Changes in oxygen and carbon dioxide environment alter gene expression of cowpea bruchids

Journal of Insect Physiology 57 (2011) 220–230

Contents lists available at ScienceDirect

Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys

Changes in oxygen and carbon dioxide environment alter gene expression of cowpea bruchids Yong Hun Chi a,b, Ji-Eun Ahn a, Dae-Jin Yun b, Sang Yeol Lee b, Tong-Xian Liu c, Keyan Zhu-Salzman a,d,* a

Department of Entomology, Texas A&M University, College Station, TX 77843, USA Division of Applied Life Sciences, Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, Republic of Korea c Key Laboratory of Applied Entomology, Northwest A&F University, Yangling, Shaanxi, China d Vegetable & Fruit Improvement Center, Texas A&M University, College Station, TX 77843, USA b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 August 2010 Received in revised form 5 November 2010 Accepted 8 November 2010

Hermetic storage is a widely adopted technique for preventing stored grain from being damaged by storage insect pests. In the air-tight container, insects consume oxygen through metabolism while concomitantly raising carbon dioxide concentrations through respiration. Previous studies on the impact of hypoxia and hypercapnia on feeding behavior of cowpea bruchids have shown that feeding activity gradually decreases in proportion to the changing gas concentrations and virtually ceases at approximately 3–6% (v/v) oxygen and 15–18% carbon dioxide. Further, a number of bruchid larvae are able to recover their feeding activity after days of low oxygen and high carbon dioxide, although extended exposure tends to reduce survival. In the current study, to gain insight into the molecular mechanism underpinning the hypoxia-coping response, we profiled transcriptomic responses to hypoxia/hypercapnia (3% oxygen, 17% carbon dioxide for 4 and 24 h) using cDNA microarrays, followed by quantitative RT-PCR verification of selected gene expression changes. A total of 1046 hypoxiaresponsive cDNAs were sequenced; these clustered into 765 contigs, of which 645 were singletons. Many (392) did not show homology with known genes, or had homology only with genes of unknown function in a BLAST search. The identified differentially-regulated sequences encoded proteins presumptively involved in nutrient transport and metabolism, cellular signaling and structure, development, and stress responses. Gene expression profiles suggested that insects compensate for lack of oxygen by coordinately reducing energy demand, shifting to anaerobic metabolism, and strengthening cellular structure and muscular contraction. ß 2010 Elsevier Ltd. All rights reserved.

Keywords: Cowpea bruchid Hypoxia Hypercapnia Microarray qRT-PCR

1. Introduction Like all aerobic organisms, terrestrial insects need oxygen to survive because it is important for generation of catabolic ATP. They are capable of intense aerobic energy metabolism. However, many insect species from Coleoptera, Diptera, Lepidoptera, Isoptera, Collembola and Orthoptera are very tolerant to oxygen deprivation, and can recover from hours to days of hypoxia and anoxia (Hoback and Stanley, 2001; Farahani and Haddad, 2003; Harrison et al., 2006). Ecological habits such as high altitudes, flood-prone soils, burrows, grain, wood, as well as mammalian alimentary canals, can cause hypoxia. Adaptation to hypoxia allows them to survive periods of oxygen lack during their life cycles. For example, the desert locust (Schistocerca gregaria) can tolerate exposure to anoxia for 8 h. The tiger beetle (Cicindela

* Corresponding author at: Department of Entomology, Texas A&M University, College Station, TX 77843, USA. Tel.: +1 979 458 3357; fax: +1 979 862 4790. E-mail address: [email protected] (K. Zhu-Salzman). 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.11.011

togata) is able to withstand seasonal flood-caused anoxia for longer than six days. Bot fly larvae (Gasterophilus intestinalis), living in horse stomachs, survive anoxia for 17–25 days. Larvae of fruit flies (Drosophila melanogaster) can tolerate extended periods of hypoxia in decaying fruits (Hoback and Stanley, 2001). Insects that feed on stored grain gradually deplete oxygen, especially in air-tight containers, thus inevitably encountering hypoxia. Although use of air-tight packaging has been successful in controlling some grain-feeding insects, a number of storage pests exhibit remarkable tolerance to hypoxia. For instance, red flour beetles, Tribolium castaneum, can survive days of low oxygen treatment (Hoback and Stanley, 2001). This cultural control measure, while saving grains by limiting oxygen required for insect development, also elicits a wide variety of physiological and behavioral adaptive responses. However, detailed studies of these responses have been done in very few species. Adaptive mechanisms to hypoxia include switching from aerobic to anaerobic metabolic pathways, down-regulating energy turnover pathways, altering behavior and enlarging the tracheal system (Hochachka et al., 1996; Hoback and Stanley, 2001).

Y.H. Chi et al. / Journal of Insect Physiology 57 (2011) 220–230

Cowpea (Vigna unguiculata) is the most economically-important grain legume grown by millions of small-holdings farmers in Africa (Langyintuo et al., 2003). Unfortunately, heavy losses often occur due to damage by storage insects, especially the cowpea bruchid, Callosobruchus maculatus. Storage insecticides cause health and environmental problems, and often are too expensive for poor farmers. Sealing the grain in air-tight plastic bags is becoming a widely adopted insect control method particularly benefiting resource-poor farmers of Africa. In addition to functioning as a protective barrier that prevents further invasion by exogenous insect pests, this hermetic storage technique works by the principle of oxygen depletion against insects already present in grain at storage. As these insects convert oxygen into carbon dioxide, decreased oxygen is accompanied by increased carbon dioxide in such systems. The effect of hypoxia and hypercapnia on cowpea bruchid survival and feeding behavior has recently been assessed by monitoring feeding under different combinations of oxygen and carbon dioxide concentrations (Margam, 2009). Feeding activity gradually decreases as the oxygen level in the container falls, with a concomitant increase in the level of carbon dioxide. When the oxygen level in the container reaches 3–6%, the insects cease feeding and growth. As a result, insect development is suppressed and population expansion arrested. Further study indicates that the effect is mostly due to varying the concentration of oxygen, rather than that of carbon dioxide. More interestingly, a portion of larvae exhibits the ability to recover from 14 days of hypoxia treatment, although subsequent development is substantially retarded (Margam, 2009), suggesting they are capable of adapting to oxygen tension, likely by modulating behavior, physiology and development. We have previously demonstrated the plasticity of the cowpea bruchid alimentary tract (Zhu-Salzman et al., 2003). cDNA microarray analyses revealed that this insect is able to adjust expression of a large number of genes in response to dietary challenge (Moon et al., 2004; Chi et al., 2009). In-depth protein function analyses provided evidence for their pivotal roles in coping with dietary anti-nutritional factors (Ahn et al., 2004, 2007, 2010; Koo et al., 2008; Ahn and Zhu-Salzman, 2009). While these studies facilitated our understanding of molecular mechanisms of insect adaptation to anti-nutritional factors, we know very little of the dynamics as well as molecular events and mechanisms behind the process of anaerobic adaptation. To assess how insects respond transcriptomically to hypoxia/hypercapnia, we took advantage of our 20,352 cDNA collection prepared from larval midgut tissue and performed microarray analyses to identify hypoxia/hypercapniaresponsive genes. 2. Materials and methods 2.1. Insect treatment and midgut dissection Cowpea bruchids were maintained on cowpea seeds (California Blackeye No. 5) at an average density of five individuals per seed at 25 8C, 35% R.H., and under normal atmospheric conditions (20% oxygen and 0% carbon dioxide). When these larvae reached midfourth instar stage, they were subjected to control and treatment conditions for periods of 4 and 24 h. In a number of species, changes of transcript and protein abundance of many hypoxiaresponsive genes become obvious within several hours of treatment and continue for days (Hochachka et al., 1996; Berra et al., 2003; Yun et al., 2005; Liu et al., 2006). For control conditions, the cowpea seeds with larvae were transferred into a 500 ml septum bottle (Industrial Glassware, Millville, NJ) whose septum was replaced with a cotton ball to allow atmospheric air to diffuse, with the concentrations of oxygen and carbon dioxide being 20%

