Overcrowding of false codling moth, Thaumatotibia leucotreta (Meyrick) leads to the isolation of five new Cryptophlebia leucotreta granulovirus (CrleGV-SA) isolates

Journal of Invertebrate Pathology 112 (2013) 219–228

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Overcrowding of false codling moth, Thaumatotibia leucotreta (Meyrick) leads to the isolation of five new Cryptophlebia leucotreta granulovirus (CrleGV-SA) isolates John K. Opoku-Debrah a,⇑, Martin P. Hill a, Caroline Knox b, Sean D. Moore a,c a

Department of Zoology and Entomology, PO Box 94, Rhodes University, Grahamstown 6140, South Africa Department of Biochemistry, Microbiology and Biotechnology, PO Box 94, Rhodes University, Grahamstown 6140, South Africa c Citrus Research International, PO Box 20285, Humewood 6031, Port Elizabeth, South Africa b

a r t i c l e

i n f o

Article history: Received 12 October 2012 Accepted 17 December 2012 Available online 28 December 2012 Keywords: Baculovirus Latent infection Cryptophlebia leucotreta granulovirus Granulin gene Egt gene

a b s t r a c t False codling moth, Thaumatotibia leucotreta (Meyrick) is a serious pest of economic importance to the South African fruit industry. As part of sustainable efforts to control this pest, biological control options that involve the application of baculovirus-based biopesticides such as Cryptogran and Cryptex (both formulated with a South African isolate of Cryptophlebia leucotreta granulovirus, CrleGV-SA) are popularly used by farmers. In order to safeguard the integrity of these biopesticides as well as protect against any future development of resistance in the host, we conducted a study to bioprospect for additional CrleGV isolates as alternatives to existing ones. Using overcrowding as an induction method for latent infection, we recovered five new CrleGV isolates (CrleGV-SA Ado, CrleGV-SA Mbl, CrleGV-SA Cit, CrleGV-SA MixC and CrleGV-SA Nels). Single restriction endonuclease (REN) analysis of viral genomic DNA extracted from purified occlusion bodies showed that isolates differed in their DNA profiles. Partial sequencing of granulin and egt genes from the different isolates and multiple alignments of nucleotide sequences revealed the presence of single nucleotide polymorphisms (SNPs), some of which resulted in amino acid substitutions in the protein sequence. Based on these findings as well as comparisons with other documented CrleGV isolates, we propose two phylogenetic groups for CrleGV-SA isolates recovered in this study. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Baculoviruses are arthropod-specific DNA viruses that have been successfully developed as biopesticides to control lepidopteran insects (Federici, 1997; Moscardi, 1999; Szewczyk et al., 2006). In South Africa one important lepidopteran pest, false codling moth, Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) can cause notable losses to the South African citrus industry if not adequately controlled (Moore et al., 2004). Since this pest does not occur in most countries to which citrus is exported from South Africa, it may cause trade disruption due to phytosanitary concerns (Moore, 2002; Hattingh, 2006). As part of management strategies to control T. leucotreta, two baculovirus biopesticides CryptogranÒ (River Bioscience, South Africa; Moore et al., 2004, 2011) and CryptexÒ (Andermatt-Biocontrol AG Switzerland; Kessler and Zingg, 2008), both formulated with Cryptophlebia leucotreta granulovirus (strain CrleGV-SA) as their active ingredient, have been registered in South Africa. The first reported case of insects developing resistance to a virus in the field was observed in the codling moth, Cydia pomonella (L.), ⇑ Corresponding author. Fax: +27 46 622 8959. E-mail address: [email protected] (J.K. Opoku-Debrah). 0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.12.008

