Aquaculture 219 (2003) 57 70 www.elsevier.

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Prevention of IHHNV vertical transmission in the white shrimp Litopenaeus vannamei

Emmerik Motte a, Edwin Yugcha a, Juan Luzardo a, Fernando Castro b, Gael Leclercq a, Juan Rodrguez a, Paul Miranda a, Oswaldo Borja a, a Javier Serrano , Manuel Terreros a, Karina Montalvo a, Alexandra Narvaez a, Narda Tenorio a, Virna Cedeno a, Eric Mialhe a, Viviane Boulo a,c,*
Concepto Azul, Cdla. Vernaza Norte, Mz.10, V.34, P.O. Box 09-02-142 A, Guayaquil, Ecuador b Emagro, Manta, Ecuador c IFREMER/CNRS/Universite de Montpellier II, UMR 5098, Defense et Resistance chez les Invertebres Marins, CC 80, 2 Pl. E. Bataillon, Montpellier 34095, France Received 23 May 2002; received in revised form 10 November 2002; accepted 17 November 2002
a

Abstract The infectious hypodermal and hematopoietic necrosis virus (IHHNV) is very pathogenic for Litopenaeus stylirostris whereas infection in Litopenaeus vannamei is known to induce development and growth abnormalities and cause economic losses that range between 10% and 50% (Lightner and Redman, 1998). In the present work, on the basis of nested-PCR analysis, IHHNV prevalences were determined to be between 47% and 63% in Ecuadorian wild and domesticated broodstocks and around 95% in Panamanian domesticated broodstock. IHHNV was regularly detected in the ovaries of infected females whereas sperm from infected males was generally free of virus. IHHNV vertical transmission from infected females was clearly established. In the case of highly infected females, embryo development may abort. IHHNV-free nauplii and larvae were produced from females and males that were found to be free of virus on the basis of nested PCR performed in the case of females after eyestalk ablation and the first spawning. The reliability of this testing process was shown to be very high since about 87% of females was confirmed IHHNV-free through a second nested-PCR analysis performed after some additional spawnings. The nauplius productivity was higher for IHHNV-free females than for the infected ones. Virus-free verification of L. vannamei broodstock

* Corresponding author. IFREMER/CNRS/Universite de Montpellier II, UMR 5098, Defense et Resistance chez les Invertebres Marins, CC 80, 2 Pl. E. Bataillon, Montpellier 34095, France. E-mail addresses: vboulo@ifremer.fr, conceptoazul@easy.pacifictel.net (V. Boulo). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(02)00631-2

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may become a general routine hatchery procedure in the near future to prevent the disease from spreading through vertical transmission. D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Litopenaeus vannamei; IHHNV; Nested-PCR; Epidemiology; Wild broodstock; Domesticated broodstock; Prevention; Vertical transmission; Nauplius productivity; Size disparity

1. Introduction The infectious hypodermal and hematopoietic necrosis virus (IHHNV) was first observed in the blue shrimp Litopenaeus stylirostris in Hawaii (Lightner et al., 1983). This virus has been classified in the Parvoviridae family according to its morphological and physico-chemical features (Bonami et al., 1990). On the basis of virus genome characteristics, IHHNV has been recently related to Brevidensovirus (Shike et al., 2000). Whereas IHHNV is very pathogenic for L. stylirostris, infection of Litopenaeus vannamei results in development and growth abnormalities (Kalagayan et al., 1991; Lightner et al., 1992). Size reduction and disparity have a significant impact on L. vannamei production with economic losses that range between 10% and 50% by comparison with IHHNV-free crops (Lightner and Redman, 1998). There is currently no information concerning the effect of virus infection on shrimp immunology and physiology, in particular related to reproduction and embryo development. Transmission of IHHNV has been performed experimentally by injection of viral suspension, by ingestion of infected material and by immersion in contaminated water (Bell and Lightner, 1984; Lotz, 1997). In farms, infection of shrimps may result from horizontal transmission through ingestion of dead individuals. Vertical transmission has been suspected since virus has been detected in ovarian tissues and ovocytes (Lotz, 1997). Vertical transmission may be a crucial factor for the increase of IHHNV prevalence in domesticated shrimps from generation to generation. As a matter of fact, broodstock gives rise to animals that had been infected through vertical transmission, with a subsequent increase of prevalence through horizontal transmission in the ponds. Since the majority of infected shrimps survive without clinical signs, many animals selected for broodstock are effectively healthy carriers of IHHNV that can transmit it vertically. Because of the negative impact of IHHNV on shrimp production of L. vannamei, prevention of this viral disease is a chief priority for shrimp farming in Latin America. Consequently, it was important, first, to demonstrate experimentally the vertical transmission of IHHNV through the egg and, later, to establish if the identification of virus-free females through nested PCR would prevent the vertical transmission of IHHNV. 2. Materials and methods 2.1. Animals Wild adult white shrimps L. vannamei were collected from October 1999 to September 2001 along the Ecuadorian coast (Provinces of Esmeraldas, Manab, Guayas and El Oro).