221

and 0% respectively. For treatment conditions, the larvae were exposed to hypoxia/hypercapnia (3% oxygen and 17% carbon dioxide) in septum bottles tightly sealed so as to prevent any diffusion of air. A 24 h-treatment caused approximately 1% decrease in oxygen concentration and 1% increase in carbon dioxide. The levels of oxygen and carbon dioxide were determined using a head space analyser (Mocon – PAC CHECK1 Model 325, Minneapolis, MN). At the end of each time point, larvae were removed from the seeds and their midguts were immediately dissected as previously described (Kitch and Murdock, 1986). Collected midguts were stored in RNAlater (Ambion, Austin, TX) and kept at 80 8C for future use. 2.2. RNA extraction and microarray probe preparation The midgut microarray platform previously established was used in this experiment (Chi et al., 2009). This microarray comprises 20,352 randomly picked cDNA clones from a normalized cDNA library constructed using cowpea bruchid larvae subjected to various treatments to increase gene representation (for details, see Chi et al., 2009). Prior to hybridization, microarray slides were processed by blocking in 1% SDS for 10 min at 25 8C, then by DNA denaturation in boiling water for 2 min, followed by 95% ethanol treatment at 20 8C for 2 min. Fifteen midguts obtained as above were homogenized. Total RNA was extracted from the dissected midguts of treated and control larvae, respectively, using a Trizol-based method (Invitrogen, Carlsbad, CA). Cy3- and Cy5-labeled probes were prepared using the 3DNA Expression Array Detection Kit for Microarray (Genisphere, Hatfield, PA). cDNAs were prepared from equal amounts of total RNA (14 mg of each biological replicate) from control or treated insects with oligo dT primer containing a proprietary capture sequence for Cy3 or Cy5. Two probes, each derived from 7 mg input RNA for each channel in each microarray, were mixed with an equal volume of hybridization buffer, added to the microarray, and covered with cover slips. For each biological replicate, two microarray hybridizations were performed. The slides were sealed in aluminum hybridization chambers (Monterey Industries, Monterey, CA) and incubated at 65 8C overnight. After washing, the Cy3 and Cy5 fluors, coupled to oligos complementary to the capture sequences, were incorporated in the second hybridization, thereby fluor-labeling the cDNA probes bound to the microarray. 2.3. Microarray scanning and data analysis The microarray slides were scanned with a Packard Scanarray 5000 four-laser confocal scanner (Packard Bioscience, Billerica, MA) using the line-scan function to balance signals, and the images were analysed with the Quantarray program (Packard Bioscience). The output data were applied to a low signal cutoff equation; i.e. if mean signal intensity <2 mean background intensity, then mean signal intensity was made equal to mean background intensity. The adjusted mean signal intensity and mean background intensity data were input into GeneSpring 7.3.1 (Agilent Technologies, Redwood City, CA). Data from 4 slides (2 technical replications  2 biological replications) were averaged and normalized using the GeneSpring Lowess algorithm for each treatment condition. Results were then filtered by expression fold change and confidence values. Expression data with mean fold changes of more than 2.0, and significance at P  0.05 (Student t-test P value, using Benjamini and Hochberg F.D.R. multiple testing correction) were retained. The corresponding clones were subjected to sequencing analyses for gene identification.

222

Y.H. Chi et al. / Journal of Insect Physiology 57 (2011) 220–230

2.4. DNA sequencing analysis, BLAST search and quantitative RT-PCR To identify hypoxia/hypercapnia-responsive genes from cowpea bruchid midgut, plasmids of selected clones based on the above criteria were extracted. Insert sequences were determined using the ABI BigDye sequencing kit (PE Biosystems) and analysed on an ABI Prism 3100 DNA sequencer. Trimming of the vector sequence from raw data and contig assembly was performed using Sequencher software (Gene Codes Corporation). The predicted biological function of each cDNA was determined based on the top tBLASTX (2.2.24+) hit in the GeneBank database with a described gene function. The E-value cutoff was set at 105. To analyse the network of hypoxia/hypercapnia-responsive genes in bruchid midgut, KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathways were determined using the BLAST2GO software suite v.2.4.4 (www.Blast2go.de) (Gotz et al., 2008). KEGG metabolic pathways were annotated from the direct mapping of Gene Ontology (GO) terms to their enzyme code equivalents. For quantitative RT-PCR (qRT-PCR), total RNA from guts was reverse transcribed with SuperscriptTM II reverse transcriptase (Invitrogen) using random hexamer primers. Primers for amplifying selected genes were designed using Primer Express (Applied Biosystems). For each gene, duplicate 10 mL qRT-PCR reactions were performed using SYBR Green mastermix (Applied Biosystems). No-template controls using untranscribed RNA confirmed that no interfering products derived from genomic DNA were present. PCR amplification of 18S rRNA was performed for normalization between treated and control samples. Amplification specificity was determined by dissociation curve analysis. To calculate mean induction fold as 2^ (DDCT), the following formulas were applied:

DDCT ¼ ðDCTcontrol cDNA Þ  ðDCTtreatment cDNA Þ

DCT ¼ ðmean CT cDNAtest primers Þ  ðmean CT cDNAribosomal primers Þ For SD range of replicate reactions, upper error bar = 2^ (DDCT + S) and the lower error bar = 2^ (DDCT  S), where

S¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 ðSD of CTtest primers Þ þ ðSD of CTribosomal primers Þ

3. Results and discussion Hermetic storage uses sealed containers to protect stored grain and reduce post-harvest losses. Environmental stimuli such as reduced oxygen tension lead to decrease in feeding activity and delayed development of cowpea bruchids (Margam, 2009), which most likely are mediated through transcriptional reconfiguration. Presumably, cowpea bruchids coordinately regulate their gene expression to cope with such an external stress and maintain homeostasis of the cells. In an air-tight container, feeding activity of cowpea bruchids causes a gradual decrease in oxygen, which is accompanied by a rise in carbon dioxide. Thus in our hypoxia treatment, we replaced depleted oxygen with carbon dioxide (i.e. hypercapnia) and maintained nitrogen constant. However, Margam (2009) discovered that when carbon dioxide levels alone were raised by maintaining ambient oxygen at 20% and proportionally replacing nitrogen with carbon dioxide, insect feeding was not impacted. By contrast, decreasing oxygen levels while keeping carbon dioxide concentration constant triggered a gradual

