2009 Vol. 30, No. 4
Comparative Analysis of Five Different Homologous Feeder Cell Lines in the Ability to Support Rhesus Embryonic Stem Cells
2009, 30(4): 345-353. doi: 10.3724/SP.J.1141.2009.04345
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In our previous study, five homologous feeder cell lines, Monkey ear skin fibroblasts (MESFs), clonally derived fibroblasts from the MESFs (CMESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFGs) cells, monkey follicular granulosa epithelium-like (MFGEs) cells, were developed for the maintenance of rhesus embryonic stem cells (rESCs). We found that MESFs, CMESFs, MOFs and MFGs, but not MFGEs, support the growth of rhesus embryonic stem cells. Moreover, we detected some genes that are upregulated in supportive feeder cell lines by semi-quantitative PCR. In the present study, we applied the GeneChip® Rhesus Macaque Genome Array of Affymetrix Corporation to study the expression profiles of these five feeder cell lines, in purpose to find out which cytokines and signaling pathways were important in maintaining the rESCs. mRNAs of eight genes, including GREM2, bFGF, KITLG, DKK3, GREM1, AREG, SERPINF1 and LTBP1, were found to be upregulated in supportive feeder cell lines, but not in MFGE. The results indicate that many signaling pathways may play redundant roles in supporting the undifferentiated growth and maintenance of pluripotency in rESCs.
Tissue Distribution of TRIM5α in Rhesus Monkey and Upregulation in Peripheral Blood Mononuclear Cell by Using Different Stimuli
2009, 30(4): 354-360. doi: 10.3724/SP.J.1141.2009.04354
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TRIM5α (tripartite motif protein 5-alpha) protein is a very important restriction factor in rhesus monkey. It can restrict the replication of retroviruses, such as HIV-1 (human immunodeficiency virus type 1), EIAV (equine infectious anemia virus) and FIV (feline immunodeficiency virus). Up to now, there is no report about the tissue distribution of TRIM5α in rhesus monkey. The influence of different stimuli on the expression of TRIM5α mRNA has also not been examined in monkey peripheral blood mononuclear cell (PBMC). In this study, total RNA was extracted from different tissues of a rhesus monkey collected in Yunnan, China, and was quantified using the β-actin gene as the internal reference. The expression levels of TRIM5α mRNA in PBMC that was treated with three conditions (the HIV-GFP-VSVG pseudotyped virus challenged, PMA and Ion-costimulated, anti-CD28 and anti-CD49d antibody-costimulated) were also evaluated at different time points. Our results showed that TRIM5α mRNA expressed broadly in all 21 studied tissues, with the highest level in tissues of immune system and urogenital system and a lower level in nervous system, such as cerebrum and spinal cord. The expression level of the TRIM5α mRNA level in PBMC was upregulated after being activated by the three treatments.
2009, 30(4): 361-368. doi: 10.3724/SP.J.1141.2009.04361
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Trichostatin A, a histone deacetylase inhibitor, could increase histone acetylation, and active gene expression in somatic cells. However, effect of TSA on histone methylation is unclear. In present study, the bovine fibroblast cells were treated with TSA, and histone acetylation and methylation were examined by using indirect immunofluorescence and scanning confocal microscopy. We found that the morphology of cells was changed from being slightly concave to more flat and elongated even at the 10 ng/mL group. Intensive H4K8ac signals in fibroblast cells were observed after TSA treatment. A similar increasing with H4K8ac in H3K4me3 was found, but no change was observed at H3K9me2 when somatic cells were treated with TSA. These result indicated that TSA was not only increasing histone acetylation, but also increasing histone methylation that was correlated with gene expression. However, TSA could not change the level of histone methylation that regulated gene silence.
