Use of subtracted libraries and macroarray to isolate developmentally specific genes from the mosquito, Aedes aegypti

Insect Biochemistry and Molecular Biology 32 (2002) 1757–1767 www.elsevier.com/locate/ibmb

Use of subtracted libraries and macroarray to isolate developmentally specific genes from the mosquito, Aedes aegypti Kendall C. Krebs a,b, Kristina L. Brzoza a, Que Lan a,∗ b

a Department of Entomology, University of Wisconsin-Madison, Madison, WI 53706, USA Present address: Department of Pathology, University of Wisconsin-Madison, Madison, WI 53706, USA

Received 9 April 2002; received in revised form 28 June 2002; accepted 1 July 2002

Abstract Subtracted cDNA libraries were screened with cDNA macroarrays to isolate larval and pupal stage-specific genes from Aedes aegypti. Of 103 partial cDNAs sequenced from the 4th instar subtracted cDNA library, 62 have counterpart genes in other organisms while 41 of them have no significant similarity to any known genes. Sequences of 116 partial cDNA clones from the pupal subtracted library revealed that 57 belong to unknown genes and 59 have homologous genes in other organisms. Results of cDNA macroarrays showed that 42–50% of randomly selected genes in the subtracted cDNA libraries were differentially expressed. Of the unknown genes, transcripts of 15–19% of the genes were detected in larval or pupal stages, respectively. The results indicate that a subtracted cDNA library in combination with a cDNA macroarray can be used effectively to identify genes expressed in a particular stage.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Aedes aegypti; Stage-specific gene; cDNA macroarray; Subtracted cDNA library; Cuticle protein gene

1. Introduction In holometabolous insects, distinct morphological and physiological events mark each developmental stage. At the cellular and molecular levels, the differences in each developmental stage are established by synthesis of stage-specific proteins. Differences in the spectrum of translatable mRNAs in 4th stadium larvae, pupae and adults were reported in Aedes aegypti (Ae. aegypti) (Wattam and Christensen, 1992). Results from those early studies support the notion that differential gene expression is correlated with different developmental stages. Several large scale Expression Sequence Tags (EST) projects have been conducted to sequence expressed genes in adult Ae. aegypti (Bohbot and Vogt, 2001; Morais and Severson, 2001a; Gill et al., 1999). However, little is known about transcription regulation of stage-specific genes during the metamorphosis, in which significant sexual dimorphism occurs in Ae. aegypti (Christophers, 1960). Only a few larval and

∗ Corresponding author. Tel.: +1-608-263-7924; fax: +1-608-2623322.

pupal genes have been identified in Ae. aegypti such as hexamerin-1 and -2 (Gordadze et al., 1999), p450 (Morais and Severson, 2001b) and intestinal mucin (Rayms-Keller et al., 2000). Most noticeable is that hexamerin-2 is more abundant in larvae and hexamrin-1 is expressed at higher levels in early pupae (Korochkina et al., 1997). Hexamerin-1 gamma subunit is the only known gene that has higher expression in female pupae (Gordadze et al., 1999). Consequently, it is necessary to identify more larval and pupal specific genes and investigate their expression regulation so that we can define the molecular pathway for their function. Methods used to identify differentially expressed genes include differential display, subtracted cDNA, cDNA macroarray and microarray. Differential display has been used successfully to isolated novel genes in mosquitoes (Ricci et al., 2002; Vizioli et al., 2001). Differential display can potentially generate high numbers of false positive results due to co-migrate of cDNA species (Dimopoulos and Louis, 1997). To eliminate false positive clones of differential display, a secondary screening step is usually required (Nagel et al., 2001; Ali et al., 2001). Subtracted cDNA is an effective technique for identifying differentially expressed genes, it is

0965-1748/02/$ - see front matter.  2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 2 ) 0 0 1 1 6 - 9

