch of maleH. assulta compared to lures containing the aldehydes alone in Korea but reduced trap catch in China. Addition of the hexadecenyl acetates to the 7.5∶1 blend of hexadecenals had no significant effect on trap catch in Thailand or China compared to the aldehydes alone. The addition of the 16-carbon alcohols to the aldehydes had a significantly inhibitory effect in all three countries, suggesting they are not pheromone components. Taken together these results indicate thatH. assulta is polymorphic with at least two populations responding to different sex pheromones.

The fine structure of the sensory hairs on the antennal flagellum of female Anopheles stephensi was studied by electron microscopy. The antennal hairs were divided into at least five different types, on the basis of hair diameter, hair wall thickness, communicating channel density, and dendritic branching in the hair lumen. Regardless of different types, however, all hairs, presumed to be olfactory receptors, were found to be innervated by two sensory cells.

Hemocytometer and cytofluorometric methods were used to count trypanosomes in the blood of neonatal and six-month-old calves infected with T. congolense. The correlation between these methods was highest in the six-month-old calves because their parasitemia was greater and the accuracy of cytofluorometric counting is reduced below 1,000 trypanosomes per microliter of blood. Fine needle aspiration biopsies showed many trypanosomes marginated in cerebral cortical capillaries, but few in muscle or liver. Quantitatively, the cerebral capillaries in the infected six-month-old calves were significantly wider than their controls with fewer endothelial nuclei suggesting capillary dilatation. There were fewer trypanosomes and no significant changes in capillary width in the neonatal calves. Several drugs were tried for activity in dispersing marginated trypanosomes into axial flow. Berenil and dexamethasone had a significant effect on trypanosome counts in blood and tissue whereas, epinephrine caused a fleeting increase in parasitemia. Hetastarch, dextran and cyclophosphamide had no specific effect. Fine needle aspiration bipsies of brain, thirty minutes after Berenil injection showed that the capillaries were cleared of trypanosomes.

It was shown by electron microscopy that antennae of male Anopheles stephensi have the same types of sensory receptors as the females, with sunken pegs (sensilla ampullacea and sensilla coeloconica), surface pegs (sensilla basiconica), hairs (sensilla trichodea), bristles and fibrillae (sensilla chaetica), and scolopidia in the Johnston's organ. Compared with the female, the male mosquito has fewer than half of the sensory cells in the flagellum, but many more in the Johnston's organ. The smaller number of flagellar sensory neurons in the male was due mainly to fewer sensory receptors of possibly olfactory types. Sensory cells of mechano- and thermoreceptors were present in almost identical numbers in both sexes, as only about 15% of the long fibrillae, abundant as whorls in the male mosquito, are innervated by a single neuron.