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These animals were sampled the same day they were caught and the hemolymph samples were subsequently analyzed by nested PCR for determination of IHHNV prevalence without previous eyestalk ablation. Domesticated adult white shrimps L. vannamei were obtained from farms and hatcheries in Ecuador and Panama. The animals were analyzed by nested PCR for determination of IHHNV prevalence, with or without previous eyestalk ablation according to the purpose. When necessary, the animals were identified with a colored and numbered eye tag. Nauplii were produced from domesticated shrimps previously analyzed by nested PCR and dot-blot to determine their status in terms of IHHNV infection. According to the purpose, females were naturally or artificially inseminated. Eggs were always disinfected using a classical iodine treatment. 2.2. DNA extraction, dot-blot and nested PCR for IHHNV diagnosis In the case of eggs and larvae, DNA extraction was performed from body homogenates using lysis buffer (NaOH, 0.05 N; SDS, 0.25%). The same DNA extraction procedure was used for hemolymph, ovaries and sperm extracted from spermatophores. This DNA extraction protocol has been previously validated for all these tissues by performing PCR with primers corresponding to penaeidin (Destoumieux et al., 2000). In the case of juveniles or adult shrimps, DNA extraction was performed using the same protocol from hemolymph samples collected with a 1 ml of tuberculin syringe and 27GX1/ 2 needle using a sodium citrate solution (10%, w/v) as anticoagulant. Detection of IHHNV was performed by dot-blot on nylon membranes positively charged (Roche biochemicals, ref. 1 417 240) using the Dig nucleic acid detection kit (Roche biochemicals, ref. 1175 041) or nested PCR. For dot-blot, the probe (815 nt) was prepared using the PCR Dig probe synthesis kit (Roche biochemicals, ref. 1 636 090) with primers corresponding respectively to nucleotides 1404 1425 and 2198 2219 by reference to the sequence AF273215 (GenBank). These primers were selected by considering sequence conservation of non-structural NS1 proteins inside the group of invertebrate Parvoviridae (Shike et al., 2000). The same primers were used for nested PCR with a second pair of internal primers (Fig. 1). Amplification was performed according to a double tube method with preheating 3 min at 94 jC and 40 cycles of 30 s at 94 jC, 45 s at 56 jC and 45 s at 72 jC. Positive controls consisted of DNA extracted from purified virus suspensions. Amplified fragments were observed after electrophoresis in agarose gel (2%) stained with Ethidium bromide. 2.3. Experimental design for demonstration of vertical transmission In order to demonstrate vertical transmission of IHHNV through egg contamination, five groups of females were established according to their infection level. Females were first analyzed by performing a nested PCR with DNA extracted from hemolymph. The females found IHHNV-negative were considered as Group 0. DNA samples extracted from females identified as IHHNV-positive through nested PCR were analyzed through dot-blot and subsequently classified in four groups (1, 2, 3 and 4) according to the

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Fig. 1. IHHNV nested-PCR with primer corresponding to a consensus region of NS1 protein. Lanes: (1) molecular marker 100 pb ladder (Gibco BRL); (2) amplicon obtained with external primers (815 pb); (3) amplicon obtained with internal primers (470 pb); (4) negative control of PCR.

increasing intensity of the dot-blots. All the females were artificially inseminated with sperm from males identified as IHHNV-negative through a nested-PCR analysis of DNA extracted from hemolymph.

