KEYWORDS
Locust, Locusta migratoria , Occupational allergy, Hexamerin-2
protein, Immunoblotting
1l INTRODUCTION
Allergy may be defined as a
hypersensitive reaction of the immune system to a definite stimulus,
termed allergen, at a dose tolerated by normal
subjects.1 Occupational respiratory allergy is a
global health problem; occupational allergic sensitization has exceeded
25%, and approximately 10% of exposed individuals develop
asthma.2,3 Occupational exposure to insects induces a
high prevalence of allergic sensitization, which can lead to
clinical
inhalant allergies and contact
allergies.4,5 Insect species of at least 12 different
orders have been implicated in the promotion of inhalant allergies in
humans, including locusts, cockroaches, and
honeybees.6,7Orthopteran
insects, including grasshoppers, locusts, crickets, and cockroaches, are
important occupational hazards because of the severe symptoms caused by
insect allergens.8,9
Locusts are not only one of the world’s most destructive agricultural
pests but also model systems in many laboratories for studying
behaviour, physiology, neuroscience, and phenotypic
plasticity.10,11 Several cases of occupational
allergies have been described as allergies to the migratory locust,
American locusts and grasshoppers, with symptoms of asthma, dermatitis,
rhinitis and even shock.12 In 1953, a survey of locust
sensitization of the personnel of several institutions for locust
research indicated that out of 34 workers in two locust research
stations, four individuals showed allergic symptoms, and 14 others
showed atopy.13 Moreover, a survey of the work-related
symptoms indicated wheezing or breathlessness accounted for 26% of the
symptoms, followed by rhinitis,
asthma and urticaria.14 Questionnaires, skin prick
tests (SPTs) and specific IgE (sIgE) measurements showed that 25%-34%
of individuals working in a research centre were allergic to
locusts.8,9 Even dead locusts can induce
severe contact urticaria in
zookeepers during feeding of reptiles with the migratory
locusts.15 Therefore, identification of the major
allergens from locusts is urgently needed to diagnose locust-specific
allergies and to assess the environmental conditions in work
environments.
Research on the identification of allergens from the migratory locusts
is highly preliminary, although many proteins are considered possible
allergens to humans. Among the proposed locust protein spectra, the most
likely allergens identified by immunoblotting were approximately 68, 43,
37, 29, and 18 kDa in molecular weight.9 However, the
allergens related to IgE from 10 locust laboratory workers were reported
to be 70, 55, 35 and 30 kDa in molecular weight.7 In
one recent study, isolation of proteins from patients allergic to
locusts showed that the allergens were approximately 130, 70, 38, 29 and
19 kDa in molecular weight.16 The above inconsistent
findings suggested that the major allergens of locusts and their
molecular nature remain unclear.
In our laboratory, the migratory locust (Locusta migratoria ) has
been studied as a model system for 30 years. The students and staff in
our team have exposure history from rearing and breeding locusts,
providing us the opportunity to investigate occupational locust allergy.
Our research
focused on the factors determining
the incidence of allergy in specific exposure settings and the patterns
of immunological responses.
2 l METHODS
2.1 l Subjects
The occupational allergic questionnaire surveys covered two parts. One
was
a
retrospective survey of locust allergy in cohort from a locust
laboratory from 2000 to 2019. In this survey, we investigated 94
students and staff (49 males, 45 females)
who worked on locust biology,
rearing and breeding locusts at the Institute of Zoology, Chinese
Academy of Sciences (Beijing, China). The other was a
cross-sectional
survey of 57 scientists and students (24 males, 33 females) in 2020 in
our two laboratories at the Institute of Zoology, Chinese Academy of
Sciences (Beijing, China) and College of Life Science, Hebei University
(Baoding, Hebei Province, China) (Table 1).
Serum samples were collected from 57 subjects with history of exposure
to locusts and 10 healthy controls without exposure history and allergy
symptoms from Beijing Tongren Hospital (Table 1). Total IgE levels and
sIgE levels for common inhalant allergens were measured by the UniCAP
allergen detection system (Pharmacia Diagnostics, Uppsala, Sweden).
