**3.1 Insect endogenous metabolites**

## *3.1.1 Lipids*

Lipids are basic cell components and play important roles in insect development and reproduction, such as maintenance of cell membrane structure and intra or extracellular signaling [39–41]. For example, glycerophospholipids, phosphatidylcholines, and phosphatidylethanolamines are basic components of cell and lysophospholipids have an important function in inflammation, abiotic stress, and biotic stress signal transmit [42]. MSI has been widely applied in many aspects in model insect *Drosophila melanogaster*, such as the neutral lipids three-dimensional spatial distribution on the surface adults [43, 44], body lipid distribution [45], brain lipid structure [46, 47], wing lipids [34, 44], Malpighian tubule phospholipid distribution [48], and phospholipids in the brain treated with cocaine [49]. Moreover, MSI detected and localized the composition and distribution of triacylglyceride in *Aedes aegypti*, phospholipid and phosphatidylcholine in *Anopheles stephensi* [42], and phospholipids in *Schistocerca gregaria* [37].

## *3.1.2 Neuropeptides*

Neuropeptides, a kind of structurally diverse signaling molecules, can control and regulate fundamental physiological functions such as growth, reproduction, and environmental stress tolerance in animals [50]. MSI detected and localized the distribution of 14 neuropeptides in coronal brain sections in all development stages

**65**

**Species** **Endogenous metabolites**

*Anopheles stephensi*

*Aedes aegypti* *Apis mellifera*

Whole-body

Ovarian

follicles

Brain Brain Brain Venom

*Drosophila melanogaster*

Body Brain Brain Collar Brain &

head

Malpighian

Lipids

MALDI

5% CMC

12

DHB、DAN

[48]

tubule

Surface Whole-body Whole-body

Wing Head to

abdomen

Whole-body

Defensive compounds

AP-SMALDI

10% Tragacanth

16

DHB

[35]

gum

*Graphosoma lineatum*

*Paederus riparius*

Lipids Non-polar compounds

DAPPI

*/*

/

Lipids

Neuropeptides

Lipids

MALDI AP-SMALDI

MALDI

SIMS

PBS

/

5% CMC 10% Gelatin

20

20

/

/

LiDHB

DHB DHB DHB

/

[43]

[29]

[45]

[34, 44]

[57]

Peptide

Lipids Phospholipid

Lipids GABA

L-arginine Venom toxins

Neuropeptides

Protein

MALDI MALDI MALDI MALDI MALDI

SIMS SIMS SIMS MALDI

4% CMC

15

10% Gelatin

10

10% Gelatin

12

Agarose 10% Gelatin

15

10

/

10

/

12

/

12

/

14

CHCA

CA DHB CHCA CHCA

/ / / CHCA

Lipids Lipids

AP-SMALDI

3D-SIMS

5% CMC

/

100

20

DHB

/

[42]

[41]

**Tissue**

**Major analyte**

**Method**

**Embedding medium**

**Thickness** 

**Matrix**

**Ref.**

**(μm)**

*Current Advances in Mass Spectrometry Imaging for Insect Physiology and Metabolism*

*DOI: http://dx.doi.org/10.5772/intechopen.92584*

[51, 52]

[32]

[53]

[54]

[55]

[47]

[49]

[46]

[56]


#### *Current Advances in Mass Spectrometry Imaging for Insect Physiology and Metabolism DOI: http://dx.doi.org/10.5772/intechopen.92584*

*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

e.A chemical matrix is applied to promote desorption and ionization. Matrix is coated by a sprayer/nebulizer or by solvent-free sublimation to acquire homo-

f. After matrix deposition, the target is inserted into the instrument, for which experimental parameters (e.g., laser energy, step size of plate movement, and a

g.A laser beam is emitted for desorption to acquire mass spectra at every x and y grid points within the scanning area, so to visualize target ions and convert the

h.Hematoxylin-eosin staining is optional for displaying tissue localization.

**3. Application of mass spectrometry imaging in entomological** 

understanding of insect physiology and metabolism (**Table 1**).