decrease in feeding activity, in a manner comparable to the combined effect of low oxygen and high carbon dioxide. It thus appears that cowpea bruchids change their feeding activity in response to the drop of oxygen, rather than the rise in carbon dioxide. Accordingly, in this study, we do not attempt to differentiate genes responsive specifically to hypercapnia. 3.1. Transcriptomic response to hypoxia and hypercapnia treatments Decreased feeding activity in hypoxic cowpea bruchids implies an impact of low oxygen on their digestive system. We thus planned to investigate how this storage pest regulates gene expression in gut tissue, which is responsible for food intake, digestion and absorption, in an oxygen-reduced environment. The midgut microarray platform we established previously offered a great advantage for us to identify hypoxia/hypercapnia-responsive genes. Of the 20,352 cDNA clones present on the microarray, 1046 cDNAs were induced or repressed two-fold or more (P  0.05) in at least one of the two treatment durations. Sequence analysis of this set of cDNAs (average 858 bp) revealed that hypoxia/hypercapnia altered expression of a wide range of midgut genes. Assembly of these sequences generated 765 unique genes (Suppl. Table, which include 607 new GenBank accession numbers; HO204634– HO205240). Among the responsive unigene set, 645 were singletons. Possible gene function was determined by sequence alignment with previously characterized genes in BLAST searches at an E value cutoff of 105. Based on their particularly strong regulation at one or both time points in microarray experiments as well as predicted gene functions, 104 genes were selected for qRT-PCR analysis to confirm microarray results. Of 208 data points, regulation direction of 91% was consistent with microarray data (Table 1), suggesting a general agreement between microarray and qRT-PCR data. Responsive genes encode proteins potentially involved in nutrient transport and metabolism, cellular signaling and structure and stress responses (Fig. 1, Suppl. Table). Hypoxic regulation of genes involved in such biological functions have been reported in other organisms such as Drosophila and Caenorhabditis elegans (Shen et al., 2005; Liu et al., 2006; Azad et al., 2009). Many genes (340) had no hits in BLAST searches, and 52 matched genes with unknown function. Those that shared sequence similarities to genes of known function were categorized according to biological process and molecular function (Fig. 1). Exposure to low oxygen and high carbon dioxide caused responses in a broad range of functional gene classes, suggesting that the insect digestive system actively reallocates genomic resources to adapt to and survive oxygen deprivation. To understand how hypoxia/hypercapnia impact gene networks, the 765 unique sequences were mapped to KEGG metabolic and regulatory pathways using the BLAST2GO software. The most abundant categories for the hypoxia/hypercapnia exposure were associated with metabolism of amino acids, carbohydrates and lipids (Fig. 2). One interesting observation was the minimal number of genes that overlapped between 4 h and 24 h time points; 12 induced and 3 repressed (Fig. 3). It seems that the gene sets responsive to hypoxia changed over time. A similar phenomenon has also been observed in Drosophila, where a very small number of regulated genes were shared between the shorter and longer periods of hypoxic treatment (Liu et al., 2006). Perhaps following an acute response to the initial crisis, longer term strategies have to be in place. Such temporal changes in gene expression suggested that different sets of genes are used by cowpea bruchids to cope with short- and long-term low oxygen stress.

Y.H. Chi et al. / Journal of Insect Physiology 57 (2011) 220–230

3.2. Hypoxic suppression of genes for protein synthesis and breakdown Under normoxic conditions, protein synthesis and breakdown is one of the major energy sinks in insects. It has been reported that terrestrial insects are able to attenuate basal metabolic rates and decrease ATP levels within minutes of exposure to oxygen deprivation (Hoback and Stanley, 2001). Regulated suppression of ATP demand in cowpea bruchids was reflected by expression changes of genes involved in protein synthesis and targeting, such as aminoacyl tRNA synthetases, SRP72 and NCL-1 (Table 1). Repressed expression of aminoacyl tRNA synthetase genes, serS and alaS, may slow down the rate of protein translation. In C. elegans, inactivation of aninoacyl-tRNA synthetases rescued animals from hypoxia. It is thought that such hypoxia resistance is partly due to reduced unfolded protein toxicity (Anderson et al., 2009) because translational repression decreases protein aggregation.

The signal recognition particle (SRP) functions to recognize emerging secretory proteins, attach the translating ribosome to the endoplamic reticulum (ER), and deliver them to the ER lumen (Utz et al., 1998). This is a highly conserved, ATP-driven process. Downregulation of CmSRP72, encoding a protein essential for translocation, presumably inhibits protein synthesis and translocation, thus contributing to reduced energy turnover. Consistently, we identified an upregulated ribosome synthesis repressor homolog, CmNCL1. In C. elegans, this zinc finger protein represses synthesis of rRNA and 5S RNA. Loss of function mutations in ncl-1 resulted in enlarged nucleoli and increased rRNA transcription and cell growth (Frank and Roth, 1998). Induction of CmNCL1 under hypoxia potentially reduces the ATP demand by restricting protein synthesis and cell growth. In agreement with decreased feeding activity under hypoxia, several genes encoding cathepsin L-like cysteine proteases, the aspartic cathepsin D protease, and exopeptidases were downregulated (Table 1). It has been established that cowpea bruchids

Table 1 Induced and suppressed transcripts in cowpea bruchid midgut by hypoxia.

Fold Change Accession #

Tribolium Homolog

Putative Function

4 hr

24 hr

µarray

qPCR

µarray

qPCR

β-Mannosidase (CmMANBA) 6-Phosphofructo-2kinase/fructose-2,6-biphosphatase 3 (CmPFKFB3) β-Glucosidase (CmBGA2) β-1,4-Mannanase (CmMAN1) UDP-glucuronosyl transferase (CmUGT3) α-Glucosidase (CmAGL1) β-Galactosidase (Cm BGL1) Lysosomal α-mannosidase (CmLAMAN2) UDP-glucose 4-epimerase (CmGALE) Glycerol-3-phosphate dehydrogenase (CmGPD) Pectate lyase (CmPEL5) α-Amylase (CmAMY4) α-1,2-Mannosidase (CmMAN1B1)

2.59

2.97Y

1.30

6.30Y

2.05

2.63Y

1.20

3.42Y

0.40 0.34

0.40Y 0.48Y

1.72 0.81

1.17Y 0.63Y

0.48

0.37Y

1.83

3.66Y

0.47 0.95

0.26Y 0.47Y

1.27 2.47

0.57 n.a. 2.16Y

1.06

1.89Y

2.11

7.82Y

1.02

0.38N

2.38

2.33Y

1.04

0.66 n.a.

2.49

3.27Y

[TD$INLE]

0.38 0.45

0.27Y 0.14Y

2.35 2.41

9.99Y 2.52Y

0.90

0.42Y

0.37

2.15N

Cathepsin D (CmCatD) Cathepsin F (CmCatF) Cathepsin L (CmCatL2) Cathepsin L (CmCatL8) Cathepsin L (CmCatL13) Mitochondrial processing peptidase α subunit (CmMPPα)

0.36 1.13 0.62 0.71 0.61

0.01Y 1.16Y 0.39Y 1.23 n.a. 0.92Y

1.44 2.94 0.50 0.12 0.01

1.96Y 1.31Y 0.47Y 0.001Y 0.001Y

1.08

0.48N

0.42

0.79Y

2.98

1.93Y

2.91

3.13Y

2.22

4.51Y

1.20

2.39Y

2.53

1.56Y

1.29

5.03Y

2.18

1.43Y

1.25

5.81Y

0.36

0.32Y

0.81

4.45N

Sugar Metabolism FK668897

XM_969290.1

HO204635

XM_968315.2

FK668905 FK668935

XM_970573.1 None

FK669185

XM_963911.1

FK668899 FK668880

XM_961929.1 XM_962554.1

FK668895

None

HO204646

XM_963523.1

HO204647

XM_970689.2

FK668999 HO204642

None XM_964141.2

HO204645

XM_965987.1

Protein Metabolism FK668963 HO204652 FK668949 FK668955 HO204651

XM_961424.1 XM_968514.2 XM_965804.1 XM_965551.1 None

HO204656

XM_965978.2

Lipid metabolism FK669040

XM_967542.1

HO204657

XM_964924.1

HO204658

XM_964420.2

HO204660

XM_00181365 9.1

HO204659

XM_968737.1

Farnesyl diphosphate synthase (CmFPPS) Acyl-coenzyme A dehydrogenase, very long chain (CmVLCAD) Acyl-coenzyme A oxidase 3, peroxisomal (CmACOX3) AMP dependent coa ligase (CmCL2) Acyl-coenzyme A dehydrogenase, C-2 to C-3 short

223

224

Y.H. Chi et al. / Journal of Insect Physiology 57 (2011) 220–230

Table 1 (Continued )

HO204665

XM_968961.2

HO204664

XM_970050.2

FK669047

XM_968853.1

HO204661

XM_968781.2

HO204666

None

HO204667 XM_962789.1 HO204671 XM_968567.1 Other metabolism

[TD$INLE]

HO204674

XM_963836.2

HO204675

XM_966816.1

HO204684

XM_961636.1

HO204688

XM_963350.1

HO204697

XM_961376.1

HO204691 HO204693

XM_961906.1 XM_963367.2

HO204698

XM_961372.1

chain (CmACADS) Phosphatidylglycerophosphate synthase (CmPGS1) 2-Hydroxyphytanoyl-coa lyase (CmHACL) Fatty acid 2-hydroxylase (CmFA2H) AMP dependent coa ligase (CmCL3) Acyl-coenzyme A dehydrogenase (CmACDH) Glycerol kinase (CmGK2) Acyl-coA oxidase (CmACO) Autophagy related 7 homolog (S. cerevisiae) (CmATG7) N-Acetyl neuraminate lyase (CmNAL) 5-Aminolevulinic acid synthase (CmALAS) Sarcosine dehydrogenase (CmSARDH1) Myo-inositol oxygenase (CmMIOX2) Arginase (CmARG) Seryl-tRNA synthetase (CmserS) Alanyl-tRNA synthetase (CmalaS)

0.45

0.32Y

0.97

1.30 n.a.

