我国中华按蚊群体分子遗传多态研究

利用RAPD-PCR技术研究了采自我国9省10个代表点的中华按蚊Anopheles sinensis自然群体的遗传多态现象,根据23个RAPD等位基因位点进行了分析。结果显示：①多态位点比例为68.2%～86.4%,期望平均杂合度为0.249～0.348,说明中华按蚊群体具广泛的遗传多态现象;②利用3种方法计算Fst和θ,平均值为0.069～0.111,相应的迁移率Nm为2.0～3.4,表明基因流水平较低;③中华按蚊各自然群体间的遗传一致性达0.8795～0.9973,平均遗传距离为0.041±0.033,属种内变异范围。聚类分析显示,群体间遗传距离与地理位置无对应关系。     

Emergence of Zika Virus in Culex tritaeniorhynchus and Anopheles sinensis Mosquitoes in China

Zika virus(ZIKV) has been isolated from mosquitoes such as Aedes, Mansonia uniformis, and Culex perfuscus; However,the isolation of ZIKV from Anopheles sinensis and Culex tritaeniorhynchus has not yet been reported. In June and July2018, 22,985 mosquitoes and 57,500 midges were collected in Jiangxi Province in southeastern China. Among them, six strains of ZIKV were isolated from mosquitoes: four from An. sinensis and two from Cx. tritaeniorhynchus. Molecular genetic analysis showed that the ZIKV isolated from An. sinensis and Cx. tritaeniorhynchus belonged to genotype 2 in the Asian evolutionary branch of ZIKV. In addition, the ZIKV strains isolated from An. sinensis and Cx. tritaeniorhynchus had amino acid substitutions identical to ZIKV strains prevalent in South America since 2015. This study is the first to isolate ZIKV from mosquito specimens collected in the wild of Jiangxi Province, China; This is also the first time that ZIKV has been isolated from An. sinensis and Cx. tritaeniorhynchus. Given that An. sinensis and Cx. tritaeniorhynchus have a very wide geographical distribution in China and even in eastern and southern Asia, the isolation of several strains of ZIKV from these two mosquitoes poses new challenges for the prevention and control of ZIKV infection in the mainland of China and countries and regions with the same distribution of mosquitoes.     

Quantifying Dispersal of European Culicoides (Diptera: Ceratopogonidae) Vectors between Farms Using a Novel Mark-Release-Recapture Technique

Studying the dispersal of small flying insects such as Culicoides constitutes a great challenge due to huge population sizes and lack of a method to efficiently mark and objectively detect many specimens at a time. We here describe a novel mark-release-recapture method for Culicoides in the field using fluorescein isothiocyanate (FITC) as marking agent without anaesthesia. Using a plate scanner, this detection technique can be used to analyse thousands of individual Culicoides specimens per day at a reasonable cost. We marked and released an estimated 853 specimens of the Pulicaris group and 607 specimens of the Obsoletus group on a cattle farm in Denmark. An estimated 9,090 (8,918–9,260) Obsoletus group specimens and 14,272 (14,194–14,448) Pulicaris group specimens were captured in the surroundings and subsequently analysed. Two (0.3%) Obsoletus group specimens and 28 (4.6%) Pulicaris group specimens were recaptured. The two recaptured Obsoletus group specimens were caught at the release point on the night following release. Eight (29%) of the recaptured Pulicaris group specimens were caught at a pig farm 1,750 m upwind from the release point. Five of these were recaptured on the night following release and the three other were recaptured on the second night after release. This is the first time that movement of Culicoides vectors between farms in Europe has been directly quantified. The findings suggest an extensive and rapid exchange of disease vectors between farms. Rapid movement of vectors between neighboring farms may explain the the high rate of spatial spread of Schmallenberg and bluetongue virus (BTV) in northern Europe.     