where field populations in Europe developed resistance to a Mexican isolate of the Cydia pomonella baculovirus biopesticide, Cydia pomonella granulovirus (CpGV-M), after repeated field applications in organic orchards had failed (Fritsch et al., 2005; Sauphanor et al., 2006; Eberle and Jehle, 2006; Berling et al., 2009). Prior to this, insect resistance to baculoviruses was observed under laboratory selection pressure (e.g. Phthorimaea operculella (Zeller) to PhopGV (Briese and Mende, 1981; Briese, 1982; Fuxa, 1993) and Anticarsia gemmatalis (Hubner) to AgMNPV (Abot et al., 1996)). As part of a resistance management strategy to solve this problem, studies conducted by some researchers showed that by challenging virus resistant insects with different virus isolates from the same virus species, one could manage resistance to a large degree (Eberle et al., 2008; Jehle et al., 2008; Berling et al., 2009). These trials led to commercial replacement of products containing CpGV-M with those containing genetically different CpGV isolates, namely MADEX PlusÒ (CpGV genotype mixture) and MADEXÒ I12 (an Iranian CpGV isolate) (Andermatt-Biocontrol AG; Kienzle et al., 2007; Besse et al., 2011; Zingg et al., 2011). In order to be prepared should a similar situation occur in the case of T. leucotreta in South Africa, this study aimed at bioprospecting for new CrleGV isolates as possible alternatives to the existing ones used to formulate Cryptogran and Cryptex.

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In the field, conventional approaches for recovering baculovirus isolates involve scouting for diseased insects showing baculovirus symptoms during epizootics (Cory et al., 1997; Rezapanah et al., 2008). As T. leucotreta is a cryptic pest, such a method would not be feasible. Other feasible methods involve subjecting laboratory reared insects to various stressors such as overcrowding, inoculation with heterologous viruses, changes in diet, varying insect rearing temperature and a host of other methods with the aim of recovering virus from a latent infection within their larval populations (Smith, 1967; Hughes et al., 1993; Kukan, 1999; Il’inykh and Ul’yanova, 2005). In this study, we report the use of overcrowding as a virus induction method for geographically distinct T. leucotreta populations reared under laboratory conditions. The results presented here provide a platform for further research and possibly the development of new biopesticides for the control of T. leucotreta in South Africa, particularly if resistance to existing CrleGV-SA products should occur. 2. Materials and methods 2.1. Host rearing Five colonies of insects were established and maintained in an insectary facility at Rhodes University. The first colony (MixC) consisted of a heterogeneous population reared by Citrus Research International (CRI), Port Elizabeth, South Africa. This colony was initially established from field-collected T. leucotreta larvae from three regions namely, Citrusdal (32°360 S, 19°010 E, Western Cape Province), Zebediela (24°160 S, 29°170 E, Limpopo Province) and the Eastern Cape (Moore, 2002; Sean Moore, pers. comm.) (Fig. 1). This colony has been maintained continuously for over 166 generations

on artificial diet. The other four laboratory colonies (Ado, Mbl, Cit and Nels), were initially established by Opoku-Debrah (2008) from field-collected larvae from Addo (33°340 S, 25°410 E, Eastern Cape), Marble Hall (24°580 S, 24°180 E, Limpopo), Citrusdal (32°360 S, 19°010 E, Western Cape), and Nelspruit (25°280 S, 30°580 E, Mpumalanga) (Fig. 1). These colonies were in their 32nd, 31st, 30th and 30th generations respectively prior to re-establishment at Rhodes University. All colonies were maintained in isolation without incidence of interbreeding or viral outbreak. The colonies were reared at 25.0 ± 2.0 °C, 50–60% relative humidity (RH) and 12-h photoperiod (L12:D12) on Moore’s artificial T. leucotreta diet (Moore, 2002), in preserve jars (380 ml, Patteson’s Glass Ltd.). 2.2. Induction of latent viruses by overcrowding The MixC, Ado, Mbl, Cit and Nels colonies were used as a stock for the execution of latent induction trials. For the control jars, approximately 400–500 eggs were selected from each colony and incubated on 40 g of Moore’s artificial T. leucotreta diet (Moore, 2002). For the treatment jars, the number of eggs was increased fourfold and larvae reared with 40 g of Moore’s diet. Eggs used in both treatment and control jars were randomly selected from each batch of laying females, per colony and reared under similar conditions as described above. All experiments were replicated three times. 2.3. Symptomology Larvae in each jar were observed periodically for symptoms of viral infection until adulthood. Symptoms of CrleGV infection involved the appearance of a milky glazed colouration in both younger and matured larvae, with matured larvae tending to maintain

Fig. 1. Map showing citrus growing areas in South Africa where T. leucotrea infested fruit were collected for establishment on artificial diet. Sample sites are indicated by shaded dots.