0.36

0.29Y

0.86

0.52Y

0.83

0.70Y

2.58

2.17Y

1.08

0.69 n.a.

2.64

2.19Y

0.96

0.78Y

2.28

2.85Y

1.02 0.48

1.42Y 0.63Y

2.26 0.37

3.85Y 2.19N

2.44

1.49Y

1.07

6.78Y

2.76

2.27Y

1.21

6.70Y

1.07

1.45Y

2.11

2.69Y

0.74

0.80Y

4.83

24.9Y

0.89

0.36Y

0.37

4.35N

1.00 0.68

0.64 n.a. 0.23Y

0.45 0.46

0.58Y 0.93Y

0.93

0.63Y

0.49

0.90Y

2.25

2.06Y

0.95

6.90N

2.50 1.10 1.01

1.93Y 2.45Y 0.88 n.a.

1.10 2.09 2.37

3.71Y 7.49Y 19.3Y

1.32

0.79 n.a.

2.53

7.66Y

1.11

1.25Y

2.61

3.24Y

1.89

4.13Y

3.80

5.34Y

1.54

1.07Y

4.08

2.85Y

1.01

0.41N

0.42

0.66Y

1.19

0.21N

0.47

0.57Y

3.52 2.69

2.88Y 2.34Y

1.74 1.21

1.37Y 2.01Y

Transport Mitochondrial dicarboxylate carrier (CmDIC) HO204714 XM_965749.1 Importin-7 (CmIPO7) FK669129 XM_965073.1 Carbonic anhydrase 2 (CmCA2-2) HO204702 XM_967593.2 Sugar transporter (CmSUT11) ATP-binding cassette, sub-family HO204720 XM_963655.1 C (CFTR/MRP), member 4 (CmABCC4-3) Phospholipid scramblase 1 HO204723 XM_963464.1 (CmPLSCR1) Calcium-binding mitochondrial HO204727 XM_965406.1 carrier Aralar1 (CmARALAR1) Sorting nexin family member 30 HO204728 XM_962003.1 (CmSNX30) FK669105 XM_963249.1 Aquaporin (CmAQP2) Signal recognition particle 72 HO204733 XM_968152.1 (CmSRP72) Signaling / Transcriptional regulation HO204734 None Reverse transcriptase (CmRT2) HO204737 XM_962221.1 Serine/threonine-protein HO204707

XM_967917.2

use cathepsin L as their major digestive enzymes (Zhu-Salzman and Salzman, 2001), while aspartic protease is a minor component, accounting for approximately 10% of the total gut proteolytic activity under normal growth conditions (Kitch and Murdock, 1986; Silva and Xavier-Filho, 1991; Zhu-Salzman et al., 2003). Taken together, the gene expression pattern we observed suggests that cowpea bruchids attempt to decrease energy input in protein biosynthesis and degradation, which is important for cowpea bruchids when metabolic depression is needed. 3.3. Promoting gene expression changes favoring anaerobic energy production Oxygen is a critical factor for catabolic ATP generation. When oxygen becomes limiting, cells shift to a glycolytic mode for energy production. The bifunctional enzyme 6-phosphofructo-2-kinase/ fructose 2,6-bisphosphatase (PFK-2)-encoding gene (PFKFB3) is known to be regulated by the hypoxia-inducible factor 1 (HIF-1) pathway (Minchenko et al., 2003). Consistent with previous

findings, CmPFKFB3 was identified as a hypoxia-induced gene (Table 1). This particular isoform has the highest kinase:bisphosphatase activity ratio (Duran et al., 2008). The resulting elevated fructose 2,6-bisphosphate levels in turn promote high glycolytic rates in the cell. Our data suggest that cowpea bruchids are able to respond and adapt to environmental hypoxia by switching from aerobic respiration to anaerobic glycolysis. It has been shown that hypoxia-responsive cells and tissues do not use anaerobic metabolism to make up energy deficits but to sustain a reduced energy turnover state instead (Hochachka et al., 1996). The observed expression pattern of polysaccharide hydrolase genes, such as those encoding a- or b-glucosidases, bgalactosidases, a-amylases, b-1,4-mannanases and a-1,2-mannosidases, supports this idea (Table 1). Down regulation of these enzymes is agreeable with reduced food intake and suggests a decrease in products that can be funneled into energy production. As with suppressed protein digestion, tuning down polysaccharide degradation can be viewed as controlled metabolic reduction by insects to adapt to hypoxic conditions. Overall, attenuated ATP

Y.H. Chi et al. / Journal of Insect Physiology 57 (2011) 220–230

225

Table 1 (Continued )

[TD$INLE]

HO204739

XM_961626.2

HO204740

XM_961368.1

HO204742

XM_963274.2

HO204749

XM_968811.1

HO204760

XM_963205.2

HO204758

None

HO204765

XM_963742.2

HO204768

XM_966409.1

HO204769

XM_961598.1

HO204771

XM_961635.1

HO204761

XM_965329.2

Neuronal function HO204794 XM_962242.1 Defense HO204797

XR_043082.1

FK669257

XM_967158.1

FK668898 XM_968097.1 FK669232 XM_964986.1 FK669235 XM_967567.1 Detoxification XM_00180956 HO204806 8.1 HO204808

XM_966519.1

HO204811

XM_970288.1

phosphatase PP-V (CmPP5) Integrin-linked protein kinase (CmILK) Anterior pharynx defective 1 (CmAPH1) PTEN-induced putative kinase 1 (CmPINK1) B-box type zinc-finger protein ncl-1 (CmNCL-1) Hypoxia-inducible factor prolyl hydroxylase 2 (CmPHD2) Interleukin 16 isoform 1 (CmPRIL-16) Phosphodiesterase 8B (Cm PDE8B) Bent (CmBT) Choline/ethanolamine kinase (CmCK/EK) Takeout like protein (CmTOL) Arylhydrocarbon receptor nuclear translocator homolog a isoform (CmARNT) Contactin (CmCNTN) Juvenile hormone esterase (CmJHE7) Juvenile hormone esterase (CmJHE5) Chitin deacetylase 1 (CmCDA1) c-Type lysozyme (CmLys-PJ) SERPINB3 (CmSERPINB3) Cytochrome P450 CYP6F1 (CmCYP6F1) Cytochrome P450 CYP4BN1 (CmCYP4BN1-1) Cytochrome P450 CYP9V1 (CmCYP9V1-2)

2.10

0.73 N

1.301

1.24 Y

2.19

2.79Y

0.91

1.22 n.a.