Written informed consent was obtained from all participants.
2.2 l Preparation of locust protein extracts
Migratory
locusts reared in cages (30 cm × 30 cm × 32 cm) with a diet of fresh
wheat seedlings were obtained from colonies maintained in the College of
Life Science, Hebei University, Hebei Province, China. Samples of frozen
migratory locust were ground to a powder in a mortar and pestle using
liquid nitrogen.
The
samples were solubilized in PBS
(0.01 M, pH 7.2), the mixture was
centrifuged at 15,000 rpm for 15 min at 4°C, and
the
supernatant was collected. The total protein content was determined by
the BCA method.
2.3 l Skin prick test
For the SPT, participants did not take any medications
(antihistamines, steroids, and other
drugs) for at least 2 weeks. SPT against
crude
protein extracts (2.0 mg/ml) of locusts and commercial extracts ofBlattella germanica (produced by Beijing Union Hospital) was
conducted in the
Beijing
Tongren Hospital, Beijing, China. Histamine was used as the positive
control, and 0.9% saline was used as the negative control. The SPT
results were recorded 15 min later. The wheal size was calculated as the
mean of the longest diameter and the length of the perpendicular line
through its middle. The ratio of the allergen wheal size to histamine
wheal size was the skin index. The skin index was expressed as 2+ when
the wheal diameter was half that of the positive control and 3+ when the
mean wheal diameter was equal to that of the positive control. Allergy
to locust was defined as skin index ≥ 2+ combined with allergic
symptoms. Atopy to locust was defined as skin index ≥ 2+ but without
allergic symptoms after locust exposure. Non-atopy was defined as skin
index < 2+ and no allergic symptoms after locust exposure.
2.4 l ELISA of sIgE for locust protein extracts
The concentration of the crude protein extracts was adjusted to 500
μg/ml
in 0.01 M PBS (pH 7.2), and the extracts were added to 96-well
polystyrene microtiter plates (100 μl/well) (BBI, Sangon Biotech, China)
and incubated overnight at 4°C. Then, the cells were blocked with
skimmed milk in PBS (5%) at 37°C
for 2 h and washed 3 times in TBS containing 0.05% Tween 20 (TBST).
Wells were then emptied and coated for 2 h with
100
μl of sera from individuals and 10 healthy controls (1:30, diluted with
5% skimmed milk in PBS)
at
37°C. The bound IgE was then reacted with 100 μl of horseradish
peroxidase (HRP)-labelled goat anti-human IgE (1:3,000 dilution).
3,3′,5,5′-Tetramethylbenzidine (TMB) was used as the substrate, and the
reaction was stopped by 50 μl of 2 M
H2SO4.
2.5 l SDS-polyacrylamide gel electrophoresis analysis
and IgE immunoblotting
The crude protein extracts were separated in triplicate by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples (50 µg) were
loaded onto a SDS-PAGE gel (10% separating gel and 4% stacking gel) at
20 μl/lane and electrically separated by the
Mini-PROTEIN
Tetra system (Bio-Rad; Hercules, CA, USA). The gels were stained with
Coomassie Brilliant Blue R-250. For immunodetection of IgE-binding
proteins, the separated proteins were electroblotted onto a PVDF
membrane (Immobilon, Merck Millipore, Germany) by applying a
constant current of 350 mA for 55
min at room temperature. Then, the membrane was
blocked with 5%
skimmed milk in TBST and incubated
overnight at 4°C with a 1:30 dilution in 5% skimmed milk of pooled
sera, the 9 individual sera and a negative control of pooled sera from
10 healthy individuals. Bound IgE antibodies were detected using
HRP-conjugated goat anti-human IgE
(Abnova, Taiwan) at a dilution of
1:10,000 in 5% skimmed milk. The membrane was incubated with
Super
Signal ECL Chemiluminescent Substrate (Thermo Scientific, USA). Finally,
immunoreactive bands were visualized using
Fusion
Fx (Vilber Lourmat, France).