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their brownish-orange colouration during infection (Moore, 2002). Larvae suspected of showing these symptoms were observed under a dissecting microscope (Leica, EZ4D) for confirmation and later placed into Schott bottles (DURANÒ) and stored at 25 °C. These larvae were then subjected to occlusion body (OB) purification. 2.4. Isolation of baculovirus OBs, OB enumeration and transmission electron microscopy (TEM) Baculovirus OBs from CrleGV infected larvae as well as Cryptogran and Cryptex, were recovered according to the methods described by Hunter-Fujita et al. (1998) and Moore (2002) with minor modifications. Approximately 2 g of larval cadavers (30–40 larvae) were macerated in 6 ml of 0.1% sodium dodecyl sulphate (SDS) and filtered through cheese cloth. An equal volume of 0.1% SDS was added and the process repeated. The resulting filtrate was placed in two JA-20 (BeckmanÒ) centrifuge tubes, in 3 ml aliquots. For OB purification from Cryptex and Cryptogran, 6 ml of formulated product was dispensed into two JA-20 tubes. JA-20 tubes filled to the brim with 0.1% SDS were centrifuged at 7840 g for 30 min at 4 °C in a BeckmanÒ Coulter centrifuge (J-E Avanti). The supernatant was discarded and the pellet re-suspended in 3 ml sterile distilled water (ddH20). A 30–80% (v/v) glycerol gradient was prepared in two SW 28 BeckmanÒ Ultra-clear centrifuge tubes using a Gradient-maker (Amersham Biosciences Inc.). With the aid of a magnetic stirrer, the two solutions were mixed to form a continuous gradient. The tubes were sealed with Parafilm and incubated overnight at 4 °C. The pellets were loaded on top of the glycerol gradients and centrifuged for 15 min at 29,774 g at 4 °C in a BeckmanÒ Coulter Optima (L-90 K) ultracentrifuge. The OBs were visualised as thick milky white or brown concentric bands appearing in the middle of the tube. The OBs were extracted using a pipette and placed into two sterile JA-20 tubes filled to the brim with ddH20 and centrifuged for 30 min at 29,774 g at 4 °C. This process was repeated 3 times in order to remove all traces of glycerol. The final pellets were re-suspended in 1 ml of ddH20, vortexed briefly, and stored at 20 °C. OBs were viewed and counted using the light microscopy method described by Hunter-Fujita et al. (1998) and Jones (2000). For viewing, approximately 5 ll of OBs were pipetted onto a Helber counting chamber (0.02 mm depth, HawksleyÒ). Prior to counting on the Helber, OBs were diluted in ddH20 and sonicated for 50 s to disperse them. The chamber was covered with a glass slip, the slide was allowed to stand for 5 min, and OBs visualised at 400 magnification under dark field microscopy. OBs obtained were visualised by transmission electron microscopy (TEM) (JEOL JEM-1210) using the method described by Dezianian et al. (2010). Approximately 5 ll of purified OBs were pipetted onto a carbon grid, incubated for 20 s and dried using filter paper. Images were captured using ScandiumÒ image analysis software and processed using MicrosoftÒ Office Picture Manager. 2.5. Genomic DNA extraction Genomic DNA was extracted from purified OBs according to a modified version of the CTAB DNA extraction protocol described by Aspinall et al. (2002). Approximately 200 ll (2.0  1011 OBs/ ml) of OBs was placed in sterile microcentrifuge tubes and the suspension clarified by addition of 90 ll of 1 M Na2CO3. The contents were incubated at 37 °C for 30 min and the suspension neutralized with 120 ll of 1 M Tris–HCl (pH 6.8) before addition of 90 ll of SDS (10% w/v) and 50 ll of Proteinase K (25 mg/ml), followed by incubation for 30 min at 37 °C. 10 ll of RNaseA (10 mg/ml) was added and samples incubated for a further 30 min at 37 °C. Samples were centrifuged at 14,000 rpm for 3 min in a tabletop laboratory centrifuge (BIO-RAD, model 16 K). The supernatant was transferred into

