Effects of UVA radiation on cuticle microstructure of Tribolium castaneum

LIU Yan-mei，YU Lin，SHAUKAT Ali，LEI Chao-liang，QIU Bao-li，SANG Wen

（1.College of Agriculture，South China Agricultural University/Key Laboratory of Bio-Pesticide Creation and Application，Guangzhou 510640，China；2.Plant Protection Research Institute，Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of High Technology for Plant Protection，Guangzhou 510640，China；3.College of Plant Science and Technology，Huazhong Agricultural University/Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory，Wuhan 430070，China）

Abstract：Ultraviolet A （UVA）is a very important abiotic environmental factor to organisms.Cuticles are always the first line of defense to UV radiation.Changes of cuticle morphology and physiology contributing to UV resistance have been found in many marine organisms，higher plants，and human.But there were few researches about the cuticle of insects influenced by UVA.In this study，we used SEM and TEM to analyze changes of ultrastructural structure of larvae of Tribolium castaneum after 2 h and 4 h of UVA exposure.It was found that with the irradiation time increasing，boundaries of scale corresponding to the underlying epidermal cells on the dorsal surface of larvae were blurred and more secretions were deposited up to epidermal cells.In addition，the exocuticle became much thicker，and electron dense outside region of the exocuticle enlarged.However，endocuticle nuclei，Golgi apparatus and mitochondria were not influenced by UVA.These results might indicate that T.castaneum could change cuticle properties to reduce UVA damages.

Key words：cuticle；dermal gland；ultraviolet A；Tribolium castaneum

Natural ultraviolet （UV）radiations emitted by the sun can be divided into three wave bands，including UVA （315-400 nm），UVB （280-315 nm）and UVC （＜280 nm）[1-2].UVA and UVB are important environment factors because they can influence the biosphere，while UVC is totally absorbed by oxygen and ozone in atmosphere[3].UV radiations have different energy levels and can cause various effects to organisms.UVB is a short-wave irradiation with high energy.It could be absorbed by DNA and lead to genetic profile loss which appeared to be single-strand breaks，cyclobutane pyrimidine dimmers （CPDs）and （6-4）photoproducts （6-4）PPs，which could inhibit transcription and replication of genes[4-6].UVA is less efficient to induce DNA damage because it has low energy level and is hardly absorbed by native DNA[5].However，UVA can impact animals and plants mainly by indirectly producing reactive oxygen species （ROS）via photochemical interactions with intracellular chromospheres and affect cellular metabolism[7-10].Through the evolutionary process，some animals，including insects can use partial waves of UV radiation for survival[11-13].The butterf l y Heliconius erato uses UV vision to distinguish wings coloration of relative spices for successful mating[14].The visit behavior of bees Apismellifera （Apidae）and Lipotriches sp.（Halictidae）is strongly influenced by UV reflectance of flowers[15].

In order to reduce UVA damagesduring process of survival，insects have developed many strategies.The antioxidantsystem of Helicoverpa armigera could be activated by UVA exposure.And SOD，CAT，POX and GST enzymeswere induced to eliminate the ROS caused by UVA[8，16].Stress response genes of Drosophila melanogaster，including Hsp22，Hsp23，Hsp26，Hsp27，Hsp40，Hsp60，Hsp68，Hsp70 and Hsp83，were significantly upregulatedby UVA exposure，which implied that these Hsps may contribute to the tolerance of UV Aradiation[17].Sang et al[18]also found thatthe expressions of detoxification enzymegenes P450（CYP6BQ4 and CYP6BQ8）were induced by UVA in a coordinated manner.Because these two genes were regulated by the same transcriptional factors that responds to UVA stress.In addition，by using RNA-seq，the MAPK，calcium，chemokine，Wnt，andinsulin signaling pathways were found to constitute a multiple and/or cascading functional regulation mechanism to resist UVA-induced stress in D.melanogaster[9].Genes involved instress response，circadian rhythm，phototransduction，olfactory transduction，taste transduction，and DNA repair participated into UVA exposure in Mythimna separate[19].Although，many studies illustrated the physiology andmolecular adaptation of insects to UVA stress，little information was about the morphological adaptation to UVA.In organisms，sunscreen compounds/photoprotective pigments such as mycosporine-like amino acids （MAAs）accumulation in the cuticle to reject or absorb solar UV[20-22].MAAs in the external mucus of reef fish Pomacentrus amboinensis acted as sunscreen by preventing the DNA damaging effects induced by UVA and UVB[23].The insect cuticle is a complex extra cellular structure.It is the first barrier for insect to resist external stresses.Morphology and physiology changes of the cuticle could affect the adaption of insect to various environments.The colorado potato beetle Leptinotarsa decemlineata increased cuticular component deposition to reduce cuticular penetration in the adult stage to adapt to high concentration insecticide[24].Melanic greater wax moth Galleria mellonella heightened resistance to the infection of entomopathogenic fungus Beauveriabassiana through thickening the cuticle，increasing basal expression of immunity- and stress-managementrelated genes，enhancing numbers of circulating haemocytes[25].

