冬青油与肉桂油对淡色库蚊的生物活性

为进一步明确冬青油（wintergreen oil）与肉桂油（cinnamon oil）对蚊虫的生物活性, 本研究采用浸液法、 “Y”型嗅觉仪法和密闭三角瓶熏蒸法, 分别测定了两种精油对淡色库蚊Culex pipiens pallens幼虫的毒杀作用以及对成虫的驱避和熏杀活性。结果显示： 肉桂油和冬青油对淡色库蚊幼虫均具有较强的毒杀作用, 处理24 h的LC₅₀分别为71.87 mg/L和 102.83 mg/L。在0.5 μL的供试剂量下, 冬青油与肉桂油在20 min内对淡色库蚊的驱避率均在80%以上, 显示了较好的驱蚊活性。冬青油对淡色库蚊具有明显的熏蒸击倒作用, 12 μL/L浓度下的KT₅₀为3.97 min; 而肉桂油则具有较好的熏蒸致死活性, 熏蒸5 h的LC₅₀ 为0.31 μL/L。冬青油与肉桂油按1∶1体积比混配后对淡色库蚊表现出较好的击倒和致死效果。冬青油和肉桂油既对淡色库蚊幼虫具有较强的毒杀作用, 又对淡色库蚊雌成虫表现出较好的驱避和熏杀活性, 具有开发成为植物源蚊虫防控剂的潜力。     

尖音库蚊复合组杂交子一代4龄幼虫形态性状的研究

对我国尖音库蚊复合组（Culex pipiens complex）中的尖音库蚊Cx. Pipiens pipiens、淡色库蚊Cx. Pipiens pallens和致倦库蚊Cx. Pipiens quinquefasciatus之间杂交的F₁代幼虫形态进行了方差和典型变量分析,结果发现杂交组F₁代4龄幼虫在呼吸管指数、1-S第1对毛和第2对毛的分枝数3个性状上与亲本有明显的差异,而且介于两亲本之间。     

Carotenoid-based colour polyphenism in a moth species: search for fitness correlates

Carotenoid‐based integumental coloration is often associated with individual performance in various animals. This is because the limited amount of the pigment has to be allocated to different vital functions. However, most of the evidence for the carotenoid‐based trade‐off comes from vertebrate studies, and it is unclear if this principle can be applied to insects. This possibility was investigated in Orgyia antiqua L. (Lepidoptera: Lymantriidae). The larvae of this species are polyphenic in their coloration, varying from a highly conspicuous combination of yellow hair tufts on black background to cryptic appearance with brown hair tufts. The conspicuous larvae are aposematic, advertising their aversive hairiness. The maintenance of different colour morphs in O. antiqua requires explanation, as an aposematic signal is expected to evolve towards monomorphism. Chromatographic analysis showed that the yellow coloration of the hair is based on the carotenoid pigment lutein (α‐carotene‐3,3’‐diol). The colour of hair tufts was dependent on their carotenoid content. This justifies an expectation of carotenoid‐based physiological trade‐offs between aposematic coloration and individual performance. To test this hypothesis, we monitored life histories of differently coloured larvae reared on various host plants, recording their body sizes, growth rates, and mortalities in each instar. There was a significant but relatively low heritability of tuft coloration, which allowed us to expect environmental effects. We found no phenotypic associations between hair tuft colour and performance indices in O. antiqua larvae, neither did the quality of host plant affect the frequency of colour morphs. However, the frequency of colour morphs differed between larval instars. Our results suggest that carotenoid‐mediated physiological trade‐offs are not involved in the maintenance of colour morphs in O. antiqua larvae, and factors other than individual condition should be responsible for the observed variability.     

The Use of Papain in Clearing Plant Tissues for Whole Mounts

Materials killed and fixed in FAA (formalin-acetic acid-alcohol) and similar fixatives frequently are difficult to clear for whole mounts because the denatured proteins will not become soluble in NaOH and other clearing agents. If tissues are washed for 3 days in running water, then incubated at 40 C for 5-7 days in 2% papain buffered to pH 7.2 and activated with 15 ml of .02 M Na₂S, cell contents are partly digested. Normal clearing with 5-10% NaOH followed by chloral hydrate (sat. aq.) can then effect complete solublility of cell contents and their removal. Permanent slides can be made after staining (1% safranin O in 50% alcohol for 12 hr is successful), by dehydration through alcohols, clearing in xylene, and mounting in resin.     

A Rapid Technic for Demonstrating mast cells in Mouse Skin

A simple rapid technic is described for demonstrating mast cells in mouse skin. The procedure requires about 60 minutes from time of specimen removal until permanent mounting. The steps comprise: (1) stretch-mounting of skin on a cardboard frame; (2) fixing and dehydrating in absolute ethanol for 15 minutes; (3) xylene washing for 10 minutes; (4) absolute ethanol washing for 10-15 minutes; (5) 3-4 minutes in a 0.1% aqueous solution of methylene blue; (6) dehydrating and differentiating in absolute ethanol; (7) clearing in xylene; (8) trimming and mounting. Cell counts may be made immediately, as well as high dry and oil immersion study of cytological detail of mast cells.     