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a new tube and the pellets discarded before addition of 400 ll of pre-warmed (at 70 °C) CTAB buffer (2% w/v, CTAB, 100 mM Tris, pH 8.0, 20 mM Na2EDTA, 1.4 M NaCl). The samples were incubated at 70 °C for 1 h with mixing every 10 min. Samples were treated with 400 ll of cold chloroform mixed and centrifuged for 10 min at 10,000 rpm. The upper aqueous phase was transferred into a new tube and 400 ll of ice-cold isopropanol was added. The tubes were incubated overnight at 25 °C and then centrifuged at 14,000 rpm for 20 min. The pellets were re-suspended in 1 ml of 70% ice-cold ethanol, centrifuged at 14,000 rpm for 5 min and the ethanol gently poured off without discarding the DNA pellet. After drying to remove all traces of ethanol, pellets were re-suspended overnight at 4 °C in 20 ll of 10 mM Tris–HCl (pH 8.0). The DNA was stored for a few days at 4 °C or for longer periods at 25 °C before use. 2.6. REN analysis of genomic DNA Single restriction endonuclease (REN) digest reactions were performed in a total volume of 30 ll, containing 20 ll (10–12 lg) genomic DNA, 3 ll 1 restriction enzyme (RE) buffer and 3 ll (30 U) of RE with ddH20 added to make the final volume. RE BamH1, Sal1, Xba1, Pst1, Xho1, Kpn1, HindIII and EcoR1 (FermentasÒ) were used in digests. The contents of the reaction were briefly centrifuged and the tubes incubated at 37 °C for 4 h. Digests were analysed by 0.6% agarose gel electrophoresis (AGE) with ethidium bromide staining in 1x TAE buffer (40 mM Tris–acetate, 20 mM acetic acid, 1 mM EDTA) at 30 V for 16 h. Four DNA markers namely k mix 19, GeneRuler High Range, DNA marker II and 1 Kb DNA marker (FermentasÒ) were used to estimate the size of the DNA fragments. The gels were photographed under a UV trans-illuminator (UvitecÒ) and gel images captured using UVI Prochemi software. 2.7. PCR amplification of granulin and egt genes Granulin and egt gene sequences of all isolates were amplified by PCR using virus-specific oligonucleotides described by Lange and Jehle (2003). Reactions were carried out in a total volume of 50 ll (36 ll ddH20, 5 ll of 10X Taq buffer, 2 lM forward and reverse primers, 0.2 mM dNTPs, 2.5 ll DNA template and 1.25 U Taq DNA polymerase (FermentasÒ). The PCR protocol was 95 °C for 5 min, followed by 30 cycles of 95 °C for 1 min, 50 °C for 1 min 30 s, 72 °C for 1 min 30 s and a final extension at 72 °C for 7 min. Amplified products were analysed by 0.6 % AGE and sequenced by Inqaba Biotechnical Industries (Pty) Ltd. (South Africa). 2.8. Multiple sequence alignment and phylogenetic analysis of the granulin and egt gene sequences Phylogenetic comparisons between isolates was conducted using the nucleotide sequences obtained from the granulin and egt genes of the seven South African (SA) isolates (five new CrleGV and Cryptogran and Cryptex isolates). The sequences were assembled and edited using DNA DragonÒ (version 1.3.0) and translated and aligned using Molecular Evolutionary Genetics Analysis (MEGA) software, version 5.0 (Tamura et al., 2007, 2011). The granulin gene sequences of the seven SA isolates were compared against another SA isolate, CrleGV-SA (GenBank ID: AY293731) reported by Singh et al. (2003). The egt gene sequences of the seven SA isolates were also compared against another isolate outside this region, the Cape Verde isolate, CrleGV-CV3 (GenBank ID: AY229987) (Lange and Jehle, 2003). The complete sequence of this isolate has been determined by Lange and Jehle (2003) and served as a reference isolate in this study. The minimum evolution (ME)

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Table 1 Virus induction in five colonies subjected to overcrowding versus untreated control larvae. T. leucotreta colony

Addo Mixed Nelspruit Marble Hall Citrusdal

Treatment: overcrowding

Control

Total number of jars

Jars with symptomatic larvae

% of jars with infected larvae

Total number of jars

Jars with symptomatic larvae

28 30 27 27 27

6 8 7 4 5

21.43 26.67 25.93 14.81 18.52

12 12 12 12 12

0 0 0 0 0

Fig. 2. BamH1 restriction endonuclease digest profiles of Cryptex (lane 3), CrleGV-SA Ado (lane 5), CrleGV-SA MixC (lane 7), CrleGV-SA Cit (lane 9), CrleGV-SA Mbl (lane 11), CrleGV-SA Nels (lane 13) and Cryptogran (lane 15) samples analysed by 0.6% AGE at 30 V for 16 h. DNA markers: 1 Kb DNA marker (lane 1), DNA marker II (lane 17) and GeneRuler High range (lane 18) were run along the outside lanes of gels. Asterisks () in yellow indicate submolar bands. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

method was used in constructing molecular phylogenetic trees (Rzhetsky and Nei, 1992).

