Artificial mating ability of
Throughout the world, 528 species of
So far, at least 1 and 2 traditional techniques have been used widely for the recognition of sibling species and/or subspecies members at post- and pre-mating barriers. For post-mating barriers; the hybridization or crossing experiment, using the artificial mating technique to determine hybrid non-viability, sterility or breakdown, is still a useful tool for recognizing
Regarding the modernized technique; molecular investigation of some specific genomic markers, e.g., ribosomal DNA (ITS2, D2, D3, IGS) and mitochondrial DNA (COI, COII, Cyt b, ND5), has been used extensively as a tool to characterize and/or diagnose cryptic members in the intra-taxa of
2. Formation of robust systematic procedures
In light of the advantages and disadvantages of the techniques mentioned above, 3 techniques, i.e., the crossing experiment, molecular investigation and cytogenetic markers (characteristics of metaphase karyotypes) were selected, and they formed the robust systematic procedures for the recognition of
By following the flow chart: (1) try to collect anopheline mosquitoes that are distinct in their behavior (e.g., biting humans or animals with relation to different microhabitats and/or locations), (2) try to record morphological variation(s) as far as possible during the species identification process of wild-caught females, (3) establish an iso-female line colony by allowing gravid females to lay eggs individually, (4) conduct molecular investigation of laid-egg feral females to obtained a robust DNA marker, with this step usually taking about 1 week. Since development of the F1-progeny usually takes about 2 weeks from first instar larvae to adults, the metaphase karyotype investigation of fourth instar larvae, newly emerged adult females and males is performed in order to (5) obtain a cytogenetic marker (karyotypic form), (6) if molecular investigation fails in the step of laid-egg feral female it will be performed in F1-progeny, (7) carry out morphometric and morphological investigations of eggs, larvae, pupal skins and adults to confirm precise species identification, and (8) perform the important step of crossing experiments among iso-female line colonies by using a karyotypic marker (or form) related to a DNA marker (large sequence divergence or very low intraspecific sequence variation) of each iso-female line colony.
Regarding techniques necessary for success in operating robust systematic procedure: 3 important techniques were developed by the authors, and they have been proven as efficient and necessary for the robust systematic recognition of sibling species and/or subspecies members within the taxon
3. Techniques for establishment of a healthy iso-female line colony of difficult-to-rear anophelines
An iso-female line colony of
4.1. Transportation of wild-caught anophelines
Wild-caught fully engorged adult females collected from human- and/or animal-baited traps in the field were kept in a plastic cup (8.5 cm in diameter and 11 cm in depth, lined inside with filter paper), with a pad of cotton wool soaked with 10% sucrose solution placed on top of the covering screen. It was covered with a translucent plastic bag in order to keep humid conditions in the cup and delay rapid drying of the soaked cotton wool (Figure 2a). It was stored in a humid chamber using a picnic foam-box (18 x 26 x 39 cm) to maintain humidity and temperature (Figure 2b). Then it was transported to the insectarium for colonization and biological studies. All of the experiments were performed in the insectarium at 27±2 ºC, 70-80% relative humidity, and illumination from a combination of natural daylight from a glass-window and fluorescent lighting was provided for approximately 12 hours a day.
4.2. Egg laying
After the engorged adult female was maintained for 4-5 days and/or until gravid in the insectarium, it was placed in a screen-topped oviposition plastic-cup (6 cm in diameter and 7 cm in depth) containing 25 ml of natural water (brought from a basin that was used for tap-water production). Wet filter paper lined the inside of the screen-topped was covered with a black plastic sheet (Figure 3a-c). The eggs attached to the moist side of the filter paper and/or floating on the water surface were rinsed and transferred to white plastic tray (25 x 36 x 6 cm) containing 1,500 ml rearing water (equal part of natural water and distilled water) with wet filter paper lining the inside. During the embryonation period, the eggs were exposed to a 40-watt light instead of sunlight, for warming the eggs until hatching (Figure 3d).