Range of Hevea brasiliensis Pollen Dispersal Estimated by Esterase Isozyme Markers

A study was carried out to estimate the distance Hevea brasiliensispollen could be dispersed under natural conditions. Seeds werecollected at varying distances from the boundary between twoadjacent fields that were each planted with a pure stand ofa genetic clone. Esterase isozyme markers were used to determineif the seeds had been derived from self- or cross-pollination.The incidence of cross-pollination was then examined in relationto the distance from the inter-clonal boundary. A logarithmicmodel gave the best fit (r²=0.864) and suggested that pollencould travel distances in the order of 0.3 to 1.1 km. Copyright1999 Annals of Botany Company Hevea brasiliensis (rubber tree), pollen dispersal, cross-pollination, self-pollination, esterase isozymes.     

Malaria transmission potential by Anopheles sinensis in the Republic of Korea.

To evaluate the factors that determine the transmission level of vivax malaria using vectorial capacity, entomological surveys were conducted from June to August, 2000. From 6 nights of human-bait collection in Paju, the human biting rate (ma) was counted as 87.5 bites/man/night. The parity of Anopheles sinensis from human baiting collections fluctuated from 41% to 71% (average 48.8%) of which the rate gradually increased as time passed on: 35.2% in Jun.; 55.0% in July; 66.2% in Aug. From this proportion of parous, we could estimate the probability of daily survival rate of An. sinensis to be 0.79 assumed with 3 days gonotrophic cycle and the expectancy of infective life through 11 days could be defined as 0.073. Blood meal analysis was performed using ELISA to determine the blood meal source. Only 0.8% of blood meals were from human hosts. We could conclude that An. sinensis is highly zoophilic (cow 61.8%). Malaria is highly unstable (stability index < 0.5) in this area. From these data, vectorial capacity (VC) was determined to be 0.081. In spite of a high human biting rate (ma), malaria transmission potential is very low due to a low human blood index. Therefore, we could conclude that malaria transmission by An. sinensis is resulted by high population density, not by high transmission potential. For this reason, we need more effort to decrease vector population and vector-human contact to eradicate malaria in Korea.     

Seed Dispersal by a Frugivore Pycnonotus sinensis and the Distribution of Sambucus chinensis

Frugivorous birds play an important role in the distribution of those plants on whose fruits they feed and affect species composition of plant communities. The fruiting phenology of Sambucus chinensis Lindl. in Wuhan area ranges from July to November, while abundant fruits are available in July and August. Although other seven species of frugivores were found in the study area, only Pycnonotus sinensis (Gmelin), a resident bird endemic to China, was observed foraging in the fruits of this plant for the field. Pycnonotus sinensis shifts from eating fruits of Broussonetia papyrifera (L.) Vent. to those of S. chinensis from July to August. Identification of seeds in a total of 658 feces disseminated by P. sinensis which were collected in the field showed that most of the seeds came from S. chinensis and B. papyrifera. Seeds in those feces collected at the end of August were all of S. chinensis. Field investigation of the associations of S. chinensis shows that its frequent companion species are B. papyrifera, Cinnamomum camphora, Ligustrum lucidum and Rubus biflorus, whose fruits are also preferred by this frugivore in different seasons. The distribution of Pycnonotus sinensis is limited to the south of Qinling Mountain in China. The distribution of S. chinensis is basically consistent with that of Pycnonotus sinensis. The result indicates that seed dispersal of S. chinensis depends on the foraging of this frugivore. Likewise, the fruits of S. chinensis are the primary food for this frugivore in autumn. As the result suggests, conservationists and restoration biologists should highlight the study of plant-disperser mutualism in the future work.     