MSI can visualize the spatial and temporal distributions of molecules. Endogenous metabolites, exogenous metabolites, and insect-plant interactions are three main aspects of MSI application to insect tissue section for *in situ* characterization. Endogenous metabolites refer to lipids, neuropeptides, proteins, and defense compounds [32–35]; exogenous metabolites are drugs and insecticides [36, 37]; insect-plant interactions are associated with the fate of plant secondary defense compounds in insects [38]. We summarize the major applications of MSI for a better

Lipids are basic cell components and play important roles in insect development and reproduction, such as maintenance of cell membrane structure and intra or extracellular signaling [39–41]. For example, glycerophospholipids, phosphatidylcholines, and phosphatidylethanolamines are basic components of cell and lysophospholipids have an important function in inflammation, abiotic stress, and biotic stress signal transmit [42]. MSI has been widely applied in many aspects in model insect *Drosophila melanogaster*, such as the neutral lipids three-dimensional spatial distribution on the surface adults [43, 44], body lipid distribution [45], brain lipid structure [46, 47], wing lipids [34, 44], Malpighian tubule phospholipid distribution [48], and phospholipids in the brain treated with cocaine [49]. Moreover, MSI detected and localized the composition and distribution of triacylglyceride in *Aedes aegypti*, phospholipid and phosphatidylcholine in *Anopheles stephensi* [42],

Neuropeptides, a kind of structurally diverse signaling molecules, can control and regulate fundamental physiological functions such as growth, reproduction, and environmental stress tolerance in animals [50]. MSI detected and localized the distribution of 14 neuropeptides in coronal brain sections in all development stages

d.A digital image of the sample with fiducials is acquired;

geneous matrix coverage over the entire tissue surface;

ion's intensity into a color scheme;

**3.1 Insect endogenous metabolites**

and phospholipids in *Schistocerca gregaria* [37].

**researches**

*3.1.1 Lipids*

selected region of the tissue) are optimized to scan the image;

**64**

*3.1.2 Neuropeptides*


*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

**Table 1.**

**67**

*3.2.1 Insecticides*

*Current Advances in Mass Spectrometry Imaging for Insect Physiology and Metabolism*

sensitivity, which can facilitate to detect peptides in low abundance.

of *D. melanogaster* [29]. These neuropeptides play important roles in physiological processes (e.g., allatostatins and tachykinin-like peptides participate in odor perception and locomotor activity). Neuropeptides can act as transmitters or neuromodulators in the central nervous system [33]. Neuropeptides in the brain of *Apis mellifera* are related to the functional division of the population and their activities. Worker bees' neuropeptide levels at the age of 0–15 d increased with the in-hive activities but decreased with out-hive activities (guarding and foraging) at 15–25 d [51]. Further study proved that allatostatin and tachykinin-related neuropeptides in the brain of worker bees were related to aggressiveness behaviors [52]. Neuropeptides distribution in the retrocerebral complex of *Periplaneta Americana* revealed the differentiation of prohormone processing and the distinctness of neuropeptides-based compartmentalization [33]. These studies proved that MSI has the advantages of

As a kind of macromolecules, proteins are fundamental compounds of organisms and take part in important cellular processes, such as DNA replication and metabolisms. MSI can simultaneously and specifically detect the spatial distribution of massive proteins and overcome antibody cross-contamination. MSI system has been used to evaluate the negative impacts in the brain of *A. mellifera* exposing to a sublethal concentration of imidacloprid. The system has successfully visualized the distribution of 24 proteins (e.g., cytochrome P450s, glutathione S-transferases, and heat shock protein 70s). Besides, 8-day exposure to imidacloprid triggered biochemical changes in *A. mellifera* brain (e.g., up-regulated acetylcholinesterase and amyloid precursorlike protein and down-regulated cytochrome P450 and disulfide-isomerase protein). This could influence the well-being of *A. mellifera* (e.g., learning and memory acquisition, maintaining neuronal integrity, detoxification, and apoptosis) [32].

In addition to lipids, neuropeptides, and proteins, MSI can also be used to visualize the distributions of defensive compounds, special proteins (e.g., venom allergens and toxins) and other small molecules (e.g., betaine and amino acids). Defensive compounds (e.g., pederin, pseudopederin, and pederon) were detected and localized in the organs of *Paederus riparius* [35]. Three venom allergens and two venom toxins were mapped in the honeybee [54]. Poison sac was the lactation of main venom proteins in *Solenopsis invicta* [59]. Nonpolar compounds (e.g., (*E*)-1-nitropentadec-1 ene and (*E*)-hex-2-enal) can be detected from the head to the abdomen in two model insects, *Prorhinotermes simplex* and *Graphosoma lineatum*. Gland openings and gland reservoirs were the most active areas in *P. simplex* and *G. lineatum* [57]. Other small molecules (e.g., betaine and amino acids) were detected in *Schistocerca gregaria* [37]. Semiochemicals were mapped on the surface of the adults of *D. melanogaster* [43]. Two male-specific sex pheromones were localized in the ejaculatory bulb of *D. melanogaster* [45]. MSI can also be used as a novel *in situ* metabonomic tool to study the metabolism

of L-arginine of the honeybee brain in response to proboscis extension [53].

