Rs) to grazing along an elevation gradient in an alpine meadow on the Tibetan Plateau. Here three fenced enclosures were established in an alpine meadow at three elevations  in July 2008. We measured Rs inside and outside the three fenced enclosures in July–September, 2010-2011. Topsoil (0–20 cm) samples were gathered in July, August, and September, 2011. There were no significant differences for Rs, dissolved organic C (DOC), and belowground root biomass (BGB) between the grazed and ungrazed soils. Soil respiration was positively correlated with soil organic C (SOC), microbial biomass (MBC), DOC, and BGB. In addition, both Rs and BGB increased with total N (TN), the ratio of SOC to TN, ammonium N (NH4+-N), and the ratio of NH4+-N to nitrate N. Our findings suggested that the negligible response of Rs to grazing could be directly attributed to that of respiration substrate and that soil N may indirectly affect Rs by its effect on BGB.Alpine meadows are one major type of pastureland on the Tibetan Plateau. However, few studies have evaluated the response of soil respiration ( Rs) is an important flux in the C cycle [Rs is the second in magnitude to gross primary production but equivalent to or even greater than net primary production. Root and microbial respiration are the two most important components of Rs; thus the factors which affect root growth and microbial activity could all influence Rs [Rs at various spatial and temporal scales [Rs and temperature could be weakened or masked by other factors  [Rs increases with respiration substrate, including labile C  and belowground root biomass (BGB) [Rs by influencing plant growth and microbial activity [Soil respiration  , 10. Press (BGB) , 10–12. activity .Rs to grazing [Rs [Rs to grazing is complex and may be dependent on the responses of respiration substrate [Grazing is a major type of land use in grasslands and previous studies have shown inconsistent results on the response of  grazing , 15. Maning [Rs , 16, 17,ing [Rs , 19. Theing [Rs –23. Thering [Rs . The resubstrate  and soil−2 at depth of 0–100 cm [Rs to grazing in an alpine meadow along an elevation gradient, although pasture for domestic sheep and yak is a common land use type on the Tibetan Plateau [Rs in an alpine meadow at three elevations  on the Northern Tibetan Plateau.Alpine meadows are a major type of pastureland on the Tibetan Plateau , 22 and 0–100 cm . Few stuRs, soil C, and N and (2) the relationships between Rs and respiration substrate  and soil N  along an elevation gradient in an alpine meadow in Tibet.The main objectives of this study were to examine (1) the effect of grazing on −2 and sunlight was 2880.9 h [ Kobresia-dominated alpine meadow [The study area (30°30′–30°32′N and 91°03′–91°04′E) was located at the Damxung Grassland Observation Station, Tibetan Autonomous Region in China. Annual mean solar radiation was 7527.6 MJ m2880.9 h . Annual 2880.9 h . The soi2880.9 h . The vege meadow .−1 [Based on meteorological observations from 1963 to 2012 at the Damxung Station , there was no significant change for annual precipitation, while annual mean air temperature increased at a rate of 0.04°C aThree sites (about 20 m × 20 m for each) were fenced in an alpine meadow on a south-facing slope on the Nyainqentanglha Mountains along an elevation gradient  in July 2008. Before enclosure, the site at elevation 4313 m was winter pasture, while the other two sites were summer pasture . A more Ts) at a depth of 5 cm, soil water content (SWC) at a depth of 10 cm, and air temperature and relative humidity at a height of 15 cm were continuously monitored using data loggers  at each elevation [Ts increased with decreasing elevation [Soil temperature  [Rs [Rs was measured between 9:00 and 11:00  in this study. Four polyvinyl chloride (PVC) collars (20 cm in diameter and 5 cm in height) were inserted into the soil to depths of about 2-3 cm on each measuring date. All the PVC collars were installed and the aboveground biomass was removed at least 12 h before Rs measurement in order to reduce disturbance [Rs measurements [Soil respiration was measured using a soil CONE, USA) , 30 duriSA) [Rs , 31; thuturbance , 12. The3−-N, NH4+-N, DOC, and DON. All the roots in the soil samples were washed, dried at 65°C for 48 h, and weighed.Topsoil samples (0–20 cm depth) inside and outside the three fenced enclosures were collected (using a soil auger of 3.0 cm in diameter) on July 7, August 9, and September 10, 