The cryptic red fl our beetle Tribolium castaneum（Herbst）（Coleoptera：Tenebrionidae），an important pest of storage products，avoids light and undergoes all stages of the lifecycle within its food substrate.The sensitive UV vision ability gives the beetle advantages of detecting brightness and avoiding more severe UV damage in sunshine[26-27].Results of our previous research showed that T.castaneum adults could tolerate short term （＜4 h）UVA exposure by inducing expression of stressresponse genes Hsps and P450s，which wonthe time for beetles to find shields and escape danger in sunshine[18].We are interested in whether T .castaneum could change morphology and physiology of its cuticle to reduce UVA damage.To address this aim，we examined microstructure changes of T.castaneum larval cuticle under UVA stress by electron microscopy in this study.

1 Materials and methods

1.1 Experimental animals

The laboratory culture of T.castaneumwas originally collected from Wuhan，Hubei province，China in 2007.The experimental population was reared on 5% yeast supplemented with whole wheat flour at 30℃ and 50（±2）% relative humidity in constant darkness.Adults （emergence less than one week）laid eggs in flour for 1 hour.Then eggs were collected and transferred to new culture by 60-mesh sieve.The larvae which developed 20 days after eggs laying were used to analyze.

1.2 UV irradiation treatments

A UVA light （X-15N，Spectronics，USA）that emits UV in the 365 nm wavelength was used as the light source for irradiating samples.Before treatments，T.castaneum larvae were removed from flour to 7 cm-diameter petri dish and slightly cleaned by a writing brush.Beetles were irradiated at 1 000 µW/cm2，and the radiation was filtered by an infrared filter to remove heat effects.The environmental temperature was at 30（±1）℃.Larvae were irradiated for 2 h and 4 h，respectively.Untreated larvae were as controls.Each treatment includes 20 larvae.

1.3 Observation of surface morphology

After treatments，the larvae were washed by phosphate-buffered （pH7.2）and fixed in 2.5%glutaraldehyde for 24 hours （4℃）.Then they were dehydrated in an ethanol series，critical point dried in liquid CO2.Specimens were attached to the copper stage with conductive double-sided adhesive tape.After coating with gold using an ion sputtering instrument，they were observed in the SEM（Model JSM-6390，Jeol Ltd.，Japan）at an accelerating voltage of 20 kV.

1.4 Observation of cell structure

Using by dissecting microscope，the larval cuticle of T.castaneum was collected and cut into 0.5-1.0 mm3tissue masses.Then tissue masses were fixed for 24 hours in 2.5% glutaraldehyde.The cuticles were then washed once with the same solution，post-fixed for 2 h in 2% osmium tetroxide phosphate-buffered （pH7.2）solution，and washed twice in the buffer for 15 min each time.Specimens were dehydrated in four changes of acetone for 15 min，embedded in epoxy resin （Epon 812），then cured for 72 h at 55 to 60℃.Ultrathin sections （40-50 nm）were prepared using a ultramicrotome （LKB，Bromma，Sweden），doublestained with uranylacetate and lead citrate （30 min each），then viewed in the TEM （Tecnai G2TWIN，FEI Company，USA）.

2 Results and ananlysis

2.1 Effects of UVA radiation on dorsal surface microstructure of T.castaneum larvae

Effects of UVA radiation on dorsal surface of T.castaneum larvae were showed in Fig.1（Inside back cover）.In the control sample，the dorsal surface exhibited an orderly arrangement of scales corresponding to the underlying epidermal cells （Fig.1A，Inside back cover）.After UVA irradiation for 2 h，the boundaries of scale were blurred and more secretions were deposited （Fig.1B，Inside back cover）.With the irradiation time increasing to 4 h，the secretions increased and covered the epidermal cells.Boundaries of scale disappeared （Fig.1C，Inside back cover）.