Studies on Anopheles sinensis, the vector species of vivax malaria in Korea

Extensive previous studies on taxonomy, behavior/bionomics and control of Anopheles sinensis are reviewed and summarized. Recent molecular identification revealed that the population of An. sinensis complex includes An. sinensis, An. pullus, An. lesteri and at least two new species, and An. yatsushiroensis is synonmy of An. pullus. An. sinensis is the main vector species of vivax malaria in Korea. Larvae of An. sinensis breed in wide range of habitats which are naturally-made clean water, stagnant or flowing; main habitats include rice fields, ditches, streams, irrigation canals, marshes, ponds, ground pools, etc. Their host preferences are highly zoophilic. Human blood rate is very low (0.7-1.7%); nevertheless An. sinensis readily feeds on man when domestic animals are not found near by. They feed on hosts throughout the night from dusk to dawn with a peak period of 02:00-04:00 hours; they are slightly more exophagic (biting outdoors); much larger numbers come into the room when light is on. Main resting places are outdoors such as grasses, vegetable fields and rice fields. A mark-release-recapture study resulted that 37.1% was recaptured within 1 km, 29.4% at 1-3 km, 21.1% at 3-6 km, 10.3% at 6-9 km and 2.1% at 9-12 km distance. An. sinensis hibernate outdoors (mostly under part of dense grasses) during October-March. At the end of the hibernation period (March-April) they feed on cows at daytime. Until today any single measure to effectively control An. sinensis population has not been found. Indoor residual spray with a long-lasting insecticide can not reduce vector population densities, but shorten their life spans in some degree, so contributes to malaria control.     

应用PCR技术检测中华按蚊蚊胃血血源

【目的】应用PCR技术检测甘肃地区中华按蚊Anopheles sinensis蚊胃血来源,研究其吸血习性。【方法】根据中华按蚊可能吸血对象(人、牛、猪、羊、鼠、鸡)的线粒体DNA细胞色素b序列的差异,设计种特异引物,建立聚合酶链反应(PCR)体系鉴定中华按蚊蚊胃血来源。同时,对人血、猪血、牛血、羊血、鼠血、鸡血及未吸血中华按蚊中提取的DNA进行检测,验证该方法的特异性;对吸饲人血后不同时间(6、12、24、36、48、60 h)的中华按蚊进行检测,测试该方法的敏感性。【结果】该方法可从已知动物血样和中华按蚊提取的DNA中分别扩增得到689 bp(人)、271 bp(牛)、453 bp(猪)、225 bp(羊)、485 bp(鼠)、266 bp(鸡)和468 bp(中华按蚊)大小的特异性条带;吸人血36 h内的中华按蚊均能扩增出特异性条带,在吸人血后48 h、60 h的中华按蚊中,均未扩出特异性条带。共检测10只现场中华按蚊,血源来自人、牛、猪分别为5只、2只、3只,其中1只蚊子兼吸人血和猪血。【结论】建立的PCR检测方法可鉴定中华按蚊蚊胃血来源,结果稳定可靠;甘肃地区中华按蚊不仅嗜吸人血,也可兼吸其他动物血。     

食果鸟白头鹎与接骨草的分布

作为种子的传播者 ,以果实为生的动物在所食植物的繁殖周期中起着关键作用。尽管动植物相互作用的研究自达尔文开始就引起了进化生物学家和生态学家的关注 ,但和传粉者与植物的相互作用的研究相比 ,种子传播者与植物相互作用的研究在 2 0世纪 70年代后才真正开始[1] 。植物学家     

First report on co-occurrence knockdown resistance mutations and susceptibility to beta-cypermethrin in Anopheles sinensis from Jiangsu Province, China

The increasing prevalence of insecticide resistance in Anopheles sinensis, a major vector of malaria in Jiangsu province in eastern China, threatens to compromise the successful use of insecticides in malaria control strategies. It is therefore vital to understand the insecticide resistance status of An. sinensis in the region. This study examined the nucleotide diversity of the para-sodium channel and knockdown resistance (kdr) in five field populations of adult An. sinensis mosquitoes collected in Jiangsu province, identifying the L1014F and L1014C substitutions for the first time. Competitive polymerase chain reaction (PCR) amplification of specific allele (cPASA) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) for resistance diagnosis were developed and validated. Comparing the results with direct sequencing revealed that the PCR-RFLP method was more sensitive and specific whereas the cPASA method was more convenient and suitable. The significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014C substitutions in the kdr gene provides a useful molecular marker for monitoring beta-cypermethrin resistance in natural populations of An. sinensis. Our results point to the L1014F substitution as the key mutation associated with beta-cypermethrin resistance. The high resistance and mutation frequency detected in the five populations also suggest cross-resistance with other pyrethroids may occur in An. sinensis, highlighting the need for further surveys to map insecticide resistance in China and the adoption of a rational management of insecticide application for resistance management and mosquito vector control.     