2011 . Five so2SO4. The K2SO4 extracts were filtered through 0.45 μm filter membrane and then soil available N  in the extracts were analyzed on a LACHAT Quickchem Automated Ion Analyzer.We extracted 20 g fresh soil samples using 100 mL Kμm filter membrane. We analyzed the extractable soil organic C and total N in the ultrapure water extracts using a Liqui TOC II elementar analyzer  and a UV-1700 PharmaSpec visible spectrophotometer (220 nm and 275 nm), respectively. Dissolved inorganic N (DIN) concentrations in the ultrapure water extracts were also determined on a LACHAT Quickchem Automated Ion Analyzer. Then, DON was calculated as the difference between DTN and DIN. Soil organic C, TN, MBC, and MBN data were obtained from Fu et al. [The methods of Jones and Willett  were useRs for each site .Repeated-measures analysis of variance (ANOVA) was used to estimate the main and interactive effects of measuring date and grazing on ach site . Repeate, and BGB. Student3−-N, NH4+-N, and SAN under grazing were 31.78% (1.95 mg kg−1), 39.14% (2.34 mg kg−1), and 35.41% (4.29 mg kg−1) lower compared with ungrazed soils across all the three sampling dates , 23.60% (1.33 mg kg−1), and 22.75% (2.75 mg kg−1) lower than that of ungrazed soils . Average NH4+-N at elevations 4313 m and 4513 m was 68.40% and 67.23% lower compared with elevation 4693 m, respectively (P < 0.05). Average NH4+-N/NO3−-N ratio at elevations 4313 m and 4513 m was 66.76% and 70.52% lower compared with elevation 4693 m, respectively (P < 0.05).No significant differences of DOC, NHP < 0.05), whereas there was no significant difference between elevations 4313 m and 4513 m. There were no significant differences of average DON among the three sites when grazing was absent.Average DON at elevations 4313 m and 4513 m across all the three sampling dates was 50.78% and 33.84% lower than that of elevation 4693 m under grazing, respectively (P < 0.05), while no significant difference between the two lower elevations was found. By contrast, average SAN increased with increasing elevation under grazing .Average SAN at elevations 4313 m and 4513 m across all the three sampling dates was 38.69% and 38.88% lower than that of elevation 4693 m when grazing was absent, respectively , 4.70% (0.11 μmol CO2 m−2 s−1), and −4.07% (−0.15 μmol CO2 m−2 s−1) at elevations 4313 m, 4513 m, and 4693 m, respectively. In contrast, there was significant seasonal variation for Rs  and BGB  across all the measuring dates.There were significant elevation effects on 4+-N, SAN, SOC/TN ratio, and NH4+-N/NO3−-N ratio, respectively (3−-N (data not shown).Belowground biomass was positively related to TN, NHectively , but notRs was positively correlated with SOC, TN, SOC/TN ratio, MBC, MBN, DOC, DON, NH4+-N, SAN, NH4+-N/NO3−-N ratio, and BGB, respectively .ectively . HoweverPrevious studies indicated that grazer urine and dung stimulated soil microbial activity and accelerated nutrient cycling in grasslands , 34. HowRs, DOC, DON, DOC/DON ratio, NO3−-N, NH4+-N, and BGB along the elevation gradient, which was in line with previous studies [Generally, grazing did not alter the distributions of  studies , 35, 36.3−-N and NH4+-N) at elevations 4313 m and 4513 m under grazing were significantly lower compared with ungrazed soils, while there were no significant differences of SOC, TN, DOC, DON, BGB, and Rs between grazed and ungrazed soils .4+-N, SAN, and NH4+-N/NO3−-N ratio, but not with NO3−-N. Therefore, the positive relationship between BGB and SAN may be mainly attributed to that between BGB and NH4+-N. In addition, the ratio of different soil available N forms could affect BGB.Previous studies showed that BGB increased with increasing TN in alpine grasslands on the Tibetan Plateau , 37. OurRs increased with TN, SOC/TN ratio, NH4+-N, SAN, and NH4+-N/NO3−-N ratio, respectively  were in accordance with previous studies which were made in alpine meadows on the Tibetan Plateau , 38 and Rs and Ts in various ecosystems [Rs increased significantly with increasing elevation, while both soil and air temperatures declined in the current study. In other words, the relationship between Rs and Ts was negative along the elevation gradient. This implied that other factors  probably regulated or confounded the positive relationship between respiration and temperature [Rs and BGB, SOC, MBC, and DOC  in Tibet during the period from July to September in 2010-2011. We found that grazing did not significantly affect soil respiration, which was probably attributed to the insignificant response of respiration substrate  to grazing. Soil N availability and the ratio of ammonium to nitrate N might also influence soil respiration by affecting belowground root growth.