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very laborious and time consuming if it is used alone as the analysis of expression profiles of hundreds of clones are required to confirm the results. A newly developed technique for identifying differentially expressed genes is the cDNA array. The array can be performed at a macro or micro scale. In macroarray, hundreds of cDNAs are arrayed on 8 × 12 cm Nylon membranes for screening. In microarray, thousands of cDNAs are spotted onto a glass slide for screening. The high-throughput cDNA microarray is costly and time consuming and it is not an affordable technique for most of the laboratories. To minimize those problems, we have developed an alternative procedure based on two methods, a subtracted cDNA library and a cDNA macroarray, to isolate stage-specific genes from Ae. aegypti. Sequence analysis was conducted for randomly selected cDNAs that are larger than 300 bp. We here report that the subtracted cDNA libraries enriched differentially expressed genes. The results of cDNA array screening showed that expression of 15–19% genes were detected only in 4th instars or pupae, respectively. Northern blotting analysis confirmed that over 90% of identified genes were differentially expressed. 2. Materials and methods 2.1. Mosquitoes The mosquitoes used in these experiments, Ae. aegypti, were from an inbred laboratory strain (Rockefeller). Larvae were reared at 26°C in 70–80% humidity with a light:dark cycle (14:10) with pellets of rabbit food. Larvae hatching during a 15-min period were collected and used in experiments. Under these conditions, development of the 4th stadium took 64 h (mostly males) to 72 h (mostly females) to complete the 4th stadium. Adults were maintained at 24°C. Pharate 4th instars were staged by using the appearance of visible dark black hairs wrapped around the body under the thorax and abdominal cuticle of the 3rd instars (Christophers, 1960). Larvae selected by this criteria ecdyse within a 1 h period. Selected larvae were transferred to a new container and newly molted 4th instar larvae were collected. Pharate pupae were identified by their physical characteristics: rudimentary respiratory trumpets that turn dark brown under the cuticle of a late 4th instar. Larvae selected by this criteria will pupate within a 1 h period (Christophers, 1960). Pharate pupae were transferred to a new container and collected as newly molted pupae. 2.2. RNA extraction Ten staged animals were washed with ddH2O, rinsed once with DEPC–H2O and excess water was removed by

blotting. Cleaned animals were put into 1.5 ml microfuge tubes and homogenized with a micropestle in 1 ml of Trizol reagent according to the manufacture instruction (Invitrogen Corporation, Carlsbad, CA). Tissues were dissected in insect saline solution (Riddiford et al., 1979) under dissecting microscope and homogenized in 0.5 ml of Trizol reagent immediately and another 0.5 ml of Trizol reagent was added into the test tube. The RNA samples were frozen at –80°C until use. At least 20 animals were used to isolate different tissues at each time point. To extract mRNAs for the construction of cDNA libraries, 1 mg of total RNA was used as starting material and mRNAs were selected using RNA separator (ClonTech, Palo Alto, CA). 2.3. Construction of subtracted cDNA libraries Two micrograms of mRNAs from early 4th instars (12–24 h post 3rd molt) and early pupae (12–24 h post pupation) were used to make tester and driver cDNAs according to the manufacture instructions (ClonTech). Libraries were constructed as described in the PCR–Select cDNA subtraction Kit. PCR products were cloned into the pT-Adv plasmid (ClonTech), transformed into TOP 10F’ E. coli competent cells and plated on LB plates under a dNTP containing cold dATP (10 mM of dTTP, dCTP, dGTP and 10 µM of dATP) Ampicillin selection. Each colony was inoculated on a master plate and cultured to generate stocks. A 4th instar subtracted cDNA library was constructed by hybridizing early pupal driver cDNA with early 4th instar tester cDNA at 2:1 ratio. A pupal subtracted cDNA library was made using driver cDNAs of early 4th instars vs early pupal tester cDNA at 2:1 ratio. The low driver vs tester cDNA ratio is designed to increase the possibility of isolating moderately differentially expression genes (Konietzko and Kuhl, 1998). 2.4. Sequence analysis Randomly selected cDNA clones were screened using PCR with nested primers of the two adaptors. Plasmid minipreps of clones containing inserts of 300 bp or more were made using QiaSpin column (QIAGEN, Valencia, CA) and sequenced in an automatic sequencer (ABI 377XL) using BigDye labeling (Amersham Pharmacia Biotech AB, Uppsala, Sweden). DNA sequences were compared to the databank Blastn (nr database) and Blastx (nr database) 2.2 programs (Altschul et al., 1997; NCBI). Sequence similarity (identical and conversed substitution of amino acid at aligned positions) above 50% and an E value less than 1 was considered a ‘positive match’. An E value of 1 assigned to a hit can be interpreted as meaning that in a database of current size one might expect to see one match with a similar score