3. Results 3.1. IHHNV prevalence in broodstock 3.1.1. Wild adult populations A large epidemiological survey was performed from October 1999 to June 2000 along the Ecuadorian coast (Table 1) with a total of 3176 wild adult shrimps analyzed for IHHNV detection based on a nested-PCR assay. All these animals were analyzed without previous eyestalk ablation. IHHNV prevalence was more than 50% in the Provinces of Esmeraldas, Guayas and El Oro and only 20% in the Province of Manabi. The average of

Table 1 IHHNV prevalence in wild adult shrimps along Ecuadorian coastal provinces as determined through nested-PCR analysis of a hemolymph sample Ecuadorian coastal provinces (from North to South) Esmeraldas Manab Guayas El Oro Total Number of analyzed shrimps 101 200 2830 45 3176 Number of IHHNV-positive shrimps 63 41 1726 23 1853 IHHNV prevalence (%) 62 20 61 51 58

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IHHNV prevalence in Ecuadorian populations of L. vannamei was at this time 58% (Table 1). In October 2001, a small epidemiological survey of 40 wild adult shrimps was performed at San Pablo (Province of Guayas) without previous eyestalk ablation. IHHNV prevalence was estimated at 22%. 3.1.2. Domesticated adult populations A preliminary study performed in an Ecuadorian hatchery consisted in analyzing L. vannamei domesticated females, either before spawning or after spawning. As a matter of fact, it has been demonstrated that spawning induces WSSV replication in Penaeus monodon (Peng et al., 2001). In the present study, all the females were previously eyestalk ablated. On the basis of nested-PCR analyses, IHHNV prevalence increased from 19% (109 females analyzed) before the first spawning to 31% (1608 females analyzed) after the first spawning. On the basis of these results, it may be assumed that spawning induces virus replication, which leads to an increase in the proportion of IHHNV-positive individuals. Consequently, IHHNV prevalence in domesticated adult female shrimps was estimated after the first spawning. In the case of domesticated males, the nested-PCR analysis for estimation of IHHNV prevalence was generally made a few weeks after the transfer to the maturation unit. Analyses of males were then considered reliable since IHHNV prevalence was similar for males and females. Domesticated adult shrimps of Colombian and Panamanian origin which were grown for the two last generations in Ecuador were analyzed to determine IHHNV prevalence. In the case of shrimps from Colombian origin, IHHNV prevalence was around 50%, without clear difference either between females and males or between the time of analysis. In the case of shrimps from Panamanian origin, IHHNV prevalence was around 50% for males and 63% for females. These results suggest that around 13% of males may be IHHNV false-negatives (Table 2). Domesticated adult shrimps from Panamanian origin, grown for the last two generations in Panama, were also analyzed. IHHNV prevalence was estimated to be 94% for females (441 analyzed animals) and 95% for males (722 analyzed animals). The similarity of IHHNV prevalence in females and males may be related to a longer time between the analysis and the transfer of animals to the maturation unit, which ranged from 4 to 6 weeks.

Table 2 Prevalence of IHHNV in domesticated adult shrimps of Colombian and Panamanian origin and grown for two generations in Ecuador Genetic origin and time of analysis Colombia (November December 2000) Colombia (May 2001) Panama (November December 2000) Sex Female Male Female Female Male Number of analyzed shrimps 437 277 1101 148 162 IHHNV prevalence (%) 52 55 47 63 50

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3.2. IHHNV detection in spermatophore-collected sperm and ovaries of infected adult shrimps Nested-PCR analysis was performed with DNA extracted from ovaries and from spermatophore-collected sperm corresponding to IHHNV-infected adult shrimps. It was shown that ovaries of IHHNV-infected females were generally infected (17/20) whereas the sperm of IHHNV-infected males was rarely positive (2/15). The quality of extracted DNA was verified by performing PCR with primers specific for penaeidin. These results are in agreement with vertical transmission of IHHNV through the eggs of infected females, and at a lesser extent through the sperm of infected males. 3.3. IHHNV detection in eggs and larvae from females with different infection levels In order to demonstrate the vertical transmission of IHHNV through infected eggs, females with different infection levels were selected and their progeny was individually cultivated and analyzed at several developmental stages. All males used for the experiment were confirmed to be IHHNV-negative on the basis of a nested-PCR assay. The females were first analyzed by nested-PCR that led to identify six classified as IHHNV-negative. These six females constituted the Group 0. The IHHNV-positive females were then analyzed by a dot-blot assay that led to classify them in four groups according to the infection levels. Five females with very low infection level were classified in the Group 1. Four females considered as lowly infected and four others with intermediary infection constituted the Groups 2 and 3, respectively. The six highly infected females corresponding to the strongest dot-blots were classified in the Group 4. The eggs, before and after disinfection, and the subsequent progeny from these females were analyzed by nested-PCR to detect IHHNV (Table 3, Figs. 2 and 3).