MSI can be applied to visualize the distribution of insecticides in insects and their negative influence on the target insects. Imidacloprid was used to study its

**3.2 Insect exogenous metabolites**

*DOI: http://dx.doi.org/10.5772/intechopen.92584*

*3.1.3 Proteins*

*3.1.4 Others*

*Overview of the application of MSI in insect sciences.*

#### *Current Advances in Mass Spectrometry Imaging for Insect Physiology and Metabolism DOI: http://dx.doi.org/10.5772/intechopen.92584*

of *D. melanogaster* [29]. These neuropeptides play important roles in physiological processes (e.g., allatostatins and tachykinin-like peptides participate in odor perception and locomotor activity). Neuropeptides can act as transmitters or neuromodulators in the central nervous system [33]. Neuropeptides in the brain of *Apis mellifera* are related to the functional division of the population and their activities. Worker bees' neuropeptide levels at the age of 0–15 d increased with the in-hive activities but decreased with out-hive activities (guarding and foraging) at 15–25 d [51]. Further study proved that allatostatin and tachykinin-related neuropeptides in the brain of worker bees were related to aggressiveness behaviors [52]. Neuropeptides distribution in the retrocerebral complex of *Periplaneta Americana* revealed the differentiation of prohormone processing and the distinctness of neuropeptides-based compartmentalization [33]. These studies proved that MSI has the advantages of sensitivity, which can facilitate to detect peptides in low abundance.

## *3.1.3 Proteins*

*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

**66**

**Species** *Periplaneta americana*

Brain Neuroendocrine

tissues

Head to

Non-polar compounds

DAPPI

/

/

/

[57]

abdomen

Venom

Venom proteins

MALDI

Gelatin

14

DHB

[59]

*Prorhinotermes simplex*

*Solenopsis invicta*

**Exogenous metabolites**

*Drosophila melanogaster*

*Schistocerca gregaria*

*Helicoverpa armigera*

**Insect-plant interaction system**

*Aphis glycines* *Athalia rosae* *Chorthippus dorsatus*

**Others**

*Acromyrmex echinatior*

Ants *Bombus terrestris*

**Table 1.** *Overview of the application of MSI in insect sciences.*

Nest Propleural

Fungus

plate

Whole-body

/

MALDI

pHPMA

12

DHB、SA

[67]

Microbial

MALDI MALDI

/

/

/

/

DHB、CHCA

DHB

[65]

[66]

Feeding leaf Whole-body

Gut, feces

Metabolites

Metabolites Glucosinolates

MALDI MALDI

LDI

1% PBS

12

Water

15

/

/

DHB、DAN

CHCA DHB、DAN、CHCA

[38]

[62, 63]

[64]

Whole-body Whole-body Whole-body

Biopesticide

Drugs

Insecticide

MALDI

DESI MALDI

/

16

5% CMC

50

10% Gelatin

15

DHB

/ DHB

[36]

[37, 60]

[61]

Neuropeptides Neuropeptides

MALDI MALDI

Gelatin Paraffin

20

14

CHCA

DHB

[33]

[58]

**Tissue**

**Major analyte**

**Method**

**Embedding** 

**Thickness** 

**Matrix**

**Ref.**

**(μm)**

**medium**

As a kind of macromolecules, proteins are fundamental compounds of organisms and take part in important cellular processes, such as DNA replication and metabolisms. MSI can simultaneously and specifically detect the spatial distribution of massive proteins and overcome antibody cross-contamination. MSI system has been used to evaluate the negative impacts in the brain of *A. mellifera* exposing to a sublethal concentration of imidacloprid. The system has successfully visualized the distribution of 24 proteins (e.g., cytochrome P450s, glutathione S-transferases, and heat shock protein 70s). Besides, 8-day exposure to imidacloprid triggered biochemical changes in *A. mellifera* brain (e.g., up-regulated acetylcholinesterase and amyloid precursorlike protein and down-regulated cytochrome P450 and disulfide-isomerase protein). This could influence the well-being of *A. mellifera* (e.g., learning and memory acquisition, maintaining neuronal integrity, detoxification, and apoptosis) [32].