Aspergillus niger I1 were optimized by response surface methodology (RSM). A significant influence of temperature, KH2PO4, and initial pH on the protease production was evaluated by Plackett-Burman design (PBD). These factors were further optimized using Box-Behnken design and RSM. Under the proposed optimized conditions, the experimental protease production (183.13 U mL−1) closely matched the yield predicted by the statistical model (172.57 U mL−1) with R2 = 0.914. Compared with the initial M1 medium on which protease production was 43.13 U mL−1, a successful and significant improvement by 4.25 folds was achieved in the optimized medium containing (g/L): hulled grain of wheat (HGW) 5.0; KH2PO4 1.0; NaCl 0.3; MgSO4(7H2O) 0.5; CaCl2 (7H2O) 0.4; ZnSO4 0.1; Na2HPO4 1.6; shrimp peptone (SP) 1.0. The pH was adjusted at 5 and the temperature at 30°C. More interestingly, the optimization was accomplished using two cheap and local fermentation substrates, HGW and SP, which may result in a significant reduction in the cost of medium constituents. Medium composition and culture conditions for the acid protease production by  Bacillus strains. Nevertheless, the potential use of fungal proteases is being increasingly realized [Microorganisms are the most important sources for enzymes production. Among these enzymes, proteases account for nearly 60% of the total industrial enzyme market , 2. A larealized . In factrealized . Fungal realized .The use of acid proteases has been increased remarkably in various industrial processes such as animal feed, cheese and food processing, and X-ray films , 7.B. subtilis was grown in media containing only sardinelle heads and viscera powder. Haddar et al. [Sardinella aurita peptone for the proteases production by B. mojavensis A21. Also, Hadj-Ali et al. [B. licheniformis NH1.It is well known that extracellular protease production by microorganisms is greatly influenced by media components, especially carbon and nitrogen sources, and physical factors such as temperature, pH, incubation time, agitation, and inoculum density –11. Medir et al.  had repoi et al.  demonstrB. cereus BG1 was stimulated by the use of S. aurita powders [The protease production by  powders .Optimization of media compounds by the traditional “one-variable-at-a-time” strategy involving changing one independent variable is the most frequently used operation in biotechnology . This stIn recent years, the use of statistical approach involving Plackett-Burman designing and Box-Behnken design has gained lot of impetus for medium optimization and for understanding the interactions among various physicochemical parameters using a minimum number of experiments. The Plackett-Burman design allows the screening of main factors, from a large number of variables that can be retained in further optimization process. Box-Behnken design is a collection of statistical techniques for designing experiments, building models, evaluating the effects of factors, and searching optimum conditions of studied factors for desirable responses . Box-BehAspergillus niger I1 has been recently isolated and identified as a producer of an extracellular bleaching stable acid protease [A. niger I1 by using low-cost fermentation medium. The optimization steps were performed as follows: selecting carbon and nitrogen sources by one-variable-at-a-time approach, screening the main factors influencing protease production using Plackett-Burman design, and assessing the optimal region of the significant variables using Box-Behnken design. protease . The enzSardinella aurita) powder (CHVSP), and Mirabilis jalapa tubers powder (MJTP) were prepared in our laboratory. All chemicals used were of analytical grade. Hulled grain of wheat (HGW) was purchased from a local industry. Shrimp flower (SF), shrimp peptone (SP), combined heads and viscera sardinelle  was determined by Manni et al. . SP was A. niger I1 producing an acid protease was used in the present study. It was identified on the basis of a 650 bp PCR amplified DNA fragment of the 18S rDNA sequence [sequence . The strain was propagated on potato-dextrose-agar plates at 30°C, and inocula were prepared from 7-days-old mycelia by flooding with 10 mL of sterile