A band at approximately 70 kDa from the locust proteins, recognized by
immunoblotting of the pooled sera of locust-allergic patients,
was analysed by LC-MS/MS, and the
peptide sequences obtained were aligned with the migratory locust
proteome
at Beijing Protein Innovation.
2.6 l Cloning, expression, and purification of the
hexamerin-2 protein from locust
Total RNA was isolated from the whole body of migratory locusts by using
TRIzol reagent (Invitrogen, Thermo, USA). RNA was reverse-transcribed to
cDNA using a reverse transcription kit (Promega, USA). Complete open
reading frames (ORFs) for the hexamerine-2 protein were amplified from
the cDNA using the PCR primers HEXA-2-F (5’-GTGCTGCATCGCGAGGACAC-3’) and
HEXA-2-R (5’-GTCGAAATGCGCGTTGGGCA-3’). A
construct
was created by cloning the hexamerin-2 gene into the plasmid pET 30a
vector and expressed in Escherichia coli BL21. Expression and
purification of the hexamerin-2 protein were then conducted by
Beijing
Protein Innovation.
2.7 l Immunological tests of the hexamerin-2 protein in
sera from patients with occupational locust allergy
For the ELISA, the concentration of the crude locust proteins was
adjusted to 500 μg/ml in 0.01 M PBS (pH 7.2), applied to 96-well
polystyrene microtiter plates (100 μl/well) (BBI, Sangon Biotech, China)
and incubated overnight at 4°C. After this, the ELISA was continued as
previously described.
For the immunoblotting assay, SDS-PAGE-resolved purified proteins were
electro-transferred onto a PVDF membrane (Immobilon, Merck Millipore,
Germany) for immunoblotting
analysis as described above. Membranes were incubated with patient sera,
diluted 1:30 (v/v) diluted in 5% skim milk-TBST, overnight at 4℃ under
constant agitation and probed with 1:10,000 (v/v) anti-human IgE
peroxidase antibody (Abnova, Taiwan) for 1.5 h at room
temperature.
Fluorescence was visualized by ECL
Chemiluminescent Substrate as described previously.
2.8 l Competitive inhibition ELISA
In inhibition experiments, coating of the microtiter plates was
performed as described above for ELISA.17 Two
individual sera that showed positive reactions against the hexamerin-2
protein were chosen for inhibition studies. Sera were first incubated
for 1 h at room temperature with an equal volume of the inhibitor
solution. The final concentrations of the purified proteins used as
inhibitors ranged from 0.005 μg/ml to 500.000 μg/ml. Mixtures containing
the sera and the inhibitor (or serum without inhibitor as a control)
were incubated in the wells for 2 h at room temperature. After this, the
ELISA was continued as previously described.
2.9 l Statistical analysis
Statistical significance was analysed by using GraphPad Prism 6.0
(GraphPad Inc., California, USA) and SPSS 24.0 software (SPSS Inc.,
Chicago, USA), and the values are presented as the mean ± S.D. (standard
deviation). A t-test was employed to analyse the nonparametric data. A P
value of less than 0.05 was considered statistically significant.
3 l RESULTS
3.1 l Clinical characteristics
According to the retrospective questionnaire survey of 94 persons with
locust exposure history from 2000-2019, we found that 13.8% (13
persons) reported locust-induced symptoms. There were 8 males (8/49,
16.3%) and 5 female patients (5/45, 11.1%) with
suspected
locust allergy (Figure 1A). The clinical symptoms after locust exposure
were different between male and female patients.
Half
of the male patients complained shortness of breath with asthma, while
all the female patients had atopic dermatitis-like symptoms, and 80% of
them suffered from nasal symptoms (Figure 1B).
