Lipase enzyme activity assay in P. xylostella larva midguts

The Lipase Enzyme Activity Assay in Plutella xylostella Larva Midguts

Zhen Tao^{1, #}, Chen Chen^{1, #}, Hongyi Chen¹, Shijie Huang¹, Peijin Li^*

¹ The National Key Engineering Lab of Crop Stress Resistance Breeding, the School of Life Sciences, Anhui Agricultural University, Hefei, China

^# These authors contribute equally to this work.

^*Corresponding author: Peijin Li, Peijin.li@ahau.edu.cn

[Abstract]

The digestive ability of Plutella. xylostella (P. xylostella), one of the most important and destructive crop pests, is affected by various internal and external environmental conditions such as temperature, pH and forage types. We describe a method of lipase activity assay with minor modifications based on the procedure from Nanjing Jiancheng Biological Institute, China, to monitor the influence of diverse forages on digestive component of P. xylostella larvae. We disinfect the surface of 3-instar P. xylostella larva bodies and dissect their midguts for lipase activity assays, the obtained results reliably coincide with the physiological and phenotypic changes of the larvae upon 48-h treatment of the natural plant product (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). This method can be applied for the quick assays of lipase activity of P. xylostella larvae or other Lepidoptera insects after various experimental treatments, to represent the digest capacity of the larvae.

[Keywords]

Insect, midgut, lipase activity assay, P. xylostella larvae

[Background]

In agriculture, crops face series of insect pests that threat the food safety across the world such as P. xylotella, one of the most important Lepidoptera pests, which causes tremendous economic loss for cruciferae crops including rape, cauliflower and cabbages, so currently it's still a great task to learn developmental characters of insects and develop effective method to reduce the detriment. Recently from model plant Arabidopsis thaliana, we identified a natural plant product-DMNT, which significantly affects the development of P. xylostella larvae and disrupts the peritrophic matrix barrier in P. xylotella  midgut¹.

      To characterize the physiological influence of DMNT on P. xylostella, we applied a method for the assaying of lipase activity, which has been used for Bombyx mori after chlorantraniliprole exposure². Lipase is one of the major digestive enzymes in insect midgut, which is highly linked to the important biological process of insect digestion, absorption and lipid reconstruction, and thus reflecting the digestive ability of insects³. This method can be applied to the analysis of various insects to represent their digestive capacity in response to internal/external stresses or experimental treatments. 

Materials and Reagents

1. P. xylotella larvae (from Keyun Biological Company, China)
2. Kit for the assay of lipase activity (from Nanjing Jiancheng Biological Institute, catalog: A054-1-1)
3. Dissection knifes, pin-pointed forceps
4. 75% Ethanol
5. DMNT (Prepared by Li`s lab)
6. Paraffin oil (Miner oil)

Equipment

1. 37 ˚C water bath (Shanghai Yiheng Company, China, DK-8AXX)
2. Spectrophotometer (DeNoVix, Ds-11+)

Procedure

Before dissecting guts, the surface of whole larva body should be disinfected with 75% ethanol, and the lipase activity of P. xylostella larvae was assayed with a commercial kit from Jiancheng Company (catalog: A054-1-1).

1.  DMNT was diluted in paraffin oil (Sigma-Aldrich: 8042-47-5) to reach the final concentration as 0.1 mg/mL.
2.  15 μL 0.1 mg/mL DMNT was injected into forages to bring the final concentration in forages to 7 μM.
3.  The forages containing DMNT was used to feed P. xylotella larvae, the forages containing the same amount of paraffin was used a control. After certain time of feeding as desired, the larva guts were collected for lipase activity assay.
4.  Before the assays, the substrate buffer needs to be warmed to 37˚C for at least 5 min. Meanwhile, set up the spectrophotometer at 420 nm. 
5.  P. xylostella larvae were dissected with forceps and knifes, to get the entire midgut of each larva. The midguts were quickly frozen in liquid nitrogen for future use.
6.  The entire midguts from 90 P. xylostella larvae were fully grounded with liquid nitrogen, and the powder was moved to a clean tube containing 0.9% normal saline solution (mass/volume= 1:4). The tissue homogenate was gently and fully mixed on a rotator at 4˚C, and the supernatant was collected after centrifugation at 13000 rpm at 4˚C for 10 mins (Eppendorf, 5427R).
7. 25 μL of the supernatant was transferred it to a new 10 mL centrifuge tube and well mixed with 25 μL buffer #4 and 2 mL substrate buffer (pre-warmed).
8.  The mixture was quickly moved to a spectrophotometer to check the absorbance at 420 nm at 30 s (designated: A1).
9.  The mixture was then transferred to a water bath set to 37˚C for incubation. After 10 min, the mixture was transferred to the spectrophotometer to measure the absorbance at 420 nm at 30 s (A2).
10.  As a control, 2 mL substrate solution was mixed with 50 μL 0.9% NaCl solution for absorbance measurement at 420 nm (As). This As value is equivalent to the absorbance value of the concentration of the standard tube (454 umol/L).
11.  Enzyme activity was finally calculated according to the equation:

[(A1–A2)/As]*(454 μmol/L)*[reaction volume (2.05 mL)/sample volume (0.025 mL)]/reaction time (10 min)/protein concentration of sample (gprot/L).

Data analysis

The data were analyzed with SPSS (SPSS Inc. Released 2007. SPSS for Windows, Version 16.0. Chicago, SPSS Inc).

References

1       Chen, C. et al. Volatile DMNT directly protects plants against Plutella xylostella by disrupting the peritrophic matrix barrier in insect midgut. eLife 10, doi:10.7554/eLife.63938 (2021).

2      Hu, J. et al. The mechanism of damage in the midgut of Bombyx mori after chlorantraniliprole exposure. Ecotoxicology 28, 903-912, doi:10.1007/s10646-019-02089-z (2019).

3      Santana, C. C. et al. Lipase activity in the larval midgut of Rhynchophorus palmarum: biochemical characterization and the effects of reducing agents. Insects 8, doi:10.3390/insects8030100 (2017).

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