2009, 30(4): 369-376. doi: 10.3724/SP.J.1141.2009.04369
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The Mest（mesoderm-specific transcript）gene has been considered an imprinting gene in human and mouse, and was also confirmed in other mammals and flowering plants. To investigate the function and evolution of this gene, the cDNA of full length Mest gene was obtained using 5'- and 3'-RACE from the Chinese Large Toad (Bufo gargarizans). The transcript is 1 325 bp in length which contains a complete open reading frame (ORF) encoding a polypeptide of 326 amino acids (GenBank accession number: ABQ10905). There is a typical α/β hydrolase fold domain in the putative gene product, and it shows high similarity to sequence of homologous protein of Xenopus tropicali (86%), mammlian (70% − 80%). RT-PCR (reverse transcriptase-polymerase chain reaction) analysis demonstrated that the Bufo gargarizans Mest (BgMest) gene is expressed widely in testis, ovary, liver, kidney, spleen, brain, stomach and lung. The conservation of the BgMest gene sequences, protein secondary structure of the BgMest protein, in addition to the expression pattern of the BgMest gene, suggested that the function of BgMest was conserved in amphibians. However, the phylogenetic tree of the imprinting gene of the mammals and other vertebrates examined in this study indicated their divergent origins.
Association Study of a Schizophrenia Susceptible Gene GULP1 with Brain Volume in Chinese Populations
2009, 30(4): 377-380. doi: 10.3724/SP.J.1141.2009.04377
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Schizophrenia is a complex mental disease which affects about 1% of the world population. Previous studies reported that the brain volumes of the schizophrenia patients were much smaller than normal people and many schizophrenia susceptible genetic variants were associated with structural brain abnormalities in patients, which is consistent with the neurodevelopmental hypothesis of the etiology of schizophrenia. Recently, two SNPs (rs2004888 and rs452256) of GULP1 were found to be significantly associated with schizophrenia. In this study, to test whether these two SNP of GULP1 are also consistent with the neurodevelopmental hypothesis, we genotyped the two SNPs in 791 Chinese individuals and measured their brain volumes.We found that the two SNPs have no association with brain volume, suggesting that the etiology of schizophrenia is much more complicated.
Venom Properties of the Spider Latrodectus tredecimguttatus and Comparison of Two Venom-Collecting Methods
2009, 30(4): 381-388. doi: 10.3724/SP.J.1141.2009.04381
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The bioactivities and proteinous compositions of venoms collected by two different methods from Latrodectus tredecimguttatus were analyzed and compared. Most of proteinous venom components were high-molecular-mass acidic proteins (>104). Compared with the venom obtained from dissected venom gland (DGV), the venom obtained by electrical stimulation (ESV) contained more high molecular mass proteins, but the venom proteins and peptides with low molecular mass (<104) in the preparations were very similar. Intraperitoneal injection of the two venoms in mice gave rise to similar poisoning symptoms and the LD50 values of ESV and DGV were (0.16±0.03) mg/kg and (0.39±0.05) mg/kg, respectively. LD50 values of ESV and DGV in cockroaches (Periplaneta Americana) were 1.87 μg/g and 2.32 μg/g, respectively. ESV was able to block neuromuscular transmission in the mouse phrenic nerve-hemidiaphragm preparation within (25.0±2.2) min at a concentration of 3.2×10-6g/mL, whereas DGV blocked neuromuscular transmission within (45.7±1.8 )min at the same concentration. The fraction of the venom containing proteins and peptides with molecular mass <104 did not obviously affect the transmission. Those results demonstrated that Latrodectus tredecimguttatus venom was a mixture rich in larger proteins rather than smaller proteins and peptides; the mammalian toxicities of the venom were due primarily to the high molecular weight acidic proteins rather than low molecular weight peptides; the active components contained in ESV and DGV were similar but the content in ESV was higher than that in DGV.
Role of Specific Synaptic Plasticity Interfering Peptides in the Expression of Morphine Induced Conditioned Place Preference in Mice
2009, 30(4): 389-395. doi: 10.3724/SP.J.1141.2009.04389
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Learned association between context and drug abuse is essential for the drug conditioned place preference (CPP), which is an animal model widely used to measure drug reward. Synaptic plasticity, in the form of long-term potentiation (LTP) and depression (LTD), is regarded as a proposed cellular substrate of learning and memory. However, the exact role of LTP/LTD in addiction is not known yet. Therefore, by bioinformatics we designed peptides aiming to interfere with LTP and LTD respectively, to study their individual role in the expression of morphine CPP. We found that the interfering peptide Pep-A2 can specifically block hippocampal LTP in CA1 region, whereas Pep-A3 can block LTD in this region. Treatment of either of their cell penetrating forms (Tat-A2 or Tat-A3) before test can block the expression of Morphine CPP in mice. These results suggested that both LTP and LTD are required in the drug-associated learning and memory.