4.3. Rearing of larvae, pupae and adults
After egg hatching, first instar larvae were transferred daily from an ovipot to a white plastic tray (25 x 36 x 6 cm) containing 2,000 ml rearing water and approximately 15 stems of garden grass (
4.4. Suitable blood-feeding condition
Comparative direct feeding ability on white rat in a 30 x 30 x 30 cm cage, and artificial feeding ability on human heparinized-blood (obtained from human volunteers whom sign the consent form) in a plastic cup (8.5 cm in diameter and 11 cm in depth, lined inside with filter paper) (Figure 5), of female
4.5. Ability of free mating in a 30 cm cubed cage and male ability to mate artificially
One of the difficulties in the colonization of anopheline mosquitoes in the laboratory might be due to adults not being capable of copulation in a small and/or standard cage (30 x 30 x 30 cm). Thus, in order to determine the adaptive stenogamy of
|1||11 (36.67)||0 (0)|
|2||23 (76.67)||18 (78.26)|
|3||23 (76.67)||18 (78.26)|
|4||30 (100)||24 (80.00)|
|5||30 (100)||26 (86.67)|
|6||28 (93.33)||23 (82.14)|
|7||28 (93.33)||24 (85.71)|
|8||28 (93.33)||23 (82.14)|
|9||26 (86.67)||16 (61.54)|
|10||23 (76.67)||11 (47.83)|
4.6. Searching for a suitable oviposition-condition
Many anopheline colonies have been reported to adapt easily to oviposit eggs in the cage on various types of simple ovipots, e.g., petridish, crystallizing dish, terra-cotta bowl, white plastic cup, black cup, etc. [35-39]. In the case of using 20 gravid adult females of
4.7. Other important factors
Throughout the larval rearing period, the number of larvae, rearing conditions in the tray, and food were the most important factors, not only for routine rearing, but also special rearing in order to obtain a high yield of metaphase and polytene chromosomes, which were necessary for population-genetic study of anophelines. Stressful rearing-conditions, e.g., the overcrowding of larvae in a rearing tray (in this study, 80 larvae per 25 x 36 x 6 cm tray was an appropriate number for
5. Techniques for metaphase and polytene chromosome preparations
5.1. Rearing condition of mosquitoes for chromosome preparations
The methods for rearing conditions were generally routine as mentioned in paragraph 3, except, 10 first instar larvae per tray were used to obtain a high yield of metaphase chromosomes from larval brains, ovaries and testes, and polytene chromosomes from larval salivary glands. Comparative outcome rates of metaphase chromosomes from larval brains and polytene chromosomes from larval salivary glands between routine (80 larvae) and special (10 larvae) rearing revealed as follows: (1) metaphase chromosomes: experiment 1 [10 larvae (87.50%) vs. 80 larvae (33.33%)], 2 [10 larvae (75.00%) vs. 80 larvae (30.00%)] and 3 [10 larvae (77.78%) vs. 80 larvae (30.00%)]; and (2) salivary gland polytene chromosomes: experiment 1 [10 larvae (80.00%) vs. 80 larvae (50.00%)], 2 [10 larvae (66.67%) vs. 80 larvae (50.00%)] and 3 [10 larvae (100.00%) vs. 80 larvae (66.67%)]. Thus, a special rearing with 10 larvae was used routinely for chromosome preparation.
5.2. Preparation of metaphase chromosomes from adult females and males and fourth instar larvae
5.2.1. Preparation of 0.5% and 1% solutions of dried Gloriosa superba seed and rhizome powders
Summarized flow chart for normal saline-extracted
*By keeping at this condition, the colchicine-like activity in the filtrate stays stable for at least 2 years.
Other benefits include a decorative plant that can be bought at many shops in Thailand’s flower-markets, and it is hoped elsewhere in tropical countries. It can be grown easily in small-spaced land and outdoors with general fertilizers (e.g., simple formula chemical fertilizer, organic fertilizer and animal manure), which are necessary to promote its growth. It takes about 5-7 months to grow from small budding-rhizomes into mature tree with flowers and green pods (Figure 7a-d).