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simply by chance. Sequences were also compared to Anopheles gambiae DNA databank (EMBL, 2001). Sequences that matched to hypothetical proteins or having low similarity to a known protein were considered as unknown genes. 2.5. Macroarray of cDNAs All sequenced clones were amplified through PCR in a 96 well plate. The PCR product (15 µl ⫽ 50–100 ng) was mixed with 200 µl of 0.4 M NaOH/10 mM EDTA and boiled for 10 min. Immediately after denaturing, PCR products were cooled on ice for 3 min and 100 µl was blotted on positively charged Nylon membrane in duplicate filters using dot-blotting apparatus (Millipore, Bedford, MA). The DNA dot blots were washed with 250 µl of 0.4 M NaOH/dot. The membrane was rinsed in 2× SSC buffer at room temperature for 5 min and airdried. DNA was permanently attached to the membrane in an UV cross-linker (Stratagene Cloning System, La Jolla, CA). On each membrane an actin cDNA of Ae. aegypti was spotted for normalization. To label cDNA probes, the SuperScript II reverse transcription kit (Invitrogen) was used as described by manufacture with some modifications. Briefly, dNTP containing unlabeled dATP (10 mM of dTTP, dCTP, dGTP and 10 µM of dATP) and 5 µl [α-32P] dATP (3000 Ci/mM) was added into the reaction to generate probes with higher specific activity. A typical reaction gave 32 – 34% incorporation of the [α-32P] dATP and generated 5– 7.5 × 107 cpm/µg mRNAs. The cDNA arrays were prehybridized at 42°C overnight in pre/hybridization solution (4× SSC, 10× Denhardt’s solution, 50% formamide, 0.1% SDS, 50 mM Na2HPO4 (pH 7.2), 1 mM EDTA and 100 µg/ml sheared herring sperm DNA). Labeled cDNA probes were added to the prehybridization solution at 2– 3 × 106 cpm/ml (8 ml for two 8 × 12 cm2 membranes) and incubated at 42°C overnight in a hybridization oven. The membranes were washed at 65°C for 15 min twice in 2× SSC/0.1% SDS, once in 1× SSC/0.1% SDS and twice in 0.1 SSC/0.1% SDS. Membranes were exposed to a Phosphor image screen for 4 h and scanned with a Storm800 (Molecular Dynamics, Piscataway, NJ). The data were analyzed using Storm800 software. Duplicate cDNA arrays were hybridized with cDNA probes of different stages, and the intensity of the hybridization signal was first corrected by the average signals of three blank dots on the membrane where no cDNA sample was applied. Then, the corrected hybridization signals were normalized with an actin D1 cDNA that was spotted on each membrane. The signal levels of actin D1 was high (Fig. 1), but it was not at the saturation level for the phosphor image screen. The arrays were not used for absolute quantitative analysis; the relative levels were used as an indicator to catalog stage-

Fig. 1. Hybridization signals of 96 cDNA clone inserts from the 4th instar subtracted cDNA library spotted on a positively charged nylon membrane. (A) Hybridization with α-32P-labeled cDNAs of early 4th instars. (B) Hybridization with α-32P-labeled cDNAs of early pupae. Arrows indicate cDNA inserts that differentially hybridized to the 4th instar cDNA probes.

specific genes. For cDNAs that generated saturated signals (Fig. 1), the relative expression levels might have been underestimated. 2.6. Northern blotting analysis Fifteen to twenty micrograms of total RNA from indicated stages were used in Northern blotting analysis as described (Ausubel et al., 1993). RNAs were transferred from the agarose gel on to a positively charged Nylon membrane and baked at 80°C under vacuum for 1 h and UV cross-linked (Stratagene Cloning System). Radioactive probes were made using either 50 µCi [α32 P] dATP (3000 Ci/mM) or [α-32P] dCTP (3000 Ci/mM) with the PrimerIT II kit (Stratagene Cloning System) or PCR reaction with DNA polymerase Tfl (Epicentre, Madison, WI). The PCR was performed using one cycle of 94°C, 3 min to denature the template, then three cycles of 94°C for 2 min, 50°C for 3 min and 72°C for 5 min. The probes were cleaned using QiaSpin column (QIAGEN). The incorporation rate of [α-32P] dCTP was between 20 and 50% with specific activity of more than 108 cpm/µg template DNA. Prehybridization and hybridization and were conducted under the same condition as described above for cDNA arrays. The membranes were washed at 65°C for

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15 min once in 2× SSC/0.1% SDS, 1× SSC/0.1% SDS, 0.5× SSC/0.1%SDS and 0.2× SSC/0.1% SDS. The blots were exposed to a phosphor image screen for 4–12 h and scanned.