3. Results 3.1. Induction of latent viruses No disease symptoms were observed in larvae in the control jars during the course of the experiment. Larvae subjected to overcrowding in treatment jars showed disease symptoms within 10– 14 days post-treatment. Asymptomatic larvae maintained their cream (early larval instars) or brownish-orange colouration (late larval instars) whereas symptomatic larvae became enlarged or distended with a shiny and milky appearance. During the late stages of infection, infected larvae were observed to climb up the inside walls of their rearing containers. The number of jars used in treatment and control experiments is shown in Table 1. For

the Nels, Mbl and Cit colonies, out of 27 jars subjected to overcrowding only 7, 4 and 5 jars were found with diseased larvae respectively. Out of 28 and 30 jars from the Ado and MixC colonies subjected to overcrowding only 6 and 8 jars were recovered with diseased larvae. Overall, the percentage of jars with diseased larvae was less than 27%. It was also observed that within the jars containing diseased larvae it was not uncommon to find some larvae showing disease symptoms whilst others were asymptomatic. Although it was not possible to count the total number of larvae, approximately 25% in each jar were asymptomatic. The asymptomatic larvae were able to pupate and successfully emerge as adults.

3.2. OB purification and TEM To confirm the presence of a GV infection in symptomatic larvae, OBs were purified from diseased insects and prepared for TEM. The particles ranged from 392.53 to 414.83 nm in length

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Fig. 3. SalI restriction endonuclease digest profiles of CrleGV-SA Ado (lane 4), CrleGV-SA MixC (lane 6), CrleGV-SA Nels (lane 8), CrleGV-SA Cit (lane 10), CrleGV-SA Mbl (lane 12), Cryptogran (lane 14) and Cryptex (lane 16) samples analysed by 0.6% AGE at 30 V for 16 h. DNA markers: GeneRuler High range (lane 1 and 19), 1 Kb DNA marker (lane 2), DNA marker II (lane 18) and were run along the outside lanes of gels. Asterisks () in yellow indicate submolar bands. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

and were similar in size and morphology to those extracted from the biopesticides, Cryptogran and Cryptex. 3.3. REN analysis of genomic DNA To identify genotypic variation between CrleGV-SA isolates, genomic DNA extracted from purified OBs was subjected to REN analysis using BamH1, Sal1, Xba1, Pst1, Kpn1, HindIII, EcoR1 and Xho1. The DNA profiles obtained for BamH1, Sal1, Xba1 and HindIII are shown in Figs. 2–5 respectively. REN profiles for Pst1, Kpn1, EcoR1 and Xho1 digests are not shown. Results from this analysis showed the presence of two unique banding patterns. Cryptex, CrleGV-SA Ado, CrleGV-SA Mbl, CrleGV-SA Cit and CrleGV-SA MixC isolates shared similar DNA profiles as did Cryptogran and CrleGV-SA Nels. Based on this observation isolates were placed into CrleGV-SA genome Groups One (Cryptex, CrleGV-SA Ado, CrleGV-SA Mbl, CrleGV-SA Cit and CrleGV-SA MixC) and Two (Cryptogran and CrleGV-SA Nels). Several submolar bands were also observed in the DNA profiles of all isolates and have been indicated by asterisks for BamH1 (Fig. 2), Sal1 (Fig. 3), Xba1 (Fig. 4) and HindIII (Fig. 5). The submolar bands observed in Group One CrleGV-SA have been denoted with yellow asterisks and that of Group Two CrleGV-SA with red aster-