A Rapid Technic for Demonstrating mast cells in Mouse Skin

A simple rapid technic is described for demonstrating mast cells in mouse skin. The procedure requires about 60 minutes from time of specimen removal until permanent mounting. The steps comprise: (1) stretch-mounting of skin on a cardboard frame; (2) fixing and dehydrating in absolute ethanol for 15 minutes; (3) xylene washing for 10 minutes; (4) absolute ethanol washing for 10-15 minutes; (5) 3-4 minutes in a 0.1% aqueous solution of methylene blue; (6) dehydrating and differentiating in absolute ethanol; (7) clearing in xylene; (8) trimming and mounting. Cell counts may be made immediately, as well as high dry and oil immersion study of cytological detail of mast cells.     

Alcoholic Thionin Counterstaining Following Golgi Dichromate-Silver Impregnation

Brains of cats that had been fixed 2 months or longer in 10% formalin were cut into 3-6 mm. slices and impregnated by Golgi's dichromate-silver procedure (6% dichromate solution, 4-6 days; 1.5% silver nitrate solution 2 days). Sections 100 µ thick were cut after embedding in low melting point paraffin. Three changes of xylene and three of absolute alcohol were followed by staining 3-5 minutes in a saturated solution of thionin in absolute alcohol. The sections were dipped quickly in absolute alcohol and cleared in xylene, then differentiation was effected by an equal-parts mixture of absolute alcohol and xylene. A final clearing in three changes of xylene and mounting in Permount completed the process. Counter-staining was most successful when applied to freshly cut sections.     

The lizard ear: Cordylus, Platysaurus and Gerrhosaurus

In further consideration of the lizard ear, the fine structure of the cochlea has been investigated and related to auditory sensitivity in members of the family Cordylidae. The ear of this group of lizards is unusual in that a tectorial membrane is present only in a modified and seemingly vestigial form, and this membrane makes no connections with the auditory hair cells. These cells are provided instead with a series of sallets, small bodies extending in a single row through the dorsal and middle regions of the cochlea, where they rest upon the tips of the ciliary tufts and evidently bring about a stimulation of the hair cells because of their inertia. At the ventral end of the cochlea this line of sallets ends, and here is a single, relatively enormous structure, the culmen papillae, that serves a similar purpose for a large group of hair cells. Consideration is given to the manner of stimulation of the auditory sense cells in these species in relation to others with the usual arrangements involving connections between the ciliary tufts and a tectorial membrane. Included also is a study of a species of Gerrhosaurus, which some have included in the cordylid family and others have placed in a family of its own. The cochlear structure in this species is similar to that of the cordylids in many respects but differs in the ventral region, where instead of the culmen there is a heavy tectorial plate, similarly covering a large number of hair cells but connected to a tectorial membrane. The functioning of these ears is assessed in terms of the cochlear potentials, and is found to vary with species from better than average to excellent in comparison with other lizards investigated. The structural differentiation also is of fairly high degree, and hence it appears that ears without tectorial connections, or with such connections only in a limited region of the cochlea, can perform in a highly serviceable manner.     

An Analysis of Methods for Permanently Mounting Ovules Cleared in Four-and-a-Half Type Clearing Fluids

Ovules cleared in benzyl benzoate-4 1/2 clearing fluid can be permanently mounted in Piccolyte or Permount by replacing the clearing fluid with absolute ethanol, upgrading the ovules in mixtures of ethanol and xylene (3:1, 2:2, 1:3, and xylene), and mounting them in either mountant under the supported coverglass of a Raj slide. Optical sagittal sections through the ovules resemble microtome sections in that the protoplasts are slightly shrunken away from the cell walls. The artifact is common in permanently mounted sections; fixation and paraffin infiltration are usually cited as the causes—its appearance in the whole-mounted ovules is caused by xylene. Although miscible with the clearing fluid, Euparal is the least satisfactory of the standard mountants for permanent preparations of cleared ovules and is best used with an equal quantity of clearing fluid for semipermanent preparations. A large quantity of Euparal in the mountant produces pronounced shrinkage. A method for permanently mounting cleared ovules with the clearing image unaltered employs a mountant which contains the ingredients of Spurr low viscosity embedding medium. Vinylcyclohexene dioxide (10 drops) is combined with diglycidyl ether of polypropylglycol (6 drops) and nonenyl succinic anhydride (26 drops). Ovules treated for 24 hr in benzyl benzoate-4 1/2 clearing fluid are passed through a graded series of clearing fluid-epoxy medium mixtures (3:1, 2:2, 1:3, and pure epoxy medium) at intervals of 15 minutes. One drop of dimethylaminoethanol, the cure accelerator, is then added to the epoxy medium and the ovules are mounted and covered immediately on a Raj slide. The preparation is cured in an oven at 60 C for 24 hr and observed with phase contrast or Nomarski interference optics.     