Table 3 Nested-PCR analyses of egg and larvae produced by females with different levels of IHHNV infection Group 0 Nested-PCR: Dot-blot: Stages # Females 1 2 3 Eggs before disinfection Eggs after disinfection Nauplius V Zoea III Mysis III PL5 PL15 PL25 Group 1 + + # Females 4 5 6 1 2 3 4 5 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Group 2 + ++ # Females 1 2 3 4 + + + + + + Group 3 + ++ + # Females 1 2 3 4 + + + + + + Group 4 + +++ + # Females 1 2 3 4 5 6 + * * * * * +

Symbols in the table: (+): tested positive; ( ): tested negative; (*): abnormalities; no symbol: not tested.

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Fig. 2. Nested-PCR analyses of eggs produced by IHHNV-infected female considered as level 1 by dot-blot assay. Lanes: (1) and (14) molecular marker 100 pb (Gibco BRL); (2 6) eggs before disinfection; (7 and 8) negative control of extraction and negative control of PCR, respectively; (9 13) eggs after disinfection. Size of specific amplicon is 470 pb.

Concerning the non-infected females of Group 0, IHHNV was not detected in any analyzed samples. The females of Group 1 transmitted IHHNV to their progeny in three of five cases, the virus being detectable as early as the egg stage (Fig. 2). Similar results were observed with females of Groups 2 and 3. However, in each case, IHHNV was detected in eggs from two females only before disinfection, which suggests that some virions are adsorbed on the egg surface. The highly infected females of Group 4 did not spawn in two

Fig. 3. Nested-PCR analyses of different larval stages produced by IHHNV-infected female considered as level 1 by dot-blot assay. Lanes: (1) molecular marker 100 pb (Gibco BRL); (2 4) zoea III; (5 and 6) Mysis III; (7 and 8) PL5; (9 and 10) PL15; (11 and 12) PL25; (13) negative control of PCR. Size of specific amplicon is 470 pb.

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cases or the eggs did not hatch in the four other cases. Nested-PCR analysis performed with the eggs of one female, before and after disinfection, confirmed the presence of virus (Fig. 2). Positive results were obtained for all the nested-PCR analyses made with DNA extracted from larvae at different stages corresponding to the females being IHHNVinfected whatever the level of infection. These results indicated that non-infected females did not transmit the virus vertically whereas the infected females did transmit it, whatever their infection level. 3.4. Quantification of vertically transmitted IHHNV in nauplii II and V In order to quantify the vertically transmitted viruses from the females to their progeny, nested-PCR analyses were performed with DNA extracted from nauplii II and V. A 10-fold serial dilution of each DNA sample was made. An initial experiment was performed with domesticated shrimps of Colombian origin grown in Ecuador for the two last generations. Nauplii produced from five IHHNVinfected females were analyzed (Table 4). IHHNV was detected in all nauplius samples. According to the female, nested-PCR assays were positive up to dilutions of 10 2 or 10 5. Considering, on one hand, that a few molecules of viral DNA can be detected by nested PCR and, on the other hand, that each nested-PCR reaction was performed with DNA equivalent to two nauplii, it may be assumed that each nauplius contains between 100 and 100,000 copies of viral DNA molecules. A second experiment was performed with domesticated shrimps of Panamanian origin grown in Panama for the two last generations. As it was previously explained, nauplii II and V produced from 15 IHHNV-infected females were analyzed. In this experiment, the level of IHHNV infection of females was estimated by performing nested-PCR analyses of 10 fold serially diluted DNA samples extracted from hemolymph (Table 5). According to the female, nested-PCR assays were positive up to dilutions of 10 4 or 5 10 . Considering, on one hand, that a few molecules of viral DNA can be detected by nested PCR and, on the other hand, that each nested-PCR reaction was performed with DNA equivalent to 4 Al of hemolymph, it may be assumed that each one of these infected females contains between 2.5 and 25 million viral DNA molecules per milliliter. This number may correspond to a few infected hemocytes.