3. Results 3.1. Identification of stage-specific genes Many techniques exist for identifying differentially expressed genes. In the present study a combination of two techniques, a subtracted cDNA library and a cDNA macroarray, enabled us to effectively isolate stage-specific genes from larvae and pupae. Subtracted cDNAs of early 4th instars from early pupal cDNAs and early pupae from early 4th instars were cloned. The 4th instar subtracted cDNA library was enriched for genes that express at relatively higher levels between 12 and 24 h post 3rd molt (Tables 1 and 2) and the early pupal subtracted cDNA library contained more genes that were transcribed between 12 and 24 h post pupation (Table 2). We sequenced 103 cloned cDNAs from the 4th instar subtracted cDNA library, 62 had counterpart genes in other organisms and 41 had no significant similarity to any known genes (Table 1). Of the 113 partial cDNA clones from the pupal subtracted library, 57 belonged to unknown genes and 59 had homologous genes in other organisms (Table 1). Only a few cDNAs were cloned from both libraries, implying that the cDNA subtraction worked well and enriched differentially expressed genes while common housekeeping genes were minimized (Table 1). The cloned cDNAs probably do not represent the entire transcripts because the double stranded cDNAs were digested with RsaI to create shorter fragments for PCR amplification during the subtracted cDNA library construction. Sequences of cDNAs were compared to data in the GenBank using both Blastn and Blastx (http://www.ncbi.nlm.nih.gov/BLAST/, 2001). All cDNAs were translated in both directions to find ORF using ORF Finder (http://www.ncbi.nlm.nih.gov/ gorf/gorf.html, 2001). DNA sequences that matched to hypothetical proteins or having low similarity to a known protein were considered as unknown genes. Some of the cDNAs represent independent clones of the same genes. For example, all 13 partial Amy II cDNA clones are 100% homologous to each other and are most likely transcribed from the same gene. Multiple copies of the same unknown genes were also found in the subtracted cDNA libraries. Expression profiles of most of the genes represented by cDNAs from both subtracted cDNA libraries were unknown. To effectively assess the stage specificity of gene expression, cDNAs were screened using cDNA macroarrays. Radioactive cDNA probes were made from

mRNAs of early 4th and pupal stages. The labeled cDNA probes were hybridized to the cDNA arrays made from the subtracted cDNA libraries. Fig. 2 shows a typical result of cDNA array screening. The panel has cDNAs from the 4th instar subtracted cDNA library, when hybridized to cDNA probes of 4th instars (Fig. 1A), most of the clones showed varied levels of signals. However, when cDNA probes from the pupal stage were used for the same panel of cDNA clones, some of the cDNAs have much lower levels of hybridization signals (Fig. 1B, dots correspond to the arrows in panel A). The results imply that some of the genes were expressed at higher levels in the larval stage. If levels of expression differed more than 2-fold between two sets of arrays that were hybridized to two different labeled cDNA probes, the gene was identified as differentially expressed (Table 2). Results of cDNA arrays showed that 42–50% of the partial cDNAs in the subtracted cDNA libraries were differentially expressed (Table 2). Genes known with high larval stage-specificity such as amylase II (McGeachin et al., 1972; Grossman et al., 1997) and hexamerin 1γ (Korochkina et al., 1997; Gordadze et al., 1999) were confirmed by cDNA arrays (Table 2). Of the unknown genes, about 15–19% were expressed only in larval or pupal stages, respectively (Table 2). Importantly, 8–21% of the partial cDNAs represent genes with moderate change in expression levels (Table 2, column ‘⬍5- but ⱖ2-fold higher level’). It has been shown that moderate changes in the level of expression of some genes can have great impact on the overall developmental progress. For example, EcR (Jindra et al., 1996), USP (Wang et al., 2000) and MHR3 (Palli et al., 1992) only have 3to 5-fold changes in their levels of transcription, and those genes are known for their effects on the developmental progress and physiological changes. The low ratio of driver vs tester cDNAs for constructing the subtracted cDNA libraries was designed to have a more favorable condition for isolating moderately differentially expressed genes (Konietzko and Kuhl, 1998). However, the low driver to tester cDNA ratio resulted in high background. In other words, 50–58% genes in the subtracted cDNA libraries represented commonly expressed genes. To efficiently identify true differentially expressed gene, DNA array was necessary to screen the subtracted cDNA libraries. 3.2. Northern blotting analysis of stage-specific gene expression Northern blotting analysis was used to confirm that genes identified by combined methods described above were differentially expressed in the larval and pupal stages. Genes that showed more than a 2-fold differential expression between larvae and pupae (Table 2) were targeted for expression analysis. Three partial cDNAs of

Larval cuticle Larval cuticle Esterase

Serine protease Chymotrypsin 1 Sphigomyelin phospdiesterase 1 Sterol carrier protein Immuneresponsive serine protease

CdK9 NTPase/helicase Fatty acidbinding protein Ae4-137, 359, 397 Hexamerin 2 alpha Ae4-50, 136, 147, 343, 389, 416, 418 Hexamerin 1 gama

Ae4-403 Ae4-408 Ae4-372

Ae4-327

Ae4-339

Ae4-23 Ae4-36, 356, 380 Ae4-341

Ae4-371A Ae4-105, 370 AeP-390 AeP-175 AeP-51 AeP-140 AeP-17

10⫺43 10⫺15 10⫺32 10⫺29 10⫺31

AeP-315 AeP-306 AeP-64

10⫺112 0.39 10⫺15 10⫺62 10⫺31

100%, Ae. aegypti 98%, Ae. aegypti

10⫺7

Late trypsin StretchinMLCK PGDH CCT Axoxia upregulated proetin

Apolipoprotein Adult cuticle Ferritin heavy chain SP71 gene

Delta-like protein

E1

Amylase

AeP-42, 49, 101, 103, 106, 107, 109, 118, Actin-7 C121, 123, 127, 134, 142, 148, 149, 150, termini 151, 226, 232, 247, 255, 266, 278, 284, 289, 293, 310, 313