The Identification of Plasmodium vivax Infected Anopheles sinensis by Enzyme-Linked Immunosorbent Assay, Republic of Korea, 1998

ABSTRACT To figure out how far spread the malaria parasite infected mosquitoes from Demilitarized Zone (DMZ). We investigated the sporozoite-infected mosquitoes in Chosan-ri and Hajiseok-ri of Paju-shi, Tonae-dong of Koyang-shi, Mudeung-ri of Yonchon-gun, Soyo-dong of Tongduchon-shi, and Tukjeong-ri of Yangju-gun, Kyonggi-do province in 1998. They were known as highly epidemic areas in the Republic of Korea. Anopheline mosquitoes were collected by human biting collection method from the 2nd week of June to the 5th week of August. A total of 3,256 anopheles mosquitoes were captured in these areas. A sandwich enzyme-linked immununosorbent assay (ELISA) for identifying Plasmodium vivax sporozoites in mosquitoes was used in this survey. Only one mosquito which was captured in Chosan-ri, on 2nd week of July showed positive reaction in this test. This mosquito was identified as Anopheles sinensis. This showed a 0.068% (1/1,465) positive rate in Chosan-ri on 2nd week of July. We could not detect positive mosquitoes the other areas. So we concluded the parasite infected mosquitoes may be highly limited near the DMZ.     

A mark-release-recapture experiment with Anopheles sinensis in the northern part of Gyeonggi-do,Korea

In order to study the range of flight and feeding activity of Anopheles sinensis, the dispersal experiment was conducted in Paju city, located in the northern part of Gyeonggi-do, Republic of Korea, during the period of 7th to 28th September 1998. Unfed females An. sinensis were collected in cowshed and released after being marked with fluorescent dye at 23:00 hours on the same day. Released female mosquitoes were recaptured everyday during 21 days using light traps, which were set at 10 sites in the cowsheds located 1, 3, 6, 9 and 12 km north-northwest and north-northeast and at 3 sites located 1, 6 and 9 km toward south-west from the release point. In addition, to study the longest flight distance in one night, we set the light traps at 16 and 20 km toward north-northeast from the release site. All the collected mosquitoes were placed on filter papers and observed on UV transilluminator after treatment with one drop of 100% ethanol. Out of 12,773 females of An. sinensis released, 194 marked females mosquitoes were recaptured, giving 1.52% recapture rate. Of 194, 72 mosquitoes (37.1%) were recaptured in light traps from three places set at 1 km from the release point, 57 mosquitoes (29.4%) from two places at 1-3 km, 41 mosquitoes (21.1%) from three places at 3-6 km, 20 mosquitoes (10.3%) from three places at 6-9 km, and 4 mosquitoes (2.1%) from two places at 9-12 km. Since 170 female mosquitoes (87.6%) out of 194 marked mosquitoes were captured within 6 km from the release point, this flight radius represents the main activity area. An sinensis was found to be able to fly at least 12 km during one night.     

Ultrastructure of the Rectum Epithelial Cells in the Mosquito Larvae, Anopheles sinensis Wiedemann

ABSTRACT The Ultrastructure of rectum epithelial cells in the mosquito larvae, Anopheles sinensis Wiedemann, was studied using electron microscope. The rectal epithelium forms rectal papillae composed of the absorptive cells and the surrounding basal cells. Moreover, rectal epithelium was covered with thin cuticular intima. Apical plasma membrane of the epithelial cells had infoldings and in between them, mitochondria developed into elongated shape were attached. In addition, the membrane infoldings reach down into the cell cytoplasm to form several layers of leaflet-like prolongations. On both sides of these prolongations were also large, well-developed mitochondria. Their formation was that mitochondria were attached to 3 μm length and 4–13 layers of membrane wrinkle lump. Many spherites, which are lamelated crystals that form an illusory structure in concentric circles inside of the cytoplasm of epithelial cell were observed. Basal plasma membrane in the epithelial cells was also wrinkled to promulgate into the cytoplasm to become basal infoldings producing canaliculi in basal labyrinth formation. There were many mitochondria scattered in these formations as well. On the bottom of the epithelial cell, basal lamina was attached and between basal lamina and muscle bundle was subepithelial space, which is connective tissue. Inside the space, tracheal and nerve cells were observed.     