2.01

2.95Y

1.68

4.44Y

4.03

2.82Y

0.87

6.56N

2.42

4.12Y

1.19

6.97Y

0.39

0.45Y

0.46

1.27N

0.88

0.91Y

3.15

2.13Y

0.96

0.98Y

6.03

16.4Y

1.28

1.00Y

4.39

2.63Y

0.93

0.66Y

3.25

3.52Y

1.49

0.99 n.a.

0.46

0.94Y

0.90

0.55Y

2.12

3.52Y

2.42

5.65Y

1.20

20.5Y

0.47

0.58Y

1.20

3.32Y

0.40 1.10 1.27

0.46Y 0.27N 1.78Y

0.88 2.54 2.78

0.85Y 2.12Y 7.06Y

2.48

1.07Y

1.81

13.7Y

0.82

0.79Y

2.60

3.39Y

1.00

0.89 n.a.

4.09

6.85Y

1.01

1.08Y

2.46

8.19Y

2.06

2.13Y

1.28

1.26Y

0.39

0.41Y

0.77

4.38N

1.00

0.88 n.a.

0.50

1.04N

1.67

0.43N

0.40

1.77N

Apoptosis XM_00181051 0.1 Ubiqiutination HO204815

HO204816

XM_962657.1

HO204817

XM_969126.1

HO204818

XM_968222.1

HO204820

XM_966399.2

Caspase 2 (CmCASP2) Ubiquitin specific protease 41 (CmUBP41) Seven in absentia 1B (CmSIA1B) Ubiquitin-like modifier activating enzyme 5 (CmUBA5) Seven in absentia (SINA) family

demand makes it possible for less efficient anaerobic metabolism to maintain cell and tissue viability. 3.4. Alteration of mitochondrial synthetic and functional genes Defense against oxygen deficiency includes a balanced suppression of ATP demand and supply (Hochachka et al., 1996). Alteration of genes associated with mitochondrial function appears to support this notion. A mitochondrial processing peptidase gene (MPPa) and a phosphatidylglycerophosphate synthase homologue (PGS) were both down-regulated (Table 1). MPP is a metallopeptidase that processes nucleus-synthesized, mitochondrial protein precursors after they are targeted to mitochondria by cleaving the extension peptides (Nagao et al., 2000). Down-regulation of MPP presumably weakens mitochondrial function. Likewise, since lack of PGS causes morphological as well as functional abnormalities in mitochondria (Kawasaki et al., 1999), lower gene expression caused by oxygen deficit may lead to reduction of properly functional mitochondria, which is consistent

with the lessened energy demand in the hypoxic condition. Synergistic to such an effect, a homolog of autophagy protein gene Atg7 involved in the clearance of defective mitochondria (Zhang et al., 2009) was induced (Table 1). It is known that starvation induces autophagy, a process that maintains cellular homeostasis in changing nutrient conditions (Zhang et al., 2009). Decreased feeding under hypoxia may mimic a starving situation, leading to induction of Atg7. Breakdown of defective mitochondria, in turn, can be an energy source when nutrients are scarce. 3.5. Induction of the key oxygen sensor HIF prolyl hydroxylase 2 In insects, oxygen reaches organs and tissues of the body directly through the tracheal system. Besides switching to anaerobic metabolism and drastically attenuating basal metabolic rates, another adaptive strategy hypoxic insects exhibit is to enlarge tracheal system volumes (Hoback and Stanley, 2001). This mechanism has recently been elegantly illustrated in Drosophila (Centanin et al., 2008). Terminal branches of the Drosophila trachea

Y.H. Chi et al. / Journal of Insect Physiology 57 (2011) 220–230

226 Table 1 (Continued )

protein (CmSINA) Stress-related HO204822

XM_965631.2

FK669241

XM_969274.1

FK669247

XM_967758.1

HO204828

XM_962179.2

DnaJ (Hsp40) homolog, subfamily A, member 2 (CmDNAJA2) Heat shock protein 27 (CmHSP27) J domain protein c21orf55 (CmC21orF55) Methionine sulfoxide reductase (CmMSRA-2)

2.03

3.18Y

1.34

3.71Y

1.01

1.04Y

2.74

6.23Y

2.22

7.83Y

5.50

18.6Y

0.90

0.88Y

0.20

0.28Y

2.25

1.90Y

1.11

5.13Y

1.00

1.20Y

2.87

7.26Y

4.64

13.1Y

1.81

1.28Y

6.03

612.3Y

0.55

0.40Y

2.28

2.55Y

1.22

4.33Y

2.55

1.59Y

2.08

2.84Y

3.09

1.84Y

1.25

4.93Y

2.32

2.74Y

1.21

3.92Y

3.25

307.8Y

0.63

1.71 n.a.

1.64

0.83Y

4.62

3.75Y

0.82 0.84 0.84

0.87Y 0.82Y 0.36Y

4.76 4.38 2.62

11.8Y 4.34Y 1.86Y

0.91

0.85Y

3.50

3.91Y

0.89

0.58Y

4.94

8.77Y

0.63 1.22

0.53Y 1.06Y

4.53 4.83

2.72Y 5.36Y

1.12

0.76 n.a.

2.40

1.65Y

1.35

0.51 n.a.

2.79

1.98Y

2.49

0.36N

2.21

1.26Y

1.67

0.75 n.a.

2.20

2.58Y

0.60

1.20 n.a.

3.75

2.36 Y

Development Heparin sulfate 2-Osulfotransferase (CmHs2st) HO204781 XM_961908.1 10G08 (Cm10G08-3) Muscle- and cell structure-related Prolyl-4-hydroxylase-αEFB FK669010 XM_964293.1 (CmPH4αEFB1) Type IV collagen α5 chain FK669303 None (CmCOL4α5) Dynein light intermediate chain HO204830 XM_963435.1 (CmDLIC) HO204832 XM_971054.1 β-Spectrin (CmSPTB) Restin (cytoplasmic linker HO204833 XM_961925.2 protein170) (CmCLIP-170) 77 kDa echinoderm microtubuleHO204834 XM_970423.2 associated protein (CmEMAP) Collagen, type III, α1 HO204835 None (CmCOL3α1) Myosin regulatory light chain 2 HO204838 XM_969595.1 (CmMLC-2) HO204839 XM_971144.1 Troponin T (CmTnT) HO204843 XM_962035.1 Tropomyosin 2 (CmTPM2) HO204844 XM_968341.2 Coracle (CmCORA) Secreted protein acidic and rich in HO204846 XM_970372.2 cysteine (CmSPARC) Striated muscle activator of RhoHO204848 XM_962043.1 dependent signaling (CmSTARS) HO204850 XM_966756.1 Kettin (CmKETN) HO204851 None Tropoelastin (CmELN) Four and a half lim domains HO204852 XM_970895.2 (CmDRAL) HO204853 XM_965568.2 Profilin (CmPFN) XM_00181423 HO204854 Katanin 60 (CmKAT60) 1.1 UNC-45 related protein HO204855 XM_968020.1 (CmUNC-45R) HO204857 XM_963921.2 Sarcalumenin (CmSAR) Cellular function Stromal membrane-associated HO204861 XM_969010.2 protein 1 (CmSMAP1) FK669290 None Cadherin-like gene (CmCDHL1) HO204779

[TD$INLE]

[TD$INLE]

XM_968535.1

2.13

2.23Y

1.00

2.46Y

0.60

0.22Y

0.19

0.95Y

Hypoxia-responsive genes, defined as having a two-fold or larger change (P  0.05) in gene expression compared to control, are shaded green (induced) and red (repressed). Y: same trend of induction or repression by both microarray and qRT-PCR. N: opposite regulation directions by the two methods. n.a.: opposite trends but regulations below the cutoff.