isks. For seven out of the eight enzymes used, notable similarities and differences could be observed in the number of submolar bands generated for each isolate. However, by comparing and contrasting their profiles each isolate appeared to have a unique submolar banding pattern. Genotypic variation was therefore evident between samples with each isolate consisting of a mixture of more than one CrleGV-SA genotype. 3.4. Comparative analysis of egt amino acid sequences Sequence analysis of the granulin and egt genes of Group One and Two CrleGV-SA isolates revealed several SNPs in their nucleotide sequences (data not shown). Multiple alignments revealed the presence of amino acid substitutions at four positions within the egt gene sequences (Table 2). There were no amino acid substitutions in the granulin gene. When the egt amino acid sequences of Group One isolates were compared with that of CrleGV-CV3, seven amino acid substitutions were observed (Table 2). Group One CrleGV-SA showed a 98% amino acid identity to CrleGV-CV3 from a BLASTx query of the egt sequence. Six amino acid substitutions were recorded in the egt sequence of Group Two CrleGV-SA when compared to that of CrleGV-CV3 (Table 2). A BLASTx query showed a 99% amino acid identity with CrleGV-CV3.

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Fig. 4. XbaI restriction endonuclease digest profiles of the CrleGV-SA MixC (lane 3), CrleGV-SA Mbl (lane 5), CrleGV-SA Nels (lane 7), CrleGV-SA Cit (lane 9), and CrleGV-SA Ado (lane 11) isolates with Cryptogran (lane 13) and Cryptex (lane 15) analysed by 0.6% AGE at 30 V for 16 h. DNA markers: 1 Kb DNA marker (lane 1) and DNA marker II (lane 17) and were run along the outside lanes of gels. Asterisks () in yellow indicate submolar bands. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.5. Phylogenetic analysis of the granulin and egt gene sequences of Group One and Two CrleGV-SA and other CrleGV isolates Phylogenetic analysis of sequence data obtained from the granulin genes of the seven SA isolates reported in this study, as well as the CrleGV-SA isolate reported by Singh et al. (2003), CrleGV-SA (AY293731), revealed the presence of two separate groups based on their evolutionary relationship. Group Two CrleGV-SA was shown to be more closely related to CrleGV-SA (AY293731) than Group One (Fig. 6). A similar analysis of egt gene sequences also placed both the Group One and Two CrleGV-SA isolates into two groups based on their evolutionary relationships. The Group One and Two CrleGV-SA isolates appeared to have originated from a common ancestor whilst the Cape Verde isolate, CrleGV-CV3 was shown to be phylogenetically distinct (Fig. 6). These results support the existence of two distinct CrleGV-SA groups based on both their REN profiles and granulin and egt gene sequences.

4. Discussion In the field, baculovirus epizootics occur during insect population explosions and have been observed with most forest lepidopteran insects (Adams and McClintock, 1991; Cooper et al., 2003; Il’inykh et al., 2004). Notable examples include the western tent caterpillar, Malacosoma californicum pluviale (Dyer) (Hoch et al., 2001; Cory and Myers, 2009) and the gypsy moth, Lymantria dispar (L.) (Elkinton, 1990; Hoch et al., 2001). These epizootics are impor-

tant for insect population suppression (Fuxa, 1993; Cory and Myers, 2003). Epizootics can also be artificially induced in the laboratory, where stressing of laboratory reared insects may lead to a resident covert baculovirus infection being brought into an overt lethal state (Il’inykh and Ul’yanova, 2005). In this study we have shown that by crowding T. leucotreta larvae reared in preserve jars, a latent GV can be brought into an overt lethal state. Increasing the population density per unit area or varying the size of the rearing chamber per unit population of insects has long been suggested as a possible precursor for the activation of latent baculoviruses in some lepidopteran insects and has been shown in larvae of the alfalfa butterfly, Colias eurytheme (Boisduval) (Steinhaus, 1958; Steinhaus and Dineen, 1960), buckeye, Junonia coenia (Hubner) (Steinhaus, 1958) and the cabbage looper, Trichoplusia ni (Hubner) larvae (Fuxa et al., 1999). The crowding effect on the larvae increases contact between infected and susceptible individuals thereby increasing the rate of viral transmission. Once the virus is induced in some larvae it is then transferred to other susceptible larvae via horizontal infection (Cory et al., 1997). This may occur due to susceptible individuals ingesting CrleGV OBs on contaminated T. leucotreta diet or frass, as well as exudates from dead or diseased larvae. In this study despite the presence of symptomatic larvae, some individuals were able to pupate and eclose successfully as adults. These larvae may have been less susceptible to the stress of the induction process or may have developed mechanisms to evade infection. Although this phenomenon was not investigated in this case, some lepidopteran insects have been noted to slough off baculovirus in-