A cross-sectional survey investigated 57 (24 males, 33 females)
scientists and students in our two separate laboratories in Beijing and
Baoding. The latency period from the beginning of exposure to the onset
of symptoms varied, ranging from approximately one month to 20 years,
with a mean interval of 3.9 years. Twenty-three out of 57 individuals
(40.4%) were sensitized to locusts according to the SPT
(Figure
2A, Table S1). Although the number of allergic patients was similar
among males (11) and females (12), males had a higher sensitization
ratio (45.8%, 11/24) than females (36.4%, 12/33) (Figure 2A). Among
the 23 locust-sensitized subjects, 18 were diagnosed with occupational
allergy, with allergic rhinitis as the most common symptom, followed by
atopic dermatitis and asthma, whereas the other 5 subjects were
diagnosed as being atopic to locusts (Figure 2B, Table S1). More male
patients complained of allergic rhinitis and asthma, while 70% of
female patients suffered from atopic dermatitis (Figure 2B). One student
(P3) had a severe allergic reaction to locusts, with difficulty
breathing and asthma and even anaphylaxis once he went into the
insectary. The total IgE level in three subjects was less than 60 kU/L,
while in others, the level ranged from 69.3 to 1920 kU/L (Table S1).
Moreover, 9 of the 23 locust-sensitized individuals were also sensitized
to cockroaches, as determined by the SPT (Table S1). The duration of
exposure was a risk factor for occupational allergy
(Figure
2D). Therefore, occupational exposure lasting for 2-3 h per day
(OR=4.225) and continuing for one and half years (OR=2.842) obviously
increased the risk of locust allergy. We also found that previous
allergy history showed an effect on allergy to locusts. Among
locust-sensitized subjects, 14 patients (14/23, 60.9%) had a previous
allergic history, whereas 9 patients (9/23, 39.1%) were not previously
allergic (P<0.001). This indicated that past history of
allergy might be a risk factor for locust allergy (Figure 2C).
3.2 l Identification of the major allergens in locusts
To further confirm the
sensitization of the persons exposed to locusts, we conducted in
vitro experiments as well. The sIgE for locust proteins in patients’
sera was detected by ELISA; the sIgE levels of the allergic
group
(absorbance at 450 nm was
1.174±0.136) and atopic group (0.603±0.198) were significantly higher
than those of the non-atopic group (0.177±0.014) and healthy group
(0.174±0.007) (Figure 3A).
To determine the molecular weights of possible locust allergens, the
locust extracts separated by SDS-PAGE were transferred to a PVDF
membrane for immunoblotting with a serum pool of 10 sera (Table S1:
P1-P10). Although the bands from the locust extracts ranged from 30 to
80 kDa, significant major bands at 56 and 70 kDa were observed (Figure
3B). When the locust extract immunoblots were probed with 9 individual
sera, only a 70-kDa band was observed in all 9 sera (Figure 3C).
3.3 l The ~70-kDa protein is the locust
hexamerin-2 protein
To identify the protein with the band at ~70 kDa, we
analysed the 70-kDa IgE-binding band of the locust extracts by LC-MS/MS.
Peptide spectrum matching gave the highest sequence coverage (43%) with
a hexamerin-2-like protein (NCBI: ACU78069.1), annotated as a storage
protein, the juvenile hormone (JH)-binding protein in locusts (Figure
4B).
Then, we cloned the cDNA of the locust hexamerin-2 protein ORF, which
covers 2031 nucleotides. The nucleotide sequence could be translated to
a protein sequence with 677 amino acids, and the first 20 amino acids
were predicted as a signal peptide. The calculated molecular mass of the
hexamerin-2 protein was 78663 Da, with a pI of 5.76. According to
sequence structure analysis, the hexamerin-2 protein has 3 domains, and
there is evidence to indicate that these domains are Hemocyanin-N
domain, Hemocyanin-M domain and Hemocyanin-C domain (Figure 4A, C).