AeP-126, 180

10⫺41

10⫺6

AeP-10 AeP-50, 285, 259 AeP-113

10⫺10 10⫺22 10⫺14

10⫺63

Similarity to gene

AeP-3, 5, 8, 15, 21, 41, 44, 61, 67, 110, Unknown 111, 112, 114, 115, 116, 122, 128, 129, 131, 132A, 136, 139, 146, 152, 155, 162, 166, 169, 170, 171, 190A, 190B, 229, 230, 234, 235, 242, 252, 256, 257, 268, 273, 275, 282, 301, 302, 304, 381, 384, 386, 391, 392, 393, 394, 396, 397, 399

E value Pupal subtracted cDNA clone#

95%, Drosophila 52%, Melanoplus 72%, Locusta

61%, Anopheles

55%, Rat

99%, Culex 58%, Ae. aegypti 54%, Mouse

Alpha esterase- 66%, Drosophila 7 Maltase 1 65%, Drosophila Aldolase 78%, Drosophila

81%, Locusta 81%, Drosophila 54%, Anisopteromalus

Amylase

Ae4-24, 57, 85, 95, 150, 344, 361, 375, 402, 405, 413, 415, 439 Ae4-4 Ae4-7, 97, 410 Ae4-77, 83, 419A, 423

Ae4-133

100%, Ae. aegypti

Unknown

Ae4-5, 8, 18, 34, 39, 43, 54, 56, 58, 61, 113, 115, 119, 121, 141, 146, 325, 326, 328, 329, 338, 340A, 358, 376, 382A, 385, 386, 387, 388, 390, 393, 404, 411, 414, 421, 424, 427, 430, 435, 441, 449

% Similarity, organism

Similarity to gene

IV instar subtracted cDNA clone#

Table 1 List of partial cDNAs sequenced and identified from 4th instar and pupal subtracted cDNA libraries

10⫺31

0.62

10⫺63

0.79

10⫺20 10⫺44 10⫺4

10⫺53 10⫺31

10⫺71

10⫺18 0.48 10⫺40

E valuea

(continued on next page)

84%, Drosophila

48%, Drosophila

100%, Ae. aegypti

92%, Drosophila

77%, rat 93%, Carassius 80%, Drosophila

79%, Ae. aegypti 70%, Drosophila

85%, Drosophila

54%, Drosophila 58%, Drosophila 100%, Ae. aegypti

% Similarity, organism

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Lipase Actin D1 Tubulin beta chain

Aminopeptidase N-like Deltaaminlevuinate Beta trypsin p450 Glucosidase

Ae4-354 Ae4-60, 349 Ae4-438

Ae4-345

73%, Drosophila 50%, Drosophila

79%, Culex

57%, Drosophila 98%, Ae. aegypti 62%, Spodoptera

86%, Drosophila

53%, Ae. aegypti

54%, Drosophila 99%, Drosophila 96%, Drosophila

75%, Anopheles

% Similarity, organism

AeP-270, 398 AeP-274

10⫺9 10⫺33 10⫺46

AeP-387 AeP-283 AeP-60, 117, 120, 124, 135, 227, 279, 288, 290

10⫺9 10⫺104 10⫺14

10⫺35 10⫺14

10⫺24

AeP-194

10⫺22

10⫺13

Membrane protein

AeP-241

10⫺74

54%, yeast

% Similarity, organism

Muscle specific protein Nit protein oxoprolinase Myosin heavy chain

68%, human 59%, rat 74%, Drosophila

59%, Drosophila

Actin D1 99%, Drosophila Receptor-like 66%, Arabidopsis serine/threonine kinase

Similarity to gene

E value Pupal subtracted cDNA clone#

10⫺35 10⫺20 10⫺74

10⫺11

10⫺34 0.24

0.68

E valuea

a Cloned PCR amplified cDNAs from 4th instar (Ae4-XXX) and pupal subtracted cDNA library (AeP-XXX). Inserts of cDNAs that are 300 bp or longer were sequenced using an automated sequencer. The sequences were compared to sequences in GenBank using the ‘BLASTX’ program. An E value of 1 ⫽ 1 match with similar score by chance in the database. Genes in bold letter are chosen for Northern blotting analysis described in the Results section.