distilled water and scraping off the agar plates.4)2SO4 5.0; CaCl2(7H2O) 0.4; KH2PO4 1.0; Na2HPO4 0.8; MgSO4(7H2O) 0.5; ZnSO4 0.1; NaCl 0.3. Media were autoclaved at 120°C for 20 min. Cultures were inoculated with 107 spores/mL in 300 mL Erlenmeyer flasks with a working volume of 50 mL and incubated in a rotatory shaker (200 rpm) for 72 h. Cultures were centrifuged at 8,000 ×g for 15 min to remove fungi mycelia, and the supernatant was used for estimation of proteolytic activities. It will be designated in the text by enzyme preparation.Initial screening of the most significant carbon and nitrogen sources allowing the maximum protease production was performed by one-variable-at-a-time approach. Seven different nitrogen sources (5 g/L), including casein peptone, meat peptone, urea, gelatine, sodium chloride, yeast extract, SP or ammonium sulphate and eight simple or complex carbon sources (10 g/L) including glucose, maltose, lactose, HGW, MJTP, SF, CHVSP, and casein, were tested. Initial M1 medium consists of (g/L): HGW 10.0;  for a low level and (+1) for a high level. The Plackett-Burman design is an efficient way to screen the main physicochemical parameters, required for elevated production, among a large number of process variables . The car2PO4, pH, and temperature) for enhancing protease production, screened by Plackett-Burman design, the three independent factors were investigated at three different levels , and the experimental design used for study is shown in A Box-Behnken design of RSM was employed to optimize the three most significant factors , was used for experimental designs and statistical analysis of the experimental data. The analysis of variance (ANOVA) was used to estimate the statistical parameters.The optimum pH of the enzyme preparation was studied over a pH range of 3.0–9.0 at 50°C using hemoglobin 1% (w/v). For studying pH stability, the crude enzyme was incubated in buffers of different pH values in the range of pH 3.0–9.0 for 1 h at 30°C. Residual proteolytic activity was then determined under standard assay conditions. The following buffer systems were used: 100 mM glycine-HCl buffer for pH 3.0, 100 mM sodium acetate buffer for pH 4.0–6.0, 100 mM potassium phosphate buffer for pH 7.0, 100 mM Tris-HCl buffer for pH 8.0, and 100 mM glycine-NaOH buffer for pH 9.0.To investigate the effect of temperature, proteolytic activity was tested at different temperatures using hemoglobin as a substrate for 5 min at pH 4.0. Thermal stability was examined by incubating the enzyme for 60 min at 30, 40, 50, and 60°C. Aliquots were withdrawn at desired time intervals to test the remaining activity at optimum conditions of pH and temperature. The nonheated enzyme was considered as the 100% control.2, MnSO4, ZnSO4, CoSO4, CuSO4, BaCl2, FeCl2, HgCl2, MgSO4, NaCl, and KCl.The effect of various metal ions at 5 mM on enzyme activity was investigated using CaClβ-mercaptoethanol, and ethylenediaminetetraacetic acid (EDTA). The enzyme preparation was preincubated with inhibitor for 60 min at 4°C, and then the remaining proteolytic activity was estimated using hemoglobin as a substrate. The activity of the enzyme without any additive was taken as the 100% control.The effects of inhibitors on protease activity were studied using phenylmethylsulfonyl fluoride (PMSF), pepstatin A, α-lactoalbumin (14.4 kDa).For zymography, casein was used as substrate. Casein-zymography was performed to estimate the number of the proteolytic activity in the crude supernatant. Zymography was performed in conjunction with SDS-PAGE according to the method of Garcia-Carreno et al.  with a sA. niger. Cultures were first conducted in medium M1 containing different carbon sources, each added at a concentration of 10 g/L. Protease activity was produced at high levels in the presence of complex organic carbon sources (−1) followed by CHVSP and MJTP . HGW and MJTP were tested as carbon sources since they contained high starch content 62 and 32.6 ± 2%, respectively. The easily assimilated simple carbon sources like maltose, glucose, and lactose resulted in weak acid protease production, 14.37, 3.33, and 1.24 U mL−1, respectively.A series of experiments was first carried out to study the effects of various simple and complex carbon and nitrogen sources on protease production by  sources . The higA. niger I1 produced high levels of protease on SP (82.3 U mL−1) followed by meat peptone, casein peptone, CHVSP, yeast extract, and gelatine . Ammonium sulphate and ammonium chloride showed weak acid protease production levels, 39.8 and 25.49 U mL−1, respectively. In general, both organic and inorganic nitrogen sources were used efficiently for protease production. In the present study, six organic  and two inorganic nitrogen sources (ammonium sulphate and ammonium chloride) were tested, at a concentration of 5 g/L, in M1 medium containing HGW at 10 g/L as carbon source . Among tA. niger I1. Thus, these substrates were selected for further optimization steps.Among the various carbon and nitrogen sources tested, HGW and SP were found to be the most suitable substrates for the production of acid protease by Analysed by design Expert, a first-order model was fitted to results obtained from the twelve experiments.t-test was used to identify the effect of each factor on protease production. 2PO4, initial pH, and temperature are the most significant factors (P < 0.05); they were selected for further optimization.The 2PO4, and initial pH were determined to be significant factors (P < 0.05) on the protease production. Among nonsignificant factors, six factors  exerted a negative effect while two variables (Na2HPO4 and SP) exerted positive effects on proteases production. The significant variables  were selected for further optimization by a response surface methodology. The nonsignificant variables with positive effect (Na2HPO4 and SP) were fixed at high levels, while variables with a negative effect  were fixed at low levels.According to 2PO4 concentration) for the protease production. A total of 17 experiments with different combinations of the three selected variables were performed. The design matrix with the corresponding results of Box-Behnken experiments, as well as the predicted results, is presented in P values for the model (<0.0001) and for “Lack of Fit” (0.0687) also suggested that the obtained experimental data was a good fit with the model. The regression equation coefficients were calculated, and the data were fitted to a first-order polynomial equation. The response of protease production (Y) by A. niger I1 can be expressed in terms of the following regression equation:Box-Behnken design was used to determine the optimum values of the three selected significant variables  was significant, as was shown by the low P value (0.0001) for the interactive terms.The ANOVA analysis of the optimization study  indicateR2 (multiple correlation coefficient) is 0.914. This value indicated that only 10.884% of the total variation was not explained by the model. The value of the adjusted determination coefficient (Adj-R2 = 0.894) further confirms the significance of the model. The value of the predicted determination coefficient pred2-R of 0.7841 is in reasonable agreement with the value of the adjusted determination coefficient Adj2-R of 0.8939. In addition, the model has an adequate precision value of 24.832. This suggests that the model can be used to navigate the design space  . In the Response surface plot was generally the graphical representation of the regression equation, from which the response (protease production) is plotted against any two variables Figures , while o2PO4 concentration. The model predicted that the maximum proteolytic enzyme production of 172.57 U mL−1 is achieved in the medium containing (g/L): HGW 5; SP 5; KH2PO4 1; Na2HPO4 1.6; CaCl2(7H2O) 0.4; ZnSO4 0.1; NaCl 0.3; MgSO4(7H2O) 0.5; pH 5.0.As shown in Figures R2 = 0.914), confirming the model's authenticity. In addition, the “Pre-R2” of 0.7841 is in reasonable agreement with the “Adj-R2” of 0.8939, indicating that this model can be used to navigate the design space  on the proteolytic activity of the enzyme preparation were studied at pH 4.0 and at 50°C by the addition of the respective cations or enzyme inhibitor to the reaction mixture. As shown in A. niger could result in a twofold benefit, namely, a substantial reduction in the cost of enzyme production and a hygienic treatment of the environment. Indeed, in the present work, shrimp peptone was found to be an excellent substrate for protease induction by A. niger I1. This may be due to the fact that shrimp wastes contain carbohydrates and minerals and a great amount (~40%) of proteins [In general, no defined medium has been carried out for the optimum production of proteases from different microbial strains . Each miproteins  and thenA. niger I1 was achieved with shrimp peptone as nitrogen source (82.3 U mL−1), followed