An analysis of methods for permanently mounting ovules cleared in four-and-a-half type clearing fluids

Ovules cleared in benzyl benzoate-4 1/2 clearing fluid can be permanently mounted in Piccolyte or Permount by replacing the cleaning fluid with absolute ethanol, upgrading the ovules in mixtures of ethanol and xylene (3:1, 2:2, 1:3, and xylene), and mounting them in either mountant under the supported coverglass of a Raj slide. Optical saggittal sections through the ovules resemble microtome sections in that the protoplasts are slightly shrunken away from the cell walls. The artifact is common in permanently mounted sections; fixation and paraffin infiltration are usually cited as the causes--its appearance in the whole-mounted ovules is caused by xylene. Although miscible with the clearing fluid, Euparal is the least satisfactory of the standard mountants for permanent preparations of cleared ovules and is best used with an equal quantity of clearing fluid for semipermanent preparations. A large quantity of Euparal in the mountant produces pronounced shrinkage. A method for permanently mounting cleared ovules with the clearing image unaltered employs a mountant which contains the ingredients of Spurr low viscosity embedding medium. Vinylcyclohexene dioxide (10 drops) is combined with diglycidyl ether of polypropylglycol (6 drops) and nonenyl succinic anhydride (26 drops). Ovules treated for 24 hr in benzyl benzoate-4 1/2 clearing fluid are passed through a graded series of clearing fluid-epoxy medium mixtures (3:1, 2:2, 1:3, and pure epoxy medium) at intervals of 14 minutes. One drop of dimethylaminoethanol, the cure accelerator, is then added to the epoxy medium and the ovules are mounted and covered immediately on a Raj slide. The preparation is cured in an oven at 60 C for 24 hr and observed with phase contrast or Nomarski interference optics.     

Pelagic eggs and larvae of Coelorinchus kishinouyei (Gadiformes: Macrouridae) collected from Suruga Bay, Japan

Pelagic eggs and larvae of the macrourid fish Coelorinchus kishinouyei, collected from Suruga Bay, southern Japan and subsequently identified by 16S rRNA gene nucleotide sequences, are described. The spherical eggs, 1.18–1.31 mm in diameter, contained a single oil globule, 0.28–0.33 mm in diameter, and had hexagonally patterned ornamentation on the chorion, 0.017–0.022 mm in width. Melanophores were present on the embryo, yolk and oil globule after the blastopore had closed. Within 1 day after hatching, the body axis of the yolk-sac larvae was bent slightly at the anterior trunk region. During this stage many melanophores formed on the head, trunk, tail, yolk and oil globule, along with small irregular wrinkles on the dorsal and ventral finfolds. Pelagic eggs (after the caudal end of the embryo had detached from the yolk) and yolk-sac larvae also developed xanthophores on the embryo and yolk, and head, trunk, dorsal and ventral finfolds just before tail tip, and yolk, respectively. The pelagic larvae had a short tail, stalked pectoral-fin base and no elongate first dorsal and pelvic-fin rays. Three clusters of melanophores were present on the tail (anterior two embedded to muscle and one just before tail tip subsequently lost with development) and a cluster around the anus (beyond 3.9 mm head length). Nucleotide sequence analyses of comparative adult specimens appeared to confirm a previous proposal that C. productus is a junior synonym of C. anatirostris.     

Protocol for quantitative shape analysis of deformities in early larval European seabass Dicentrarchus labrax

This study established an optimized protocol for quantifying the shape variation of newly hatched larvae of European seabass Dicentrarchus labrax, focusing on the effect of fixatives and mounting on body shape from hatching until 14 days post hatching, while also minimizing the error introduced by handling. This assessment was performed based on both biometric and geometric shape data, with the latter relying on outline based elliptic Fourier analysis. The fixation and mounting effect on the total length and shape of newly hatched larvae of D. labrax was tested for four fixation treatments: (1) 8% formalin, (2) 70% ethanol, (3) 8% formalin for 48 h and then to 70% ethanol and (4) 3% phosphate‐buffered glutaraldehyde. The analyses showed that no significant size and shape effect was observed on anaesthetized specimens 5 months post‐fixation in glutaraldehyde, and that the glycerol mounting process of specimens fixed in glutaraldehyde provided the best results for further ontogenetic qualitative and quantitative analysis. The protocol proved to be sufficiently sensitive to even quantify and visualize subtle pre‐fixation shape differences originating from a different egg batch.     