5.2.2. Preparation of the metaphase chromosomes from adult females and males and fourth instar larvae
Metaphase chromosomes for adult females and males were prepared using the modified techniques described by . The newly emerged adult females and males aged up to about 6-12 hours were starved, anaesthetized with ether and placed on their side on a slide under a binocular microscope. A needle was made by drawing out a glass capillary tube in a flame until the pointed end was approximately 80-100 µm in diameter; the shorter the needle the easier it was to handle. An inoculation was made into the post-spiracular area of the mesothorax, and a filtrate of 0.5% solution of dried
The inoculated mosquitoes were dissected in a small drop of 1% hypotonic sodium citrate solution on a siliconized slide by pulling out the last abdominal segment to obtain the ovaries or testes under a binocular microscope. The organs obtained were left in 1% hypotonic sodium citrate solution for 10 minutes, and then transferred to a small drop of Carnoy’s fixative on a siliconized slide for at least 2 minutes. Then, a drop of 60% acetic acid was added, and the organs were torn and mixed well with dissecting needles. A drop of cell suspension was placed on a clean microscopic slide on a warming plate at about 45–50°C. Droplets of cells were released slowly from a Pasteur pipette to form a circular trail of monolayer cells. The dried slides were stained with 20% Giemsa in phosphate buffer pH 7.2 for 1 hour, rinsed with deionized water, air-dried at room temperature, mounted in Permount® (Fisher, Fairlawn, NJ, USA) and examined under a green filter compound microscope. Metaphase karyotypes were identified by following the standard descriptions (Figure 9) [59-60].
The techniques for metaphase chromosome preparations in fourth instar larvae mainly followed those described above, except for the 5 fourth instar larvae that were incubated with a 1 ml filtrate of 0.5% dried
5.3. Preparation of the polytene chromosome from larval salivary glands
Salivary gland polytene chromosomes were prepared using the slightly modified published techniques [26, 61]. The early fourth instar larvae were removed from the rearing tray by a dropper and rinsed in clean distilled water. A healthy larva with flared-thorax in appearance was picked up with forceps, attached to filter paper to remove excess water, placed on a siliconized slide filled with a drop of 1% hypotonic sodium citrate solution, and then dissected under a binocular microscope. The head was cut off, and one dissecting needle was inserted through the anterior end of thorax to posterior end. Then, another dissecting needle was scratched along the line of the inserted needle to tear the thorax integument, open the thorax and take out the internal organs before the thorax and abdomen were transferred into a drop of 15% acetic acid on a siliconized slide. The bilobed salivary glands were removed from the thorax using dissecting needles, and only the whitish anterior lobe of each salivary gland was transferred into a small drop of 45% acetic acid on a siliconized slide and left for 1 minute. After that, one drop of 2% aceto-lactic orcein stain was added. After 15 minutes of staining, a grease-free 22 mm2 coverslip was placed on the stained salivary glands. The preparation was wrapped firmly in filter paper and gently pressed with a thumb to squash and spread the chromosomes. Then, the coverslip edges were sealed with transparent nail varnish. The prepared chromosomes were scrutinized under a green filter compound microscope. The arm of the polytene chromosomes was identified by following the standard map (Figure 11) .
The formation of robust systematic procedures is highly anticipated, based on the crossing experiments between iso-female lines using cytological markers (characteristics of metaphase chromosomes/karyotypic forms). Together with this information, the data on comparative sequence analyses of some specific genomic regions (rDNA and mtDNA) would bring success in recognizing and reliably identifying sibling species and/or subspecies members within the taxon of other
Sincere thanks are extended to the Thailand Research Fund (TRF Advanced Research Scholar: BRG/14/2545 and BRG5380021, and TRF Senior Research Scholar: RTA5480006), the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0044/2546, PHD/0052/2548, PHD/0082/2549, PHD/0031/2550, PHD/0297/2551 and PHD/0356/2552), Biodiversity Research and Training Program (Grant No. BRT R_249004, 250009 and 252005: 2006-2009), and Faculty of Medicine Research Fund, Chiang Mai University, Chiang Mai, Thailand, for their continuous financial support in the population-genetic study of
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