by casein peptone (71.63 U mL−1) compared to the control with ammonium sulphate as nitrogen source (39.80 U mL−1). The addition of ammonium chloride or urea to the medium containing HGW as carbon source decreased protease synthesis to 25.49 and 3.66 U mL−1, respectively. Through these results, it can be concluded that shrimp peptone is an excellent source of nitrogen for production of extracellular proteases by A. niger I1.In general, both organic and inorganic nitrogen sources were used efficiently for protease production. The optimum proteolytic activity produced by A. clavatus ES1 is substantially influenced not only by carbon and nitrogen sources, but also by initial pH and temperature of the growth.Hajji et al.  studies 2PO4 concentration, temperature, and initial pH were found to influence enzymes synthesis. The optimization of these three parameters  resulted in a proteolytic activity of 183.13 U mL−1, with a 2.2 increase comparing to 82.3 U mL−1 obtained on the unoptimized medium containing 10.0 g/L HGW and 5 g/L SP.Indeed, in line with these studies, our study showed that KH2PO4 concentration was the most influential variable on proteolytic enzyme production by Cellulosimicrobium cellulans.Our findings are in accordance with those of Luciana and Suto  who demo2PO4, 1, Na2HPO4, 1.6, CaCl2, 0.4, ZnCl2, 0.1, NaCl, 0.3, MgSO4(7 H2O), 0.5; other salts were taken at their low levels as shown in Plackett-Burman design. By optimizing the medium composition and the culture conditions, not only the production of proteases was enhanced by 4.25-fold from 43.13 to 183.13 U mL−1 but also the cost of enzyme production was reduced since two cheap and readily available complex fermentation substrates HGW and SP were used.Finally, the maximum proteolytic enzyme production was achieved at the following conditions: temperature 30°C, agitation speed of 150 rpm, pH 5.0, and (g/L) HGW, 5, SP, 5; KHA. awamori [A. terreus [The use of statistical models to optimize culture medium components and conditions has increased in present-day biotechnology, due to its easy applicability, reliability, and validity. In the present study, the significant variables necessary for the enhancement of proteolytic enzyme production were selected using the Plackett-Burman design. The RSM applied to the optimization of proteases production in this investigation suggested the importance of a variety of factors at different levels. RSM was successfully applied in the production of proteases by  awamori  and A. t terreus .A. niger I1 was found to produce at least only one acid protease as revealed by casein zymography. Van den Hombergh et al. [A. niger. Nevertheless, other works reported the production of only one proteolytic enzyme by Aspergillus oryzae MTCC 5341 strain [In this study, h et al.  reportedPenicillium camemberti (pH 3.5) [Rhizopus oryzae (pH 5.5) [A. niger NRRL 1785 protease exhibited an optimum at pH 4.0 [The enzyme has its optimum activity at pH 4.0, and then protease activity decreased significantly below and above pH 4.0. Optimum pH values between 3.0 and 5.5 have been reported for other fungi proteases, such as the one of (pH 3.5)  and Rhiz(pH 5.5) . The A. t pH 4.0 .A. niger I1 was similar to those from other fungi proteases, such as the one of Neosartorya fischeri var. spinosa IBT 4872 [R. oryzae [P. duponti K1014 [P. oxalicum [Cryptococcus albidus [The crude enzyme showed maximum activity at 50°C on hemoglobin. The optimum temperature of IBT 4872 . Proteas. oryzae , P. dupoti K1014 , P. oxaloxalicum , and CryA. niger I1 protease were studied at pH 4.0 and 50°C. A. niger I1 acid protease was practically insensitive to the most metallic ions. So this property is in the view of potential food industry applications. None of the metallic ions enhanced protease activity. In this respect, the A. niger I1 enzyme resembles to the acid proteases of Hebeloma crustuliniforme [Mucor pusillus [A. niger strains [The effects of various metal ions at a concentration of 5 mM on the activity of iniforme  and Muco strains , 51, 52, strains .A. niger I1 on two local and low-cost substrates, HGW and SP.In conclusion, this work attempted to demonstrate the efficient use of Plackett-Burman and RSM approaches to determine the conditions leading to enhance extracellular protease production by