Hippocampal Combinatorial Plasticity Induced by Conditioning of Converging Schaffer-CA1 Pathways in Rat in vivo
2009, 30(4): 396-400. doi: 10.3724/SP.J.1141.2009.04396
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Hippocampal plasticity in two conditioning pathways, i.e. hippocampal combinatorial plasticity, can be induced by conditioning of converging Schaffer-CA1 pathways in Sprague-Dawley rats under pentobarbital sodium anesthesia. During conditioning, paired pulses were delivered at 5 Hz（600 pairs）and the inter-peak interval of the field excitatory postsynaptic potentials (fEPSP) of the paired-pulse stimulation was 10 ms. Our results showed that long-term potentiation (LTP) and long-term depression (LTD) might be induced simultaneously by conditioning of two pathways whether two conditioning pathways were independent or not. Thus hippocampal combinatorial plasticity, i.e. LTP/LTD, can be induced successfully. Our results demonstrated that conditioning paradigm could enable the induction of bidirectional hippocampal synaptic plasticity and the direction of synaptic plasticity was determined by the state of synapses. Our results indicate that hippocampal combinatorial plasticity induced by cellular conditioning paradigm may encode hippocampus dependent learning and memory much better compared with LTP and LTD induced by low frequency stimulation or high frequency stimulation independently and may reflect the flexibility and stability of hippocampal synaptic plasticity.
2009, 30(4): 401-405. doi: 10.3724/SP.J.1141.2009.04401
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Hair provides a suitable sample inassessing long-term changes in hypothalamic-pituitary-adrenocortical axis (HPA) system activity. The procedure used for hair analysis is therefore very important. To ensure that our procedure is reliable, liquid nitrogen was used to deepfreeze the samples before powdering them. Experimental trials used three treatment types: blank group (phosphate buffered saline (PBS)) (n=3), old tube group (n=10) and new tube group (n=10). The potential effects of PBS and tubes was then investigated. Additionally, both the relationship between the results of two samples of the same individuals (n=16) yielded by the same procedures but at different times, and the relationship between hair cortisol concentrations and the number of received intense aggressions (n=16) were also investigated. The results showed that the cortisol concentration of the blank group was 0 pg/mg; new tubes did not differ from old tubes（F（1，19）=0.041, P=0.843，one-way ANOVA); and there was a high association between the results of two samples of the same individuals, yielded by same procedures operated at different times (r=0.893，P=0.000). Moreover, hair cortisol concentrations were moderately correlated with the number of received intense aggressions (r=0.591，P=0.008). Therefore, PBS and tubes did not affect the results significantly, therefore, our procedure was deemed reliable.
2009, 30(4): 406-410. doi: 10.3724/SP.J.1141.2009.04406
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The most infected species of H5N1 highly pathogenic avian influenza（HPAI）broke out in Qinghai Lake in 2005 was bar-headed goose (Anser indicus). To investigate the immune status to H5N1-AIV of bar-headed geese in Qinghai Lake National Nature Reserve, 68 ejected eggs and 125 in-nest eggs of the breeding colony were collected in spring 2008, and haemagglutination inhibition (HI) was applied to analyze their yolk MAb (IgY) to H5N1-AIV. The results revealed, three years after the outbreak of HPAI in 2005, 26.5% to 35.2% of breeding pairs of the bar-headed geese breeding on Bird Island in Qinghai Lake may have acquired anti-H5N1 AIV immunocompetence. In addition, we analyzed the relationship of MAb titers of the nest-eggs and the nest density, and found the significant correlation between them (r=0.736, P=0.000). It is likely to be more adaptive that the MAb transmission pattern in the breeding colony with high breeding density.