3.4 l Immunological characterization of the hexamerin-2
protein
To determine the sensitization of the hexamerin-2 protein, we used IgE
against the purified hexamerin-2 protein from locusts. The levels in the
sera of 18 patients with occupational locust allergy (absorbance at 450
nm was 1.546±0.198) were markedly higher than those in 5 atopic patients
(1.239±0.099), as determined by ELISA. Hexamerin-2 could not be detected
by non-atopic sera (0.520±0.083) and controls (0.524±0.044)
(Figure 5A). Immunoblotting was
performed using individual sera from 9 locust-allergic patients, and the
individuals reacted positively to the hexamerin-2 protein, while the
control reacted negatively (Figure 5B).
To determine whether the hexamerin-2 protein is the major allergen in
locusts, we chose two patients’ sera (P2 and P3) with positive reactions
to the hexamerin-2 protein for ELISA inhibition assays. The maximal
inhibition of the binding of patients’ IgE antibodies to the coated
hexamerin-2 protein was approximately 60%. Therefore, the hexamerin-2
protein is a major sensitive protein source in locusts (Figure 5C, D).
3.5 l Conservation of thehexamerin-2
protein in insects
We constructed a phylogenetic tree of the hexamerin-2 protein of
different grasshoppers, and the peptide sequences obtained were aligned
with those of four grasshopper species (Chondracris rosea,
Heteropternis respondens, Oedaleus asiaticus, Orinhippus tibetanus )
(Figure S1). The similarity of the sequences of the hexamerin-2 protein
of different grasshopper species was more than 93% (Figure S2). The
hexamerin-2 protein of locusts shares 36.1% similarity with the
allergen Bla g 3 (Blattella germanica ); it also shares 53.3%
similarity with the allergen Cr-PI and 49.6% allergen similarity with
Per a 3, both of which were identified from Periplaneta americana(Figure S3). By sequence
alignment between the locust
hexamerin-2 protein and shrimp hemocyanins, we found that their sequence
similarity was only approximately 29% (Figure S4). Therefore, the
hexamerin proteins of locusts are species specific, despite being
conserved in insects and arthropods.
4 l DISCUSSION
Locusts are an important insect group inducing occupational allergies,
causing sensitization and the development of allergic symptoms in a high
proportion of exposed persons. However, little information is available
on the prevalence of the allergic diseases. To date, this is the largest
study on occupational locust allergy. In this study, a retrospective
survey of 94 individuals and cross-sectional survey of 57 individuals
confirmed that locusts can cause allergic reactions during laboratory
exposure. In 57 students and staff of our laboratory, 18 subjects with
occupational allergy and 5 atopic subjects were identified by SPT. This
indicates the risk of potential allergy in the exposed subjects, and the
occupational allergy should be integrated into practitioner training
priorities.
In our study, a long-term retrospective survey indicated that the
frequency of suspected occupational allergy was relatively low (13.8%).
In particular, males were more sensitive than females to occupational
allergy. Although the allergic disorders included
atopic dermatitis, allergic rhinitis
and asthma, most male patients with allergies exhibited difficulty
breathing, as in asthma, while female patients showed a prevalence of
atopic dermatitis in both the retrospective survey and cross-sectional
survey. However, our cross-sectional survey showed a similar frequency
of occupational sensitization (~50%) to locusts as that
observed in previous
studies.7,9,14For the retrospective survey, we
investigated persons who had worked in the locust laboratory for 20
years (long term); some subjects had perhaps forgotten their atopic
symptoms, or at that time, the allergy symptoms were not obvious and
were not emphasized. However, no association could be demonstrated
between atopic status and sensitivity to locusts, probably because of
the small sample size of workers investigated. On the other hand,
methods of immunological tests are more sensitive and can obtain the
higher relevance ratio. Our results demonstrated that
occupational exposure for 2-3 h
per day and continuing one and half years obviously increased the risk
of locust allergy, especially exposure exceeding 2-3 h per day.
Identification of the major allergen is necessary to diagnose locust
allergies. Although some studies have shown that locusts can induce
allergic reactions, no
native
allergen has been purified and characterized from migratory locust.