A neural network prediction of environmental determinants of Anopheles sinensis knockdown resistance mutation to pyrethroids in China

Selection pressure caused by long‐term intensive use of insecticides is the key driving force in resistance development. Additional parameters such as environmental conditions may affect both the mosquito response to insecticides and the selection of resistance mechanisms. In this context, we analyzed the environmental determinants of kdr prevalence in Anopheles sinensis across China. We collected kdr frequency from 48 sites across central and southern China, together with key environmental factors including long‐term climatic data, topographic features, main crops, and land cover types. Trend surface analysis found that the distribution of kdr frequency can be partitioned into three regions, namely central China (kdr frequency >80%), western China (kdr frequency varies from 0% to 60%), and southern China (kdr frequency <10%). Seven predictor variables were selected based on a radial basis function neural network model. A multilayer perceptron (MLP) network model revealed that the number of crops in a year was the most important predictor for the kdr mutation rate. Topography, long‐term mean climate and land cover all contributed to the kdr mutation rate. The observed mean kdr frequency was 53.0% and the MLP network model‐predicted mean was 52.6%, a 0.1% relative error. Predicted kdr frequencies closely matched the observed values. The model explained 92% of the total variance in kdr frequency. The results indicated that kdr was associated with the intensity of pesticide usage. Crop cultivation information, together with environmental factors, may well predict the spatial heterogeneity of kdr mutations in An. sinensis in China.     

Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus,Anopheles sinensis,Armigeres subalbatus,Culex pipiens and Cx. tritaeniorhynchus in China

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.     

Spatial-Temporal Variation and Primary Ecological Drivers of Anopheles sinensis Human Biting Rates in Malaria Epidemic-Prone Regions of China

Background

Robust malaria vector surveillance is essential for optimally selecting and targeting vector control measures. Sixty-two vector surveillance sites were established between 2005 and 2008 by the national malaria surveillance program in China to measure Anopheles sinensis human biting rates. Using these data to determine the primary ecological drivers of malaria vector human biting rates in malaria epidemic-prone regions of China will allow better targeting of vector control resources in space and time as the country aims to eliminate malaria.

Methods

We analyzed data from 62 malaria surveillance sentinel sites from 2005 to 2008. Linear mixed effects models were used to identify the primary ecological drivers for Anopheles sinensis human biting rates as well as to explore the spatial-temporal variation of relevant factors at surveillance sites throughout China.

Results

Minimum semimonthly temperature (β = 2.99; 95% confidence interval (CI) 2.07- 3.92), enhanced vegetation index (β =1.07; 95% CI 0.11–2.03), and paddy index (the percentage of rice paddy field in the total cultivated land area of each site) (β = 0.86; 95% CI 0.17–1.56) were associated with greater An. Sinensis human biting rates, while increasing distance to the nearest river was associated with lower An. Sinensis human biting rates (β = −1.47; 95% CI −2.88, −0.06). The temporal variation (σt02=1.35) in biting rates was much larger than the spatial variation (σs02=0.83), with 19.3% of temporal variation attributable to differences in minimum temperature and enhanced vegetation index and 16.9% of spatial variance due to distance to the nearest river and the paddy index.

Discussion

Substantial spatial-temporal variation in An. Sinensis human biting rates exists in malaria epidemic-prone regions of China, with minimum temperature and enhanced vegetation index accounting for the greatest proportion of temporal variation and distance to nearest river and paddy index accounting for the greatest proportion of spatial variation amongst observed ecological drivers.