Ae4-139 Ae4-336, 384, 451

Ae4-394

Ae4-357, 429 Ae4-42 Ae4-440

Binary toxinbinding alphaglucosidase Catalase Protease

Hexamerin A

Ae4-378, 379A

Ae4-409

Similarity to gene

IV instar subtracted cDNA clone#

Table 1 (continued)

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Table 2 Screening of cDNA arrays with cDNA probes of early 4th larvae (top panel) and pupae (bottom panel)a Genes cloned from the early 4th instar

Expressed only in 4th instar larvae vs pupae

Early IV vs early pupa, Early IV vs early No change ⱖ5-fold higher level pupa, ⬍5- but ⱖ2-fold higher level

Total number of screened

Unknown genes Known genes Amylase II Hexamerin 1γ Aldolase 4c Ae4-4 (LCP-1) P450

15% 13% + ⫺ + + ⫺

6% 13% ⫺ + ⫺ ⫺ ⫺

33 29

Genes cloned from the early pupae

Expressed only in pupae vs early 4th instar

Early pupa vs. early IV, Early pupa vs No change ⱖ5-- fold higher level early IV, ⬍5- but ⱖ2-fold higher level

Total number of screened

Unknown genes Known genes Delta-like protein Cuticle protein-1 Myosin Ferritin

19% 15% + ⫺ ⫺ ⫺

15% 21% ⫺ ⫺ + ⫺

52 19

21% 13% ⫺ ⫺ ⫺ ⫺ ⫺

8% 15% ⫺ ⫺ ⫺ +

58% 61% ⫺ ⫺ ⫺ ⫺ +

58% 49% ⫺ + ⫺ ⫺

a The arrays were first corrected with blank spots on the membrane and then normalized with an Ae. aegypti actin cDNA (see Materials and Methods).( ⫺) Levels of expression are not in this category. (+) Levels of expression are in this category.

genes that had higher levels of expression in the early 4th instars than in the early pupae were studied in more detail. Nine partial cDNAs from early pupal subtracted cDNA library were chosen for detailed expression analysis based on the results of cDNA macroarray assay indicated these were potentially pupal stage-specific genes. All three tested partial cDNAs isolated from the 4th instar subtracted cDNA library showed much higher levels of transcription in the early 4th instars than in the early pupal stages (Fig. 2A). Two of them, Ae4-4 and Ae4-427 are larval stage-specific genes. The mRNA of Ae4-427 was not detected in the head, weakly in the gut/Malpighian tubules, and strongly in the body wall including epidermis, fat body, muscle, peripheral nerves, and trachea (Fig. 2B). Ae4-411 was expressed at high levels in the early larvae (Fig. 2A, 3rd 1 h and 4th 0– 24 h) and transcription decreased to low levels during the later periods (Fig. 2A, 3rd 17–27 h and 4th 27–66 h). Ae4-411 transcripts were detected in the early pupae but not after 24 h in the pupae and first day adults (Fig. 2A). Ae4-411 is only weakly expressed in the head and gut/Malpighian tubules of 4th instars (Fig. 2B). Other larval stage-specific genes are reported elsewhere (Vyazunova and Lan, in preparation). Therefore, the 4th instar subtracted library has enriched genes expressed at higher levels in larval stages, which is consistent with the results of the cDNA macroarray assay. Eight of the nine genes chosen from pupal library had relatively higher levels of transcription in early pupae

than that of in early 4th instars (Fig. 3). AeP-242 and 162 were the only two genes which showed exclusive pupal stage-specific expression profiles (Fig. 3). Transcripts of AeP-132A were observed in late larvae, early pupae and day 1 adults (Fig. 3). Expression of AeP-64, AeP-126, AeP-67 and AeP-170 was detected in late larval and early pupal stages (Fig. 3). One gene did not show any stage-specific expression pattern (data not shown). Therefore, developmentally specific genes were identified at about a 90% successful rate using the combination approach. The expression of AeP-242 was weak in the gut/Malpighian tubules and strong in the head and rest of the body parts (Fig. 4). AeP-162 transcripts were observed in the head and rest of the body except gut and Malpighian tubules in early pupae (Fig. 4). Transcript of AeP-126 was not observed in the gut/Malpighian tubules of either larvae or pupae and the expression was strong in the rest of the body parts including the head (Fig. 4). AeP-67 was not expressed in the gut/Malpighian tubules of larval or pupal (Fig. 4).

4. Discussion To identify and isolate stage specific genes from Ae. aegypti, we have made two subtracted cDNA libraries from the mRNAs extracted from individuals collected 12–24 h post 3rd molt and 12–24 h post-pupal stages.