have the capacity to sprout projections toward oxygen-starved areas. HIF prolyl hydroxylase (PHD2) is one of the determinant genes conferring adaptive plasticity by regulating the abundance of HIF-1 protein in response to oxygen lack (Fandrey et al., 2006; Centanin et al., 2008). PHD2 is a key oxygen sensor. It has been shown to regulate HIF complex, a master regulator mediating the adaptive genetic response, by promoting ubiquitin-mediated proteolysis of its asubunit in normoxia. Under hypoxia, proline hydroxylation is inhibited. Non-hydroxylated HIF-1a heterodimerizes with b subunits and recruits co-activators to form a transcriptionally active complex regulating gene expression in response to oxygen deprivation (Berra et al., 2003; Fandrey et al., 2006). Interestingly,

the cowpea bruchid homolog CmPHD2 transcripts were upregulated by hypoxia (Table 1). This transcriptional activation initially seemed paradoxical, as the hypoxic reactions depend on repressed PHD2 functions. The hypoxic induction of PHD2, however, can be explained by an auto-regulatory mechanism driven by oxygen tension (Berra et al., 2003); in mammals, PHD2 proteins accumulate progressively during hypoxia, but in an inactive form. Reoxygenation activates PHD2, triggering HIF-1a degradation. 3.6. Induction of muscle- and cell structure-related genes A striking feature of the microarray data is that a group of genes encoding proteins specific for muscle development and/or cell

[()TD$FIG]

Y.H. Chi et al. / Journal of Insect Physiology 57 (2011) 220–230

227

Fig. 1. Sequence annotation of differentially expressed genes in cowpea bruchids in response to hypoxia/hypercapnia based on (A) biological process and (B) molecular function. Cowpea seeds infested with cowpea bruchids (5 insects per seed) were maintained under conditions described in Section 2 till mid-fourth instar stage. The larvae were then exposed to 3% oxygen and 17% carbon dioxide for 4 h or 24 h, while the control bruchids were subjected to 20% oxygen and 0% carbon dioxide treatment. At the end of each time point, larvae were removed from the seeds and their midguts were dissected, and total RNA from midgut was extracted from hypoxic and control larvae, respectively, followed by microarray hybridization and BLAST search on differential genes. The E-value cutoff was set at 105.

structural proteins are all strongly induced by the hypoxia/ hypercapnia. Genes of several protein components associated with the actin-based contractile apparatus, such as titin, kettin, myosin light chain (MLC), UNC-45, tropnin T (TnT), tropomysin, DRAL and sarcalumenin (SAR) are strongly induced (Table 1). In Drosophila, titin is believed to function as a molecular scaffold and is instrumental for correct assembly of myofibrils and providing

[()TD$FIG]

elasticity (Machado and Andrew, 2000; Zhang et al., 2000). Myosin, the core of the thick filaments of muscle, represents the molecular motor of muscle contraction. Its light chain MLC regulates motor function via controlling the rate of tension development (Morano, 1999). UNC-45 has been shown to interact with myosin and is required for normal thick filament assembly (Etheridge et al., 2002; Price et al., 2002). TnT, tropomyosin and kettin interact with

Fig. 2. KEGG pathways. The hypoxia/hypercapnia-responsive genes were mapped to KEGG pathways using the BLAST2GO software. Only KEGG pathways with at least two genes mapped are shown.

[()TD$FIG]

228

Y.H. Chi et al. / Journal of Insect Physiology 57 (2011) 220–230

Fig. 3. Venn diagrams showing numbers of overlapping and unique midgut genes significantly induced or suppressed by hypoxia/hypercapnia treatment at 4 h and 24 h time points. U: unknown function protein; N: no hits in BLAST search. Results were based on the mean fold-change of four slides and two biological replicates. The major functional classes of genes are also indicated.

actin to act as a regulatory switch for muscle contraction (Hanke and Storti, 1986; Hakeda et al., 2000; Nongthomba et al., 2007). DRAL, the four and half LIM-only protein, binds to titin and couples metabolic enzymes to sites of high energy consumption in the sarcomere (Lange et al., 2002). Ca2+-binding sarcalumenin (SAR), although not essential for fundamental muscle functions, is important for muscle relaxation after contraction (Yoshida et al., 2005). Apparently, the cowpea bruchid midgut has undergone a process involving changes in contractility under hypoxia/hypercapnia. Among the induced genes are also those that are involved in cytoskeletal maintenance and those that mediate cell adhesion and proper cell–cell contact (Table 1). A homologue of a vertebrate actin-binding protein called striated muscle activator of Rho signaling (CmSTARS) was induced five-fold in our experiment by hypoxia. STARS is involved in cytoskeletal organization by stimulating actin polymerization and stress fiber formation under extracellular stimuli (Arai et al., 2002). An integrin-linked protein kinase (ILK), important for cell–cell adhesion and muscle attachment in Drosophila and C. elegans (Yasunaga et al., 2005), was up-regulated. Microtubules also constitute an important part of the cytoskeleton. The microtubule-associated proteins EMAP and dynein and the cytoplasmic linker protein CLIP-170 have been shown to modify the assembly dynamics of microtubules such that microtubules are slightly longer but more dynamic (Hughes et al., 1995; Lepley et al., 1999; Akhmanova et al., 2001). CLIP proteins may facilitate dynein for organelle movement. b-Spectrin, a major component of the membrane skeleton, stabilizes the cell membrane and is important in neuron and muscle development (Hammarlund et al., 2000). A membrane skeleton protein 4.1 gene (coracle) links transmembrane proteins with the underlying spectrin/actin cytoskeleton (Fehon et al., 1994). Genes for major extracellular matrix (ECM) protein collagen (COL) and non-collagenous SPARC were identified in cowpea bruchids. Both proteins play dominant roles in maintaining the structure of digestive tissues (Damja-

novski et al., 1992; Myllyharju and Kivirikko, 2004; Canty and Kadler, 2005). All collagen molecules consist of three polypeptide chains and their supramolecular assemblies serve as scaffolds for the attachment of other macromolecules. Prolyl 4-hydroxylaseaEFB gene (PH4aEFB1), encoding the key enzyme for collagen biosynthesis (Myllyharju, 2003), is also induced (Table 1). This gene has been recognized as an HIF1-dependent gene and is important to fertility and embryonic viability of C. elegans (Shen et al., 2005). The cytoskeleton interacts intimately with the cellular membrane, not only maintaining cell shape but enabling cellular movement, intracellular organelle transport, and muscular contraction. Hypoxia/hypercapnia apparently has a profound influence on the dynamics of the cytoskeletal network in the bruchid midgut based on gene expression patterns. Many of the cell structural genes are shown in mammalian and invertebrate studies to be involved in cell proliferation, migration, cytoskeletal dynamics, and myogenesis (Kuwahara et al., 2005). Perhaps hypoxic tissues are undergoing morphological changes, including even growth and differentiation of certain cells. For instance, hypoxia stimulates local tracheoles to form new branches toward oxygen-lacking tissue (Hoback and Stanley, 2001; Harrison et al., 2006). Formation and migration of the new branches may be facilitated by upregulation of these structural genes. In another scenario, lower feeding activity resulting from hypoxia may resemble starvation. It is known that fasting causes decreases in the length of intestinal microvilli (Avella et al., 1992). Microvilli of the insect midgut increase digestive tract surface area for enzyme secretion and for absorption of digested products. Shorter microvilli could reduce metabolic activity thus increasing adaptation under low oxygen stress. To achieve this, rapid reorganization of the cytoskeleton into different configurations would become necessary. Upregulation of katanin seems to support such a hypothesis (Table 1). Katanin is microtubule-severing ATPase and it uses nucleotide hydrolysis energy to disassemble microtubules (Hartman and Vale, 1999). This activity enables recycling and