Monomorium Mayr occur worldwide and are considered important urban pests. The larvae of only a few species of this genus have been described, and these descriptions are either superficial or incomplete. This study aimed to determine the number of larval instars and describe the immature stages of the ant Monomorium floricola Jerdon (Formicidae: Myrmicinae). Specimens were analyzed and measured using light and scanning electron microscopy. Three larval instars were found, and all larvae had pheidoloid bodies with ectatommoid mandibles, consistent with other Monomorium species described previously. Five types of body hairs were described, and their distribution was instar-specific. Body and mandible dimensions of the larvae also were constant for each instar. Like other Myrmicinae, the larvae did not create a cocoon. Some of differences among the hair types and sensilla were observed by comparing the samples with larvae of other species in the genus, and these differences may have taxonomic utility.Some ant species of the genus  Monomorium Mayr is a cosmopolitan ant genus that includes some 586 described species and subspecies  and M. pharaonis are considered of even greater importance as they may be active carriers of pathogens inside hospitals  and Rio Claro (22°24′25.88″S and 47°33′31.37″W) in São Paulo, Brazil, and later reared in the laboratory (temperature 25±2°C and 60±10% relative humidity). From these colonies, immatures were collected for this study.Nests of The present work focused on describing worker larvae only, as reproductive larvae can be very similar to worker larvae and thus difficult to distinguish. Knowing the general morphology of worker larvae will help identify reproductive larvae. Reproductives are usually produced during intermittent periods in much lower numbers. Therefore, care was taken to collect larvae only during periods when no reproductive forms were being produced. Workers of this species are monomorphic.Voucher specimens of eggs, larvae, and pupae were deposited in the “Adolph Hempel” entomological collection of Centro de Pesquisa e Desenvolvimento de Sanidade Vegetal of Instituto Biológico, São Paulo, Brazil. All terminology used in our larval descriptions follows Wheeler and Wheeler .n = 344) and plotted in a frequency distribution graph, wherein every distinct peak was considered to correspond to a different larval instar; the obtained number of larval instars was then tested against Dyar's rule  and a scanning electron microscope , using ten larvae of each instar for each method. The ten larvae were selected among the ones presenting the most frequent head width found for the respective instar. With a stereomicroscope  equipped with a micrometric eyepiece, the length and width of eggs (n = 159) and larvae (n = 50 for each instar), and length of pupae (n = 50) were rapidly measured. Also, body length between spiracles, a mode of measuring larvae devised by Wheeler and Wheeler . Dried specimens were then attached to aluminium stubs with double-faced conductive, adhesive tape and were goldsputtered with a Balzers SCD/050 sputterer. Observations and images were obtained as soon as possible from sample preparation. Prior to analysis under the light microscope, the larvae were warmed for 10 min in KOH 10%) and placed in a small drop of glycerine on a microscope slide.All collected samples were fixed in Dietrich's solution  for 24h. The samples were then transferred and conserved in 80% alcohol. For scanning electron microscope analysis, the samples were dehydrated in an acetone graded series , and critical-point dried . Mean growth rate through all instars was 1.23, while the growth rate was 1.23 between the first and second instars, and 1.22 between the second and third instars. Descriptions of the immature stages are as follows.The frequency distribution of the maximum head widths of the larvae formed a multimodal curve with three distinct peaks , suggestMorphological description of the immature forms Egg Ovoid in shape; chorion thin and transparent; length 0.29 ± 0.01 mm, varying 0.26–0.34 mm; width 0.18 ± 0.02 mm, varying 0.15–0.20 mm; length : width ratio was 1.61.BODY: Whitish, slender, anterior somites clearly visible, anus subterminal ; profilen = 5) uniformly distributed and organised in rows following body segmentation: most are type A hairs  and others are type B hairs  (n = 100); first thoracic pair larger than the others, which are all of roughly the same size. Body length through spiracles 0.57 ± 0.17 mm, varying 0.33 – 0.76 mm (n = 10).Body hairs   Table 2n = 5 uni; n = 6)  measurinn = 32); subcircular, with a