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Fig. 2. Northern blotting analysis of expression patterns of Ae4-4, Ae4-411 and Ae4-427 genes. (A) Developmental expression profiles. Estimated molecular weight of mRNA is marked on the right side of the picture. Twenty micrograms of total RNA is loaded in each lane. (B) Tissue distribution of the transcripts. Fifteen micrograms of total RNA is loaded in each lane. Hour post certain stage is indicated as ×h. F ⫽ females; M ⫽ males; Mt ⫽ Malpighian tubules; Body ⫽ body wall (peripheral nerves, tracheae, epidermis, muscle and fat body). Ethidium bromide stain of the rRNA in the agarose gel is show under the Northern blot image.

The choice of 12–24 h post 3rd molt was based on the 20–hydroxyecdysone (20E) titer which rises from 18 to 27 h post 3rd molt (Jenkins et al., 1992). In the second period of choice, i.e., the first 12–24 h post-pupation, there are peaks of 20E in both male and female pupae that have been shown to be critical for pupa–adult development (Whisenton et al., 1989). Certain genes such as amylase and hexamerin known to be highly expressed in larvae are present in the 4th instar subtracted cDNA library in high numbers. Amylase II activity is high in the gut of 4th instars (McGeachin et al., 1972; Grossman et al., 1997) and 12% of sequenced cDNAs (13/103) from the 4th instar subtracted cDNA library were amylase II gene (Table 1). Only one copy of amylase II was found in 113 of sequenced pupal cDNAs. Hexamerin genes are

Fig. 3. Northern blotting analysis of the developmental expression profiles of AeP-64, AeP-175, Aep-126, AeP-67, AeP-132A, AeP-170, AeP-242 and AeP-162 genes. Twenty micrograms of total RNA is loaded in each lane. Hour post certain stage is indicated as ×h. F ⫽ females; M ⫽ males. Ethidium bromide stain of the rRNA in the agarose gel is show under the Northern blot image.

Fig. 4. Tissue distribution of transcripts of AaSP71, KCK, AeP-162 and AeP-242 genes. Fifteen micrograms of total RNA is loaded in each lane. Hour post certain stage is indicated as ×h. FP ⫽ female pupae; MP ⫽ male pupae; F ⫽ females; M ⫽ males; Mpt ⫽ Malpighian tubules; Body wall ⫽ peripheral nerves, tracheae, epidermis, muscle and fat body. Ethidium bromide stain of the rRNA in the agarose gel is show under the Northern blot image.

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expressed at high levels in the 4th instars and the expression decreases in pupae in Ae. aegypti (Korochkina et al., 1997; Gordadze et al., 1999), hexamerin cDNAs represented 11% of sequenced 4th instar cDNAs (12/103); however, none was found in sequenced pupal cDNAs (Table 1). Digestive enzymes and esterases are represented in high numbers in the 4th instar subtracted cDNA library. On the other hand, cDNAs of actin and myosin heavy chain are abundant in the pupal subtracted cDNA library (Table 1). These results indicate that the subtracted cDNA libraries enriched stagespecific transcripts. However, results from cDNA arrays clearly indicated that subtracted cDNA library alone was not sufficient enough for isolating stage-specific genes. Many of the clones (50–61%) represent genes expressed in both stages (Table 2, column ‘no change’). The high percentages of background (i.e. genes that were not differentially expressed) in the subtracted cDNA libraries might have resulted from the quantity of the driver cDNA vs tester cDNA. If the ratio of driver cDNA to tester cDNA is low, the efficiency of subtraction will be lower. We used a 2:1 ratio of driver vs tester to obtain a higher number of moderately differentially expressed genes (Konietzko and Kuhl, 1998), which was much lower than the commonly used ratio of 10:1 to 100:1. We did identify 8–21% of genes as moderately differentially expressed (Table 2, column ‘⬍5- but ⱖ2-fold higher level’). The drawback of low driver vs tester cDNA ratio is the higher background (i.e. false positive clones). The efficiency for identifying stage-specific genes was significantly improved by screening the cDNA clones using DNA arrays. Three cDNAs from larval and nine from pupal subtracted cDNA libraries were selected based on results of cDNA array. One of them was false positive based on Northern blotting analysis (data not shown). Therefore, the overall efficiency for identifying differentially expressed genes using our combined methods was greater than 90%. The identity of 40–49% of the cDNAs is unknown (Table 1). Many of those unknown genes have counterpart hypothetical gene products in the genomes of other organisms. High numbers of unknown genes in our cDNA libraries may be a reflection of the lack of knowledge on the function of many genes. It should also be pointed out that the high numbers of unknown genes from both libraries might have resulted partially from fragmented cDNAs by restriction digest prior to PCR amplification. Because partial cDNAs of a gene may not present the conserved functional domains characterizing the protein; therefore, when a fragment is separated from the complete cDNA, the identity of the partial cDNA becomes unknown if it does not reside in the conserved domain(s). This shortfall can be overcome by rapid amplification of cDNA ends (RACEs). If two non-overlapping partial cDNAs are from the same transcript,