Conclusions

Targeted vector control measures based on these findings can support the ongoing malaria elimination efforts in China more effectively.     

Population structure of the malaria vector Anopheles sinensis (Diptera: Culicidae) in China: two gene pools inferred by microsatellites

Background

Anopheles sinensis is a competent malaria vector in China. An understanding of vector population structure is important to the vector-based malaria control programs. However, there is no adequate data of A. sinensis population genetics available yet.

Methodology/Principal Findings

This study used 5 microsatellite loci to estimate population genetic diversity, genetic differentiation and demographic history of A. sinensis from 14 representative localities in China. All 5 microsatellite loci were highly polymorphic across populations, with high allelic richness and heterozygosity. Hardy–Weinberg disequilibrium was found in 12 populations associated with heterozygote deficits, which was likely caused by the presence of null allele and the Wahlund effect. Bayesian clustering analysis revealed two gene pools, grouping samples into two population clusters; one includes six and the other includes eight populations. Out of 14 samples, six samples were mixed with individuals from both gene pools, indicating the coexistence of two genetic units in the areas sampled. The overall differentiation between two genetic pools was moderate (F _ST = 0.156). Pairwise differentiation between populations were lower within clusters (F _ST = 0.008–0.028 in cluster I and F _ST = 0.004–0.048 in cluster II) than between clusters (F _ST = 0.120–0.201). A reduced gene flow (Nm = 1–1.7) was detected between clusters. No evidence of isolation by distance was detected among populations neither within nor between the two clusters. There are differences in effective population size (Ne = 14.3-infinite) across sampled populations.

Conclusions/Significance

Two genetic pools with moderate genetic differentiation were identified in the A. sinensis populations in China. The population divergence was not correlated with geographic distance or barrier in the range. Variable effective population size and other demographic effects of historical population perturbations could be the factors affecting the population differentiation. The structured populations may limit the migration of genes under pressures/selections, such as insecticides and immune genes against malaria.     

Long-Distance Dispersal of a Female Cougar in a Basin and Range Landscape

Abstract: We used Global Positioning System technology to document distance, movement path, vegetation, and elevations used by a dispersing subadult female cougar (Puma concolor) through the fragmented habitat of the Intermountain West, USA. Over the course of 1 year, female number 31 moved 357 linear km, but an actual distance of 1,341 km from the Oquirrh Mountains, Utah to the White River Plateau, Colorado, constituting the farthest dispersal yet documented for a female cougar. This cougar successfully negotiated 4 major rivers and one interstate highway while traversing portions of 3 states. Our data suggest that transient survival, and therefore total distance moved, may be enhanced when dispersal occurs during the snow-free season due to low hunting pressure and greater access to high elevation habitats. Long-distance movements by both sexes will be required for the recolonization of vacant habitats, and thus inter-state management may be warranted where state boundaries do not coincide with effective dispersal barriers.     

Studies on the spatial distribution pattern of larvae of the mosquito,Anopheles sinensis,in rice fields

The spatial distribution patterns of the population of Anopheles sinensis larvae were studied in the rice field area in the suburb of Urawa city in Japan, during the summer seasons in 1973 and 1974. The distribution pattern of the larval population within the field, analysed by the m−m regression method, indicated that the basic component of larval distribution was not a group of individuals but a single individual and such components were distributed contagiously over the field. This basic pattern did not change significantly according to developmental stage, census date or field. Therefore, we could describe the distribution pattern of the population in a rice field by the single linear regression, x=0.021+1.339x(r²−0.912). Also, the relation for the whole population in the field area including the five fields could be shown by the linear regression, x=0.049+1.749x(r²−0.959). The value of α remained to be nearly equal to zero, but the value of β became larger than the value for the single-field relation. Such a change in distribution pattern seemed to reflect the greater heterogeneity in conditions among the fields than within individual field. Using the information on the distribution patterns mentioned above, some considerations were given on the sampling plans for mosquito larvae, including samplesize determination and application of sequential methods to estimate population size as well as to classify population level.