2009, 30(4): 411-417. doi: 10.3724/SP.J.1141.2009.04411
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The influencing factors of birds captured at night were investigated by light trapping at Jinshan Yakou （23º57' N, 101º30'E) of Ailao Mountain, Xinping County, Yunnan Province from 5th September to 5th November 2007 and from 3rd September to 7th November 2008. The influencing factors were analyzed by using hygrothermograph and qualitative analysis. Geography conditions had a great effect on the bird species captured at night. Weather was also an important factor influencing the number and species of birds captured at night. Statistical analysis showed that wind direction, wind force, fog and lunar phase had a significant effect on the species and number of birds captured at night（P<0.01）. However, the effect of wind direction was more obvious than that of wind force. More birds and species were captured during nights with a southwestern wind, fog, crescent moon and old moon. In addition to light is a necessary condition for trapping. But temperature and humidity had no significant effect on the number and species of birds captured at night.
Analysis on Trophic Structure of Main Waterbirds in Qinghai Lake Based on Stable Isotopic Technology
2009, 30(4): 418-422. doi: 10.3724/SP.J.1141.2009.04418
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Stable carbon and nitrogen isotopes were measured to analyze trophic level of main waterbirds in Qinghai Lake in May−June, 2008, based on the model of isotopic enrichment and the fractionation factor. Our results indicated that δ15N was significantly enriched in the muscle of waterbirds, but δ13C showed no obvious variation. We obtained a fractionation factor between Gymnocypris przewalskii and Phalacrocorax carbo, i.e., 1.78‰. Phytoplankton, zooplankton, Gammarus suifunensis, G. przewalskii and waterbirds were estimated at the trophic levels of 1.64-2.06, 2.35-2.83, 2.63, 3.64-;4.20 and 3.35-4.93 respectively. P. carbo, Larus ichthyaetus, Larus brunnicephalus and Podiceps cristatus occupied the highest trophic level due to their primary feeding on G. przewalskii. However, Tadorna ferruginea, Sterna hirundo, and Tringa totanus occupied the relatively high trophic levels. Anser indicus, mainly relying on plant materials as their food supply, was at the lowest trophic level. Our results predicted that the future resource situation of G. przewalskii would have great influence on the growth, reproduction and community structure of main waterbirds, especially for P. carbos, L. ichthyaetus, L. brunnicephalus and P. cristatus, consequently on the stability and diversity of the whole ecosystem.
Bill Morphology Does Not Influence Vocal Performance in Darwin’s Small Tree Finch on Floreana Island
2009, 30(4): 423-428. doi: 10.3724/SP.J.1141.2009.04423
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Bird song is used for mate attraction and is important for establishing reproductive isolation. Current research highlights performance constraints on song production that may be influenced by variation in bill morphology. Darwin’s finches are a model system for studying the relationship between morphology and song performance, with previous studies suggesting that lower vocal performance is correlated with larger bill size. Here, we tested for a relationship between bill morphology and vocal performance in Darwin’s Small Tree Finch (Camarhynchus parvulus) on Floreana Island. We found no evidence of a correlation between bill morphology and vocal performance. This finding is in agreement with prior study of the Small Tree Finch, but contrasts a greater body of work addressing song in Darwin’s Finches. We discussed our findings in the context of ecological divergence, and ecological variation across species.
2009, 30(4): 429-432. doi: 10.3724/SP.J.1141.2009.04429
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By the method of ad libitum sampling and all-occurrence recording, breeding behaviors of Blue Tailed Bee-eater in the Agricultural Region of Nujiang Valley were observed from 26th March to 17th July, 2007. The result showed the most common foods that male birds fed to female birds are Odonata and Hymenoptera insects, accounting for 83.56%. The frequency of females accepting the food provided by males was 96.83%, and the frequency of copulation was 17.49% following food acceptance. During the daytime of the incubation period, comparison of time on nest between male and female showed no significant difference. Male mean on nest time was (23.5±21.6)min and female (25.8±11.5)min. Once hatched, a decrease in time spent warming the young was seen until the 20th day where the parents bird ceased warming. The nursing period of Blue Tailed Bee-eater was found to be 29 - 33 days, with the presence of a helper significantly improving the success rate of reproduction.