In
our study, we identified the
hexamerin-2
protein as a major allergen of occupational allergy to locusts. We
proved for the first time the presence of the hexamerin-2 protein with
IgE binding ability in locusts. ELISA inhibition assays indicated that
maximal inhibition by the hexamerin-2 protein was approximately 60%,
suggesting that the locust hexamerin-2 protein was a major allergen
accounting for IgE binding in the two patients whose sera were used for
the inhibition experiment. Based on the results, we cannot exclude the
idea that the other patients were sensitized against other locust
allergens. Therefore, further research to identify other allergens from
locusts is necessary.
The hexamerin-2 protein, which is a storage protein in the hexamerin
family in locusts, appears to be closely related to locust hemocyanin
and associated with JH secretion.18 On the other hand,
the hexamerin-2 protein widely exists in various species of
orthopterans.19 The phylogenetic tree of the
hexamerin-2 protein of different grasshoppers and peptide sequence
alignment of four grasshopper species (C. rosea, H. respondens, O.
asiaticus,O.
tibetanus ) showed that the similarity of the sequence of the
hexamerin-2 protein in different grasshopper species was more than 93%.
These results indicated that humans are allergic to other locust and
grasshopper species as well. The current study assumed that people who
are allergic to shrimp might have allergic reactions to locusts, because
sera from subjects allergic to shrimp could be used to identify
allergens from Bombay locust.20 In our study, only one
subject was allergic to aquatic products among the 23 subjects with
locust allergy, so we considered there to be low cross-reactivity
between shrimp and locusts. Because the sequence similarity between the
hexamerin-2 protein in locusts and hemocyanin in 3 shrimp species was
only approximately 29%, cross-reactivity between them could not exist.
Because locusts and cockroaches belong to Orthopteroida within Insecta,
allergies to locusts and cockroaches in workers are associated with
cross-reactivity. In our study, 39.1% of the locust-allergic subjects
tested positive against cockroach (B. germanica ) in the SPT, and
the sequence similarity of the
hexamerin-2
protein in locusts and the allergen Blg 3 of B. germanica was
36.4%. Moreover, the sequence similarity between
the
hexamerin-2 protein in locusts and the allergen of P. americanawas approximately 50%. Therefore, the hexamerin-2 protein of locusts is
species specific, despite cross-reactivity between locusts and
cockroaches.
Locusts are consumed as food by
humans worldwide, in Africa, South America, and Asia, in both rural and
urban areas.21 Commercial farming of migratory locust
for food and feed is growing in Southeast Asia, China, Japan, and Korea.
They are also served on skewers in some Chinese food
markets.22 Approximately 27 cases of anaphylactic
shock caused by consumption of grasshoppers and 27 cases caused by
consumption of locusts were reported from 1980 to
2007.23 In addition, BBC news reported that eleven
people died in the locust swarm in Sudan in 2003, and approximately
sixteen thousand people went to a hospital because of asthma induced by
locust swarms.24 Therefore, the development of
approaches for the identification of allergens and antibody therapy is
urgently needed at the clinical level. In addition, because genomic and
transcriptomic information available,10,25 the
hexamerin-2 protein as storage proteins how to be synthesized and
function in locusts needs to be further investigated.
It is significant that the
hexamerin-2
protein can be utilized to prepare diagnostic reagents in the future.
The commercial production of the hexamerin-2 protein as a diagnostic
reagent and further applications for detection are expected to improve
the diagnosis rate and reduce the rate of misdiagnosis. On the other
hand, desensitizers prepared with the hexamerin-2 protein
will be beneficial to patients
allergic to locusts, effectively relieving symptoms and reducing the
health hazards of occupational exposure or other causes of allergies to
locusts.
5 l CONCLUSION
We conducted a retrospective survey and cross-sectional survey of
occupational allergy to locusts and demonstrated that occupational
exposure is an important risk factor for allergies to locusts. We
identified the hexamerin-2 protein as a major allergen associated with
occupational locust allergy. Natural molecules can be used for
component-resolved diagnosis and to investigate locust allergies in the
laboratory, to locusts used as food, and to locusts breeding in farms
and fields.