slit-like opening just under the border between the gena and the first thoracic segment (n = 10), but in some specimens there was a type B hair (0.010 mm long) on the gena, instead of a type A hair. Well defined clypeus with no sensilla.HEAD CAPSULE: Cranium 0.12 mm wide ( segment . Twenty-n = 3) over the anterior surface. Mandibles 0.040 ± 0.003 mm long, varying 0.035 – 0.045 mm (n = 10); profile ‘ectatommoid’ as defined by Wheeler and Wheeler  uniformly distributed in rows following body segmentation; most were type C hairs  (n = 5) (n = 2) (n = 100); first thoracic pair being larger than others, which are of similar size. Body length through spiracles was 0.77 ± 0.07 mm, varying 0.68 – 0.87 mm (n = 10).BODY: Whitish; slender and pheidoloid in profile; distinct anterior somites; anus subterminal . Body ha n = 15)  Table 2n = 5 uni (n = 5) , type E  (n = 2) , and som (n = 2)  (Table 2n = 38); subcircular. Antennae with three basiconic sensilla over a slight elliptical elevation (0.006 mm wide and 0.004 mm high) (n = 4) and type D hairs 0.015 – 0.023 mm long (n = 9). Well defined clypeus with no sensilla.HEAD CAPSULE : Craniummm high) . Twenty-n = 9) over its anterior surface, 4 setaceous sensilla  and 2 basiconic sensilla over the ventral surface, always a basiconic one in association with a setaceous one. Mandibles 0.051 ± 0.002 mm long varying 0.048 – 0.055 mm (n = 10), ectatommoide in shape, moderately sclerotized and bearing three teeth, with a basiconic sensillum at the base. Maxilla conoidal (n = 9); in some specimens one of the hairs was replaced by a setaceous sensillum (0.003 mm long). Maxillary palpus a ‘skewed peg’  with 2 setaceous sensilla ; galea digitiform  with 2 setaceous sensilla  on the top. Labium elliptical, 0.05 mm long and devoid of spinules; digitiform labial palpus culminating with a setaceous sensillum  and with 4 type A hairs  on its ventral border. There was a basiconic sensillum beneath each extremity of the opening of sericteries, which is a horizontal slit.MOUTHPARTS : Labrum conoidal , 0.033 mn = 5) uniformly distributed and organized in rows following body segmentation; most are type C hairs , but also type B hairs , type D hairs  and type E hairs  are present and located on ventral region, thorax (n = 100); first pair bigger than others, which were all of roughly same size. Body length through spiracles 1.16 ± 0.22 mm, varying 0.84 – 1.46 mm (n = 10).BODY: Whitish; slender and pheidoloid in profile; distinct anterior somites; anus subterminal . Body ha, thorax  and arou, thorax . Body le, thorax  measurinn = 89); subcircular. Antennae are slight elliptic 0 elevations (0.007 mm wide and 0.004 mm high) with three basiconic sensilla (n = 8) and type D hairs 0.019 – 0.022 mm long (n = 4). Well-defined clypeus with no sensilla.HEAD CAPSULE: Cranium 0.18 mm wide (sensilla . Twenty-n = 11) over the anterior surface, 4 setaceous sensilla  and 2 basiconic sensilla over the ventral surface; each basiconic sensillum came in association with a setaceous sensillum. Mandibles 0.056 ± 0.005 mm long, varying 0.048 – 0.063 mm (n = 10); ectatommoid in shape (n = 4); maxillary palpus a skewed peg with 2 setaceous sensilla ; galea digitiform culminating with 2 setaceous sensilla . Labium elliptical, 0.05 mm long; smooth surface; labial palpus digitiform with 1 setaceous sensillum on the top  and 4 type A hairs ; a basiconic sensillum placed on each extremity of the opening of sericteries .M. floricola was the same as in M. pharaonis .From comparing the body measures of haraonis , it was M. floricola larvae confirm the statement made by Wheeler and Wheeler  of the genera, spiracles are relatively small and uniformly sized . The firarcticum ; this coarcticum , but othM. floricola fit this pattern.As stated by Wheeler and Wheeler , all antM. floricola, previously described by Wheeler and Wheeler (Monomorium larvae described so far and confirming general similarities, e.g. the body and mandible formats. Also, the number of instars for this species was established. The different types of hair described and other findings that have direct application for instar separation with this species are particularly important. Some of the information herein may aid future systematic and taxonomic studies with the group, as well as help clarify some aspects of the biological and social organization of this ant.Finally, this study presented a deeper analysis of the morphology of the larvae of  Wheeler , while c