Fig.1 Effects of different UVA radiation exposure time on the larvae dorsal surface microstructure of T.castaneum（SEM）

2.2 Ultrastructural effects of UVA radiation treatment

The epicuticle of T.castaneum larvae is composed of the inner cuticulin layer and the outer envelope that could carry a deposit of wax layer and cement layer.Close to inside epicuticle，there were many layers of exocuticle which was arranged tightly and uniformly （Fig.2A，Inside back cover）.However，after UVA exposure，the exocuticle became much thicker.The electron dense outside region of the exocuticle enlarged，which might be due to epicuticle and outside regions of exocuticle densification or some pigments depositing（Fig.2B，Inside back cover）.With the exposure time increasing，this high electron-dense region became thicker （Fig.2C，Inside back cover）.The morphology of insect endocuticle cells was also observed （Fig.3，Inside back cover）.Nuclei，Golgi apparatus and mitochondria can be clearly observed in control larvae （Fig.3A，Inside back cover）.No significant changes with organelle were observed after UVA irradiation for 2 h or 4 h （Fig.3B and C，Inside back cover）.

Fig.2 Effects of different UVA radiation exposure time on the larvae cuticle ultrastructure of T.castaneum（TEM）

Fig.3 Effects of different UVA radiation exposure time on the larvae endocuticle cell ultrastructure of T.castaneum（TEM）

3 Discussion

UV radiation is well known as ubiquitous environmental hazard in nearly all ecosystems and has been given a great deal of attention.UV could directly and indirectly damage many organisms，including insect[5，9，28].Although UVA is with lower energy compared with UVB and UVC，ROS induced by UVA could cause membrane disruptions and apoptosis[29-30].Like other organisms，insects also developed many strategies to reduce UVA damage，such as antioxidant system，Hsp proteins and so on[16-17].In the present study，we investigated the influences of cuticle microstructure under UVA stress in T.castaneum larvae.It was found that arrangement of scales of dorsal epidermis blurred after UVA irradiation exposure.A large amount of secretions attached to the surface of the epidermis.In addition，exocuticle became much thicker after UVA irradiation exposure，but morphology of endocuticle cells was not impacted.These results indicated that the morphological changes of cuticle probably help T.castaneum resist to UVA radiation.

The cuticle of insect consists of the epicuticle in the outer，the exocuticle in the middle and the endocuticle in the inner layer.The epicuticle could be divided into cement，wax，polyphenol and cuticulin layers.The cement layer was lastly produced by dermal glands and attached outside of wax layer to protect the wax to reduce the water loss after molting.When the cement layer was damaged by mechanical or physical injuries，dermal glands would replenish loss of it[31-32].In this study，it was found that a large amount of secretions attached to the surface of the epidermis when T.castaneum was exposed to UVA.This phenomenon was likely hypersecretion of dermal glands.The secretion of dermal glands was regulatedby endocrine hormone，such as ecdysone[33].In our previous study，it was found that UVB could influent ecdysone synthesis and downstream signaling pathway of T.castaneum larvae[34].In addition，Periodically research reported that UVA could also influence signaling pathway of the other hormone，such as gonadotropin-releasing hormone.Therefore，we speculated that increasing of secretions attached to the surface of the epidermis was related to the influenceson hormone by UVA radiation.The hypersecretion of dermal glands would increase the protection of insect.

ROS production causedby UVA can impact the structure of proteins[18，35].Proteins are the main composition of cuticle.Changes of cuticle proteins could result to changes of cuticle[36].Periodically research reported that conductive heat led to a disorder arrangement of cell on the surface of T.castaneum，which was similar to our results.However，heat could not result in the secretions deposition.These differences might be due to the variation in action modes on proteins and liquids of cuticle by the two physical factors.

By TEM analysis，it was clearly showed that after UVA exposure，high electron-dense regionin exocuticle became thicker which was similar to pigment deposition.It has been reported that the accumulation of pigments could protect insects from harmful effects of UVA.The pigment melanin in epidermal cells was as a shield against damaging UV light and its likely scavenging or quenching activity against ROS induced by UV[37].In addition，the exocuticle was thickened after UVA exposure in this study.This phenotype could increase the distance from epdidermal to hypoderma cells and reduce transferring of UVA and UV damage.

In conclusion，the UVA irradiation had significant effects on the outer structure of the epidermis of T.castaneum，which mainly affected the arrangement of epidermal cells and secretion of dermal glands，as well as the accumulation of electron dense outside region of exocuticle.It was probably a strategy of T.castaneum to reduce UV damage.