Feeding Changes and Growth Performance of Sinocyclocheilus grahami (Pisces, Barbinae) Larvae and Juveniles in Farm Environment
2009, 30(4): 433-437. doi: 10.3724/SP.J.1141.2009.04433
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From March 2008 to March 2009, comparative observations of feeding and growth were undertaken on 35,000 larvae cultivated in 3 pools, with respective densities of P1 208 larvae/m3；P2 167 larvae /m3；P3 41 larvae/m3. Identical daily management processes were applied to the 3 pools. Data was collected on 5-8 larvae everyday at 08:00 and 18:00 within 15 days of hatching. 30 days from first dieting, the total length and body weight were measured on 15 juveniles collected from each pool. The first dieting of S. grahami larvae occurred in the 5th day after hatching, with a mixed nutrition phase of 3-5 days. The larvae within mixed nutrition conditions were vulnerable to death. In order to promote the survival in mixed nutrition, the larvae and juveniles were raised with jelly-rotifers-compound feed. The mean survival rate of juveniles S. grahami was 82.7%. After one year of cultivation, body weight increased from （0.027±0.01） g（0.003 -1.22）to（8.83±0.54） ) g（3.7-.7）, net growth being 0.73 g per month. Total length increased from 15.8±0.30 mm（11.5-20.0）to 91.6±1.67 mm（75.0 -116.0），net growth being 6.32 mm per month. Linear growth in total length can be described by the equation: y=9.82+7.05 month（R2=0.9891）. Index growth in body weight can be described by equation: y=0.0158 month2.54（R2=0.9939）.
2009, 30(4): 438-444. doi: 10.3724/SP.J.1141.2009.04438
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A new species of the subfamily Labeoninae, Garra nujiangensis Chen and Yang, sp. nov. is recognized from a secondary branch of Nujiang River (upper Salween) in Zhenkang county, Yunnan province, China. It can be distinguished from other beardless Garra species by the following combination of characters: 12 -14 circumpeduncle scales; 48-50 lateral- line scales; a pair of rudimentary rostral barbels present in little individuals; 8 -9 branched dorsal-fin rays; 5 -6 scales between dorsal-fin origin and lateral line and 3 -4 scales between pelvic-fin origin and lateral line; caudal peduncle relatively stouter; length between snout tip to posterior margin of eye longer than body depth at anal fin origin; head length longer than body depth at dorsal fin origin; prepelvic length 50.0%-53.7% of standard length; pre-anus length 58.9-63.9% of standard length; body width 16.7%- 19.6% of standard length; body depth at dorsal fin 19.2%-22.6% of standard length. The present study also verified that the presence of tiny barbells is a juvenile character in some beardless Garra species, such as G. nujiangensis and G. alticorpora.
Oreonectes macrolepis, A New Nemacheiline Loach of Genus Oreonectes (Balitoridae) from Guangxi, China
2009, 30(4): 445-448. doi: 10.3724/SP.J.1141.2009.04445
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A nemacheiline loach, Oreonectes macrolepis sp. nov. has been recognized based on collections from a Pearl River Drainage in Maonan County, Huanjiang City, Guangxi, China, in June 2008. Oreonectes macrolepis is a new member of the O. furcocaudalis group. It can be distinguished from all its congeners in this group by the following characters: body entirely covered by scales, except between pectoral fins and pelvic fins; lateral line present, with 5–12 pores; cephalic lateral line system with 3+7 infraorbital pores and 7–8 supraorbital; Head length 2.3–2.7 times head width in nose; head length 1.4–1.8 times maximum head width; Standard length 7.0–10.8 times caudal-peduncle depth.