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.     

Replacing xylene with n-heptane for paraffin embedding

Abstract

In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.     

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.     

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent.

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.     

Blood feeding of Ornithodoros turicata larvae using an artificial membrane system

An artificial membrane system was adapted to feed Ornithodoros turicata (Ixodida: Argasidae) larvae from a laboratory colony using defibrinated swine blood. Aspects related to larval feeding and moulting to the first nymphal instar were evaluated. A total of 55.6% of all larvae exposed to the artificial membrane in two experimental groups fed to repletion and 98.0% of all fed larvae moulted. Mortality rates of first instar nymphs differed significantly depending on the sorting tools used to handle engorged larvae (χ² = 35.578, P < 0.0001): engorged larvae handled with featherweight forceps showed significantly higher mortality (odds ratio = 4.441) than those handled with a camel‐hair brush. Differences in the physical properties of the forceps and camel‐hair brush may affect the viability of fragile soft tick larvae even when care and the same technique are used to sort them during experimental manipulations. The current results represent those of the first study to quantify successful feeding to repletion, moulting and post‐moulting mortality rates in O. turicata larvae using an artificial membrane feeding system. Applications of the artificial membrane feeding system to fill gaps in current knowledge of soft tick biology and the study of soft tick–pathogen interactions are discussed.     

Conventional xylene and xylene-free methods for routine histopathological preparation of tissue sections

Abstract

Xylene customarily has been used as a clearing agent for routine tissue processing. Because xylene is a relatively hazardous solvent, laboratories are under pressure to seek less toxic alternatives for routine use. We prepared 30 paired soft tissue specimens for routine histopathological evaluation using conventional xylene and xylene-free methods to evaluate and compare their efficacy for fixation, processing, embedding, staining and turnaround time. All specimens were measured before and after processing. Three pathologists evaluated and scored the histological sections. Tissue shrinkage was greater when using the xylene method compared to the xylene-free method. The quality of tissue sections including tissue architecture; quality of staining; preservation of epithelial, fibrous, glandular, muscle and adipose tissue; inflammatory cells; and vascular tissue was better after using the xylene method, but differences were not statistically significant. Xylene-free method produced adequate results that nearly equaled the xylene method. Added advantages included cost effectiveness, better working atmosphere and decreased toxicity.     

Use of a Silicone Rubber (Clear Seal) as a Section Adhesive Resistant to Acid, Alkali and Heat

An optically clear silicone rubber adhesive is recommended for use in histochemical procedures in which detachment of tissue sections is likely. Procedure: Cut paraffin sections and float on a 45-50 C water bath; leave frozen sections on the microtome knife in the cryostat; spread the silicone rubber thinly and evenly over 2/3 of the slide (Clear Seal—General Electric, was used); pick up paraffin sections directly from the floatation water and frozen sections from the microtome knife with a warm slide; dry for 1.5 hr at 25 C; place paraffin sections in a 60 C oven for 0.5 hr, deparaffinize through xylene and hydrate through alcohols to water. Stain sections as desired, but avoid clearing agents before mounting after strong acid or alkaline treatment, and mount rapidly if a synthetic resin is used because of the solvent effect on the silicone rubber. Of the adhesives tried, silicone rubber is the only one capable of withstanding boiling 10% HCl for any period of time without detachment of sections.     

A novel xylene substitute for histotechnology and histochemistry

Abstract

Propylene glycol methyl ether (PGME) exhibits excellent solvent and coupling properties. A toxicity database provided evidence suggesting that PGME might be a useful substitute for xylene in histotechnology and histochemistry applications. Tissue specimens were fixed, cleared in either PGME or xylene, embedded in paraffin wax, then dewaxed in either PGME or xylene. Sections were treated with the following stains: hematoxylin & eosin (H & E), three special stains of the Gordon/Sweet silver staining method, PAS, and Masson's trichrome, and immunostains including actin, CD3, CD34, CK, CK7/CK9, Ki-67, and ER/PR. The sections were mounted in a resinous medium consisting of PGME and pinene copolymer, then examined under a microscope. Variables such as water tolerance, dimension change, organic solvency, and anti-fading efficacy also were assessed. Depending on the application, PGME performance was equal to or exceeded that of xylene. PGME provided better optical clarity and nuclear detail, did not harden the tissue samples, conserved tissue antigenicity, and was amenable to resinous mounting. Tissues not dehydrated with absolute ethanol also were processed properly. Tissues treated with PGME did not warp or contract compared to those treated with xylene (p < 0.0001). PGME, however, exhibited less organic solvency than xylene. There was no discernible change in the colors of stains in sections processed with PGME even after storage for two years. These results suggest that PGME is a novel xylene substitute for applications in histotechnology and histochemistry.