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Fig. 5. (A) HindIII restriction endonuclease digest profiles of Cryptogran (lane 4). (B) HindIII profiles of CrleGV-SA Cit (lane 2) and Cryptex (lane 4). (C) HindIII profile of CrleGVSA Ado (lane 3). (D) HindIII profiles of CrleGV-SA Nels (lane 1), Cryptex (lane 3), CrleGV-SA MixC (lane 5) and CrleGV-SA Mbl (lane 7). Samples analysed by 0.6% AGE at 30 V for 16 h. DNA markers: GeneR DNA marker, 1 Kb DNA marker and DNA marker II were run along the outside lanes of gels. Asterisks () in yellow indicate submolar bands. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 2 Amino acid substitutions between the egt genes of the Groups One and Two CrleGV-SA and CrleGV-CV3 (AY229987) isolates. CrleGV isolate

CrleGV-CV3 Group One CrleGV-SA

Group Two CrleGV-SA

Amino acids Position

(AY229987) isolate CrleGV-SA Ado CrleGV-SA Cit Cryptex isolate CrleGV-SA Mbl CrleGV-SA MixC Cryptogran isolate CrleGV-SA Nels

96

152

196

225

241

310

315

373

V A A A A A A A

M I I I I I M M

N N N N N N H H

R Q Q Q Q Q Q Q

V L L L L L V V

K R R R R R G G

I F F F F F F F

M L L L L L L L

fected midgut cells as a way to evade infection (McNeil et al., 2010). No single induction method can independently result in the recovery of a latent baculovirus from its host, since the process is dependent on a multitude of factors relating to the biological activity of the virus and its multiplicity of infection, biological properties and physiological state of the host and environmental factors which play a significant role in enhancing the infection process when insects are subjected to stress (Il’inykh, 2007). Since no incidence of CrleGV infection in laboratory colonies was observed during rearing, it is assumed that the virus maintained itself in the colonies for several generations via vertical transmission. Similar observations have been reported for an NPV infection in the cabbage moth, Mamestra brassicae (L.) which was able to persist in a laboratory colony due to vertical transmission

of the virus over several generations (Hughes et al., 1993; Burden et al., 2003). These insects infected with a nucleopolyhedrovirus (NPV) or granulovirus (GV) may carry several mixed wild-type isolates consisting of a mixture of different genotypes (Parnell et al., 2002; Cory et al., 2005; Erlandson, 2009; Murillo et al., 2011). At present, CrleGV isolates from three different countries have been identified in association with T. leucotreta. These include the Ivory Coast isolate (CrleGV-IC) (Angelini et al., 1965), the Cape Verde isolate (CrleGV-CV) (Muck, 1985) and the South African isolate (CrleGV-SA) (Fritsch, 1989; Singh et al., 2003). All three isolates were shown to be genetically different (Fritsch, 1989). In this study, we have recovered five CrleGV-SA isolates (CrleGV-SA Ado, CrleGV-SA Mbl, CrleGV-SA Cit, CrleGV-SA MixC and CrleGVSA Nels) from geographically distinct T. leucotreta populations,

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A 95

CrleGV-SA MixC CrleGV-SA Mbl CrleGV-SA Cit Cryptex CrleGV-SA Ado CrleGV-SA (AY293731) Cryptogran CrleGV-SA Nels

0.0005

B

100

Cryptex CrleGV-SA Mbl CrleGV-SA Cit CrleGV-SA Ado CrleGV-SA MixC

Cryptogran 99

CrleGV-SA Nels CrleGV-CV3 (AY229987)

0.002

Fig. 6. (A) Molecular phylogenetic trees (using Minimum evolution, ME) showing relationship between the granulin gene of Group One and Two CrleGV-SA isolates and that of CrleGV-SA isolate (AY293731) reported by Singh et al. (2003) (A). (B) The relationship between the egt gene of Group One and Two CrleGV-SA isolates and that of the CrleGV-CV3 isolate (AY229987) reported by Lange and Jehle (2003). Numbers next to the branches indicate percent of support.