By serial histological sections, embryos at different development stages were sampled and fixed to investigate the relationship between the organogenesis of yolk sac, hepatopancreas and the yolk utilization during the embryonic development of swimming crab, Portunus trituberculatus. The results showed that: (1) the formation of yolk island and yolk sac were in gastrula stage and egg nauplius stage, respectively, while the yolk islands could be found in the yolk sac from egg gastrula to egg ZoeaⅠstage. (2) From egg Zoea Ⅱstage, the yolk sac was divided into two parts because of further development of the midgut while the hepatopancreatic cells (columnar epithelium) were appearing in the inner of yolk sac membrane. This indicated the starting formation of hepatopancreas. (3) At egg Zoea Ⅲ stage, the yolk sac had been developed into a pair of hepatopancreas while the yolk from different yolk islands were combined, then all yolk islands disappeared from this stage. Due to yolk utilization, many vacuoles could be found in the hepatopancreatic lumen. (4) When the embryo developed into pre-hatching stage, the yolk in the lumen of hepatopancreas had been nearly depleted while for newly-hatched ZoeaⅠ, the yolk had been completely utilized and hepatopancreas seemed to be a pair of saccate structure. These results indicated that both the yolk islands and hepatopancreatic cells play important roles for the disintegration and utilization of egg yolk from gastrula stage to pre-hatching stage in P. trituberculatus.
2009, 30(4): 457-462. doi: 10.3724/SP.J.1141.2009.04457
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Predation is the principal source of nesting mortality in Galliform species. Therefore, it is very important to select suitable nest-sites for nest survival and reproduction success. Nest-site selection of Red Jungle Fowl (Gallus gallus jabouillei) in Datian National Nature Reserve of Hainan Province was studied using plot method based on field observations between March and July 2008. We systematically searched for fowl nests throughout all habitats by random lines and consulting workers in the Reserve. Sixteen nests were recorded. They were at the base of fasciculate grass or stunt bushes, ten of which (62.5%) were below grass, one was (6.25%) below bushes, five were (31.3%) below grass and bushes. Mann-Whitney U test showed that Red Jungle Fowl preferred nesting in relatively open places near roads and forest edge, with less leaf litter. Principal component analysis showed that the vegetation cover factor and space location factor accounted for the most variations of the habitat variable, which were the main factors affecting the nest-site selection of the fowls.
2009, 30(4): 463-467. doi: 10.3724/SP.J.1141.2009.04463
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The Fuxian golden line (Sinocyclocheilus tingi) is a famous fish endemic to Fuxian Lake. From March to April 2009, four breeding experiments were undertaken. 40 females and 20 males were injected with hormones. 19 females and 15 males bred successfully, accounting for 47.5% of females and 75.0% of males. A total of 25 547 eggs were obtained and subjected to dry-fertilization, with 16 810 eggs being fertilized with a 65.8% success rate. 6 040 larvae were obtained with a 23.6% hatching rate. 3 056 larvae survived 20 days of cultivation with a 50.6% survival rate. Reproductive dysfunctions are major constraining factors to low Induced spawning of S. tingi in a captive environment. Artificial conditions that did not reflect the necessary natural environment of the fish may have also been a factor. This paper also addresses the issue of massive larvae mortality. Determining the constraints of artificial propagation is critical to the transfer of this species into cultivation fish. The presented success in propagation of S. tingi will contribute to understanding its biology and protection from extinction.
2009, 30(4): 468-472. doi: 10.3724/SP.J.1141.2009.04468
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Discovery of anticancer drugs that must kill or disable tumor cells in the presence of normal cells without undue toxicity is an extraordinary challenge. Cytotoxicity of plant or fungal materials is considered as the presence of antitumor compounds. Brine shrimp lethality for larvae (nauplii) is used as prescreening test for the antitumor compounds. In this study, culture filtrates of eight strains of Fusarium solani isolated from seeds of various crops were tested for the toxic effect on brine shrimp. Five of the strains (TS, S-29, B-17, C-10, W-5) showed highest toxic effect and three of the strains (SR, T-9, L-25) showed low toxic activity on brine shrimp. Toxic activity reduced when culture filtrates were diluted. However, F. solani strains TS, B-17, SR, T-9 and L-25 caused more than 30% mortality at 1:10 dilution. Toxic activity was slightly reduced when the filtrates were neutralized with sodium hydroxide indicating possible role of pH of culture filtrate on toxicity. Lyophilized filtrates of these strains showed less activity as compared to un-lyophilized filtrates. n-Hexane soluble fraction was obtained only in three strains which showed mild toxicity whereas chloroform soluble fraction was obtained in negligible quantity and could not further be proceeded. Toxic effect of these strains showed variation from strain to strain. Compounds from F. solani could be exploited for the development of toxic compounds.