Y.H. Chi et al. / Journal of Insect Physiology 57 (2011) 220–230

rearrangement of the microtubules to accommodate changes in cellular functions. 3.7. Maintaining cellular homeostasis under hypoxia/hypercapnia Consistent with the study in C. elegans and Drosophila, we found that hypoxia induced the expression of many transporter genes (Table 1; Suppl. Table). Broad reprogramming of gene expression to adapt to oxygen deficit no doubt requires massive reallocation of cellular resources. This is in agreement with the observation of transcript changes for a substantial number of transport proteins and proteins that facilitate molecular movement. It is noteworthy that while the majority of genes in this category are up-regulated, indicating their activation, aquaporin, the water transporter gene was down-regulated. Like many other storage insects, cowpea bruchids live and develop on dry food materials containing low water content. They have to obtain necessary water from products of food metabolism. Although it is not immediately apparent, hypoxia treatment could seriously limit the water supply for insect development due to lower metabolic activity. Shifting to anaerobic glycolysis not only furnishes less ATPs relative to complete oxidation, but also less water molecules, i.e. two versus six for each glucose molecule. Changing gene expression of this midgut aquaporin may have a role in facilitating water preservation, although this hypothesis remains to be elucidated. 4. Conclusions The current study represents our first attempt to understand the molecular responses of cowpea bruchids to hermetic control. It appears that hypoxic insects alter suites of genes in the alimentary tract, which leads to profound changes in their feeding behavior, digestive functionality, and rate of development. Results from our gene expression studies support the notion that cowpea bruchids attempt to cope with lack of oxygen by reducing energy demand, shifting to anaerobic metabolism, and strengthening cellular structure and muscular contraction. These changes presumably have prevented immediate insect death and contributed to higher tolerance to low oxygen conditions. A better understanding of insect response is crucial for development of more effective strategies aimed at substantial reduction of post-harvest losses, complementary to continued efforts to increase food production, through plant breeding and genetic engineering. Acknowledgments We thank Dr. Venu Margam at Purdue University for performing hypoxia/hypercapnia treatment and dissection of the midguts of the treated cowpea bruchids. We appreciate thoughtful discussions with Drs. Larry Murdock (Purdue) and Margam. We would also like to thank Dr. Ron Salzman for technical guidance in microarray analysis and for reviewing the manuscript. This project was supported by the USDA National Research Initiative grant# 2007-35607-17887, USDA Cooperative State Research, Education and Extension Service, grant# 2009-34402-19831, and by the KOSEF/MOST to the Environmental Biotechnology National Core Research Center, grant# R15-2003-012-01001-0, and a grant from the Biogreen 21 project of the Rural Development Administration 20070301034030. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinsphys.2010.11.011.

229

References Ahn, J.E., Guarino, L.A., Zhu-Salzman, K., 2010. Coordination of hepatocyte nuclear factor 4 and seven-up controls insect counter-defense cathepsin B expression. The Journal of Biological Chemistry 285, 6573–6584. Ahn, J.E., Lovingshimer, M.R., Salzman, R.A., Presnail, J.K., Lu, A.L., Koiwa, H., ZhuSalzman, K., 2007. Cowpea bruchid Callosobruchus maculatus counteracts dietary protease inhibitors by modulating propeptides of major digestive enzymes. Insect Molecular Biology 16, 295–304. Ahn, J.E., Salzman, R.A., Braunagel, S.C., Koiwa, H., Zhu-Salzman, K., 2004. Functional roles of specific bruchid protease isoforms in adaptation to a soybean protease inhibitor. Insect Molecular Biology 13, 649–657. Ahn, J.E., Zhu-Salzman, K., 2009. CmCatD, a cathepsin D-like protease has a potential role in insect defense against a phytocystatin. Journal of Insect Physiology 55, 678–685. Akhmanova, A., Hoogenraad, C.C., Drabek, K., Stepanova, T., Dortland, B., Verkerk, T., Vermeulen, W., Burgering, B.M., De Zeeuw, C.I., Grosveld, F., Galjart, N., 2001. Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts. Cell 104, 923–935. Anderson, L.L., Mao, X., Scott, B.A., Crowder, C.M., 2009. Survival from hypoxia in C. elegans by inactivation of aminoacyl-tRNA synthetases. Science 323, 630–633. Arai, A., Spencer, J.A., Olson, E.N., 2002. STARS, a striated muscle activator of Rho signaling and serum response factor-dependent transcription. The Journal of Biological Chemistry 277, 24453–24459. Avella, M., Blaise, O., Berhaut, J., 1992. Effects of starvation on valine and alanine transport across the intestinal mucosal border in sea bass, Dicentrarchus labrax. Journal of Comparative Physiology B-Biochemical Systemic and Environmental Physiology 162, 430–435. Azad, P., Zhou, D., Russo, E., Haddad, G.G., 2009. Distinct mechanisms underlying tolerance to intermittent and constant hypoxia in Drosophila melanogaster. PLoS ONE 4, e5371. Berra, E., Benizri, E., Ginouves, A., Volmat, V., Roux, D., Pouyssegur, J., 2003. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia. EMBO Journal 22, 4082–4090. Canty, E.G., Kadler, K.E., 2005. Procollagen trafficking, processing and fibrillogenesis. Journal of Cell Science 118, 1341–1353. Centanin, L., Dekanty, A., Romero, N., Irisarri, M., Gorr, T.A., Wappner, P., 2008. Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting. Developmental Cell 14, 547–558. Chi, Y.H., Salzman, R.A., Balfe, S., Ahn, J.E., Sun, W., Moon, J., Yun, D.J., Lee, S.Y., Higgins, T.J., Pittendrigh, B., Murdock, L.L., Zhu-Salzman, K., 2009. Cowpea bruchid midgut transcriptome response to a soybean cystatin – costs and benefits of counter-defence. Insect Molecular Biology 18, 97–110. Damjanovski, S., Liu, F., Ringuette, M., 1992. Molecular analysis of Xenopus laevis SPARC (Secreted Protein, Acidic, Rich in Cysteine). A highly conserved acidic calcium-binding extracellular-matrix protein. The Biochemical Journal 281 (Pt 2), 513–517. Duran, J., Navarro-Sabate, A., Pujol, A., Perales, J.C., Manzano, A., Obach, M., Gomez, M., Bartrons, R., 2008. Overexpression of ubiquitous 6-phosphofructo-2-kinase in the liver of transgenic mice results in weight gain. Biochemical and Biophysical Research Communications 365, 291–297. Etheridge, L., Diiorio, P., Sagerstrom, C.G., 2002. A zebrafish unc-45-related gene expressed during muscle development. Developmental Dynamics 224, 457–460. Fandrey, J., Gorr, T.A., Gassmann, M., 2006. Regulating cellular oxygen sensing by hydroxylation. Cardiovascular Research 71, 642–651. Farahani, R., Haddad, G.G., 2003. Understanding the molecular responses to hypoxia using Drosophila as a genetic model. Respiratory Physiology and Neurobiology 135, 221–229. Fehon, R.G., Dawson, I.A., Artavanis-Tsakonas, S., 1994. A Drosophila homologue of membrane-skeleton protein 4. 1 is associated with septate junctions and is encoded by the coracle gene. Development 120, 545–557. Frank, D.J., Roth, M.B., 1998. ncl-1 is required for the regulation of cell size and ribosomal RNA synthesis in Caenorhabditis elegans. Journal of Cell Biology 140, 1321–1329. Gotz, S., Garcia-Gomez, J.M., Terol, J., Williams, T.D., Nagaraj, S.H., Nueda, M.J., Robles, M., Talon, M., Dopazo, J., Conesa, A., 2008. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Research 36, 3420–3435. Hakeda, S., Endo, S., Saigo, K., 2000. Requirements of Kettin, a giant muscle protein highly conserved in overall structure in evolution, for normal muscle function, viability, and flight activity of Drosophila. Journal of Cell Biology 148, 101–114. Hammarlund, M., Davis, W.S., Jorgensen, E.M., 2000. Mutations in beta-spectrin disrupt axon outgrowth and sarcomere structure. Journal of Cell Biology 149, 931–942. Hanke, P.D., Storti, R.V., 1986. Nucleotide sequence of a cDNA clone encoding a Drosophila muscle tropomyosin II isoform. Gene 45, 211–214. Harrison, J., Frazier, M.R., Henry, J.R., Kaiser, A., Klok, C.J., Rascon, B., 2006. Responses of terrestrial insects to hypoxia or hyperoxia. Respiratory Physiology and Neurobiology 154, 4–17. Hartman, J.J., Vale, R.D., 1999. Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin. Science 286, 782–785. Hoback, W.W., Stanley, D.W., 2001. Insects in hypoxia. Journal of Insect Physiology 47, 533–542. Hochachka, P.W., Buck, L.T., Doll, C.J., Land, S.C., 1996. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving