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RACEs will reveal that they reside within a single mRNA species. It is noticed that some genes are homologs of known genes with different expression patterns from their counterpart genes. For example, AeP-390 has a 79% similarity to Ae. aegypti later trypsin gene (Table 1) that is only expressed in the female adults (Barillas-Mury and Wells, 1993). Clearly, AeP-390 is not the same gene as the late trypsin gene; but they may share enough similar functional domains so that they can be grouped within the same family of genes. Another example is the AeP126 gene that has 85% similarity to the C-terminal part of Drosophila SP71 gene (Table 1). Because Drosophila SP71 expression occurs in embryo and differentiating eyes (Chung et al., 2001), it is thought that SP71 gene product is involved in eye formation. The punctured expression profiles of AeP-126 during the larval stages and broad tissue distribution (Figs. 3 and 4) imply that AeP-126 is involved in other processes besides a possible role in the differentiation of compound eyes in mosquitoes. Most of the tested genes were differentially expressed with regard to the perspective stages of their cDNA libraries, only some of them were exclusive to larval or pupal stages such as Ae4-4, Ae4-427, AeP-162 and AeP242 (Figs. 2A and 3). This might have occurred because only a very short developmental time period in the early part of the 4th stadium was chosen as driver cDNAs for the construction of pupal subtracted cDNA libraries, which did not exclude genes expressed during the late period of the larval stage. In fact, genes from the pupal library that did show expression in both larval and pupal stages seemed to be transcribed in the late period of larval development such as AeP-126, AeP-67, AeP-170 and AeP-132A (Fig. 3). Several genes from the pupal subtracted cDNA library also showed continued expression into adult stages such as AeP-175, Aep-132A and AeP162 (Fig. 3). Because common transcripts between early pupae and adults were not subtracted from the pupal library, pupal onset adult genes were not excluded. Interestingly, three out of four of the tested genes from pupal subtracted cDNA library were not expressed in pupal gut/Malpighian tubules (Fig. 4). Pupal gut/Malpighian tubules are nonfunctional because pupae do not ingest food (Christophers, 1960), therefore, it is possible that many genes are not active in pupal gut/Malpighian tubules. However, genes such as AeP67 and AeP-126 were not active in gut/Malpighian tubules in either larval or pupal stages (Fig. 4), which implies that AeP-67 and AeP-126 were not globally activated and at least were not expressed in certain tissues. Expression of three of the unknown genes, AeP-242, AeP-132A and AeP-162, were detected in the first day male but not in the first day female adults (Fig. 4). Further study on the expression profiles in adults is war-

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ranted before any conclusion can be reached on whether those genes are adult male specific. Based on our screening results, it is possible to efficiently isolate stage-specific genes. High-throughput DAN microarray requires isolation of thousands of cloned cDNAs (i.e. ESTs) which is labor intensive and expensive. Use of a single technique such as differential display or subtracted cDNA can be adequate but not efficient. The drawback of differential display is the comigrating DNAs that can generate a high number of false positive clones. To eliminate the false positives in differential display, a secondary screen is usually required (Nagel et al., 2001; Ali et al., 2001). The subtracted cDNA library enriches differentially expressed genes in the cDNA library depending on how the subtraction is conducted. If the ratio of driver vs tester cDNA is high, most of the moderately differentially expressed genes will be lost in the subtraction process (Konietzko and Kuhl, 1998). On the other hand, if the ratio of driver vs tester cDNA is low, the number of false positive will increase as our study shows that 50–61% genes in the libraries are commonly expressed (Table 2, column of ‘no change’). The advantage of our subtracted cDNA libraries is that they contained genes of moderate changes in expression levels (Table 2, column ‘5- but ⱖ2-fold higher level’). To overcome the shortfall of our cDNA subtraction, we used cDNA macroarray to screen the subtracted cDNA libraries. DNA macroarray is a relatively low efficiency method for screening differentially expressed genes, because only hundreds of clones are screened at a time. By combining subtracted cDNA library and DNA macroarray techniques, we were able to reduce cost/labor and increase efficiency in identifying differentially expressed genes. We postulate that DNA macroarray can also be combined with differential display to increase the possibility of identifying differentially expression genes.

Acknowledgements We would like to thank Drs Ann M. Fallon and Walter G. Goodman for critical comments on the manuscript. This work was supported by the University of Wisconsin-Madison College of Agriculture and Life Sciences’ USDA-CSREES Hatch project 04487 to Q.L.

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