with two major groups being identified. Based on REN analysis of genomic DNA, Cryptex, CrleGV-SA Ado, CrleGV-SA Mbl, CrleGVSA Cit and CrleGV-SA MixC isolates were placed in Group One and Cryptogran and CrleGV-SA Nels in Group Two. Within each genome group, genotypic variation was observed between isolates as shown by the presence of submolar bands. Similar observations of high levels of genotypic variation in betabaculoviruses from the same insect host species, from different geographic locations, have been previously reported for CpGV (Rezapanah et al., 2008) and PhopGV (Espinel-Correal et al., 2010). The different DNA profiles observed for Cryptex and Cryptogran isolates was unexpected and suggests that these biopesticides may be formulated with several genotypic variants. This may have occurred as a result of mass production or multiple passage and replication in vivo in insect host systems, which can lead to increased genetic variation (Possee and Rohrmann, 1997). Quite commonly wild-type isolates appear to be more virulent than pure genotypes (Lopez-Ferber et al., 2003). Therefore the presence of multiple genotypes in these biopesticides may present some added benefits in terms of improved virulence. The identification of two distinct groups of CrleGV-SA isolates recovered in this study is interesting and may be explained by a high level of genetic diversity in host insect populations (Timm, 2005). The relationship between isolates may in part be explained by the origin of the host population from which they were isolated. For instance, initial colony material from which the Cryptogran isolate was isolated, was collected from two regions, namely Zebediela (Limpopo Province) and Citrusdal (Western Cape Province) (Sean Moore, pers. comm.). Zebediela is only about 200 km from Nelspruit (Mpumalanga Province), possibly explaining the similarity between the Cryptogran and CrleGV-SA Nels isolates. The position of the CrleGV-SA Cit isolate in Group One might be considered strange, as Citrusdal is separated from all of the other regions in this group by between 620 km (Addo) and 1300 km (Marble Hall). However, this could be explained by T. leucotreta not being indigenous to the Western Cape Province. The host was detected in the Citrusdal region for the first time in 1974 (Newton, 1998). Its origin is unknown but it could well have been introduced in infested fruit

carried by travellers. The position of CrleGV-SA Cit in Group One might provide some indication of the origin of this starter population of T. leucotreta in Cirusdal. Further comparative analysis between Group One and Two CrleGV-SA isolates and between other documented CrleGV-SA isolates revealed some similarities. For example, BamH1, HindIII and EcoR1 profiles from Group One CrleGV-SA isolates resembled those of the CrleGV-SA isolate reported by Fritsch (1989). On the other hand, Group Two CrleGV-SA isolates were phylogenetically related to that reported by Singh et al. (2003) based on granulin gene sequences. According to Moore et al. (2011) this isolate reported by Singh et al. (2003) was used to formulate Cryptogran. By contrasting CrleGV-SA isolates reported in this study to the CrleGV-CV3 isolate reported by Lange and Jehle (2003) both Group One and Two CrleGV-SA appeared to have originated from a common ancestor whilst CrleGV-CV3 was shown to be phylogenetically distinct. From an evolutionary perspective, the presence of two CrleGVSA genome groups in geographically diverse T. leucotreta populations as shown in this study may indicate that more isolates exist. This genome diversity has been previously reported for NPVs (Takatsuka et al., 2003; Redman et al., 2010) and more recently for GVs in C. pomonella, where four CpGV genome groups were described (CpGV genome type A, B, C and D), with genome type C being the most dominant group recovered so far (Eberle et al., 2009). The high genetic diversity between baculoviruses appears to be a common phenomenon and is an important avenue for future research. The SNPs which resulted in amino acid substitutions in the protein sequencing of the egt genes of Group One and Two CrleGV-SA isolates may have possible implications for viral virulence. In one study, Eberle et al. (2009) reported differences in virulence between CpGV genome groups, with CpGV viruses belonging to CpGV genome type C displaying a lower virulence than both type A and D against C. pomonella. However, these studies were conducted with one given population. It would be interesting, in this case, to investigate the possibility that geographically diverse T. leucotreta populations differ in their susceptibility to different CrleGV-SA genome groups.

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