230

Y.H. Chi et al. / Journal of Insect Physiology 57 (2011) 220–230

oxygen lack. Proceedings of the National Academy of Sciences of the United States of America 93, 9493–9498. Hughes, S.M., Vaughan, K.T., Herskovits, J.S., Vallee, R.B., 1995. Molecular analysis of a cytoplasmic dynein light intermediate chain reveals homology to a family of ATPases. Journal of Cell Science 108 (Pt 1), 17–24. Kawasaki, K., Kuge, O., Chang, S.C., Heacock, P.N., Rho, M., Suzuki, K., Nishijima, M., Dowhan, W., 1999. Isolation of a chinese hamster ovary (CHO) cDNA encoding phosphatidylglycerophosphate (PGP) synthase, expression of which corrects the mitochondrial abnormalities of a PGP synthase-defective mutant of CHO-K1 cells. The Journal of Biological Chemistry 274, 1828–1834. Kitch, L.W., Murdock, L.L., 1986. Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus-Maculatus F. Archives of Insect Biochemistry and Physiology 3, 561–575. Koo, Y.D., Ahn, J.E., Salzman, R.A., Moon, J., Chi, Y.H., Yun, D.J., Lee, S.Y., Koiwa, H., Zhu-Salzman, K., 2008. Functional expression of an insect cathepsin B-like counter-defence protein. Insect Molecular Biology 17, 235–245. Kuwahara, K., Barrientos, T., Pipes, G.C., Li, S., Olson, E.N., 2005. Muscle-specific signaling mechanism that links actin dynamics to serum response factor. Molecular and Cellular Biology 25, 3173–3181. Lange, S., Auerbach, D., McLoughlin, P., Perriard, E., Schafer, B.W., Perriard, J.C., Ehler, E., 2002. Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2. Journal of Cell Science 115, 4925–4936. Langyintuo, A.S., Lowenberg-DeBoer, J., Faye, M., Lambert, D., Ibro, G., Moussa, B., Kergna, A., Kushwaha, S., Musa, S., Ntoukam, G., 2003. Cowpea supply and demand in West and Central Africa. Field Crops Research 82, 215–231. Lepley, D.M., Palange, J.M., Suprenant, K.A., 1999. Sequence and expression patterns of a human EMAP-related protein-2 (HuEMAP-2). Gene 237, 343–349. Liu, G., Roy, J., Johnson, E.A., 2006. Identification and function of hypoxia-response genes in Drosophila melanogaster. Physiological Genomics 25, 134–141. Machado, C., Andrew, D.J., 2000. D-Titin: a giant protein with dual roles in chromosomes and muscles. Journal of Cell Biology 151, 639–652. Margam, V.M., 2009. Mode of action of hermetic storage of cowpea grain. PhD Dissertation (Purdue University). pp. 103–133 (Chapter 5). Minchenko, O., Opentanova, I., Caro, J., 2003. Hypoxic regulation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene family (PFKFB-1-4) expression in vivo. FEBS Letters 554, 264–270. Moon, J., Salzman, R.A., Ahn, J.E., Koiwa, H., Zhu-Salzman, K., 2004. Transcriptional regulation in cowpea bruchid guts during adaptation to a plant defence protease inhibitor. Insect Molecular Biology 13, 283–291. Morano, I., 1999. Tuning the human heart molecular motors by myosin light chains. Journal of Molecular Medicine 77, 544–555. Myllyharju, J., 2003. Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis. Matrix Biology 22, 15–24. Myllyharju, J., Kivirikko, K.I., 2004. Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends in Genetics 20, 33–43.

Nagao, Y., Kitada, S., Kojima, K., Toh, H., Kuhara, S., Ogishima, T., Ito, A., 2000. Glycine-rich region of mitochondrial processing peptidase alpha-subunit is essential for binding and cleavage of the precursor proteins. The Journal of Biological Chemistry 275, 34552–34556. Nongthomba, U., Ansari, M., Thimmaiya, D., Stark, M., Sparrow, J., 2007. Aberrant splicing of an alternative exon in the Drosophila troponin-T gene affects flight muscle development. Genetics 177, 295–306. Price, M.G., Landsverk, M.L., Barral, J.M., Epstein, H.F., 2002. Two mammalian UNC45 isoforms are related to distinct cytoskeletal and muscle-specific functions. Journal of Cell Science 115, 4013–4023. Shen, C., Nettleton, D., Jiang, M., Kim, S.K., Powell-Coffman, J.A., 2005. Roles of the HIF-1 hypoxia-inducible factor during hypoxia response in Caenorhabditis elegans. The Journal of Biological Chemistry 280, 20580–20588. Silva, C.P., Xavier-Filho, J., 1991. Comparison between the levels of aspartic and cysteine proteinases of the larval midguts of Callosobruchus-Maculatus (F) and Zabrotes-Subfasciatus (Boh) (Coleoptera, Bruchidae). Comparative Biochemistry and Physiology B-Biochemistry and Molecular Biology 99, 529–533. Utz, P.J., Hottelet, M., Le, T.M., Kim, S.J., Geiger, M.E., van Venrooij, W.J., Anderson, P., 1998. The 72-kDa component of signal recognition particle is cleaved during apoptosis. The Journal of Biological Chemistry 273, 35362–35370. Yasunaga, T., Kusakabe, M., Yamanaka, H., Hanafusa, H., Masuyama, N., Nishida, E., 2005. Xenopus ILK (integrin-linked kinase) is required for morphogenetic movements during gastrulation. Genes Cells 10, 369–379. Yoshida, M., Minamisawa, S., Shimura, M., Komazaki, S., Kume, H., Zhang, M., Matsumura, K., Nishi, M., Saito, M., Saeki, Y., Ishikawa, Y., Yanagisawa, T., Takeshima, H., 2005. Impaired Ca2+ store functions in skeletal and cardiac muscle cells from sarcalumenin-deficient mice. The Journal of Biological Chemistry 280, 3500–3506. Yun, H., Lee, M., Kim, S.S., Ha, J., 2005. Glucose deprivation increases mRNA stability of vascular endothelial growth factor through activation of AMP-activated protein kinase in DU145 prostate carcinoma. The Journal of Biological Chemistry 280, 9963–9972. Zhang, J., Randall, M.S., Loyd, M.R., Dorsey, F.C., Kundu, M., Cleveland, J.L., Ney, P.A., 2009. Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation. Blood 114, 157–164. Zhang, Y., Featherstone, D., Davis, W., Rushton, E., Broadie, K., 2000. Drosophila Dtitin is required for myoblast fusion and skeletal muscle striation. Journal of Cell Science 113 (Pt 17), 3103–3115. Zhu-Salzman, K., Koiwa, H., Salzman, R.A., Shade, R.E., Ahn, J.E., 2003. Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor. Insect Molecular Biology 12, 135–145. Zhu-Salzman, K., Salzman, R.A., 2001. Functional mechanics of the plant defensive Griffonia simplicifolia lectin II: resistance to proteolysis is independent of glycoconjugate binding in the insect gut. Journal of Economic Entomology 94, 1280–1284.