Table 4 Detection of IHHNV by nested-PCR in serially diluted DNA extracted from nauplii II and V corresponding to five IHHNV infected females Females Nauplii II (NII) 10 1 2 3 4 5 + + + + +
0

Nauplii V (NV) 10 + + + + +
2

10 + + + + +

10 + + + +

10 + +

10 +

100 + + + + +

10 1 + + + + +

10 2 + + + +

10 3 + + + +

10 4 + +

10 5 +

(+): tested positive; ( ): tested negative.

Table 5 Detection of IHHNV by nested-PCR in serially diluted DNA extracted from nauplii II and V corresponding to 15 IHHNV infected females of Panamanian origin # Females Female hemolymph 100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 + + + + + + + + + + + + + + + 10 1 + + + + + + + + + + + + + + + 10 2 + + + + + + + + + + + + + + + 10 3 + + + + + + + + + + + + + + + 10 4 + + + + + + + + + + + + + + + 10 5 + + + + + + + + + + + + Nauplius II (NII) 100 + + + + + + + + + + + + + + + 10 1 + + + + + + + + + + + + + + + 10 2 + + + + + + + + + + + + + + + 10 3 + + + + + + + + + + + 10 4 + + + + 10 5 Nauplius V (NV) 100 + + + + + + + + NA NA NA NA NA NA NA 10 1 + + + + + + + + NA NA NA NA NA NA NA 10 2 + + + + + + + + NA NA NA NA NA NA NA 10 3 + + + + + + + + NA NA NA NA NA NA NA 10 4 + + + + + NA NA NA NA NA NA NA 10 5 + + NA NA NA NA NA NA NA E. Motte et al. / Aquaculture 219 (2003) 5770

(+): tested positive; ( ): tested negative; NA: no analyzed.

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Concerning the nauplius II, the number of viral DNA molecules can be estimated between 100 and 10,000/nauplius, whereas this number increases between 1000 and 100,000 for the nauplius V. The increase of viral DNA, 10 100 times, detected by nested PCR suggests an effective replication of IHHNV during the nauplii stages. Some deformities were observed in nauplii, in particular when the IHHNV infection was maximal. 3.5. Reliability of female selection through nested PCR A study was performed to determine the time reliability of IHHNV detection through nested PCR. Females, initially found negative for IHHNV after the first spawn, were analyzed the second time after some supplementary spawns. The first study consisted of a group of 18 females of Panamanian origin located in a hatchery of the Guayas Province (Ecuador) and a group of 20 females of Colombian origin located in a hatchery of the Manabi Province (Ecuador) (Table 6). In the first group, only one female (5%) was IHHNV-positive with the analysis being made after five spawns. In the second group, four females (20%) were IHHNV-positive, with the analysis being made after 1, 2, 4 or 5 spawns. Finally, it was estimated that 13% of females was falsely tested IHHNV-negative on the basis of the first nested-PCR analysis. However, the reliability of the assay is relatively high with 87% of females confirmed IHHNV-negative after a second nested-PCR analysis. A second batch of 127 females (which had tested negative for IHHNV) from Panama was analyzed and showed a similar rate of reversion (11%) (Table 7).

Table 6 Reliability of detection of IHHNV in female L. vannamei using nested-PCR Number of spawning between the first test analysis and the second analysis Hatchery in Guayas (Ecuador) 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Number of IHHNV-negative females after the second analysis Number of IHHNV-positive females after the second analysis

18 females certified IHHNV-free Hatchery in Manab (Ecuador)

1 8 4 3 1 3 2 4 4 2 1 33 (87%)

1 1 1 1

20 females certified IHHNV-free Total: 38

5 (13%)

E. Motte et al. / Aquaculture 219 (2003) 5770 Table 7 Reliability of detection of IHHNV in female L. vannamei using nested-PCR Number of spawning between the first test analysis and the second analysis 1 2 3 4 5 6 7 8 Total Number of IHHNV-negative females after the second analysis 24 36 18 20 6 5 3 1 113 (89%)

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Number of IHHNV-positive females after the second analysis 6 4 2 1 1

14 (11%)

3.6. Comparison of nauplius productivity between IHHNV-infected and IHHNV-free females From November 2001 to January 2002, the nauplius productivity was compared between IHHNV-infected (about 200) and IHHNV-free (about 500) females previously separated from the same group of animals on the basis of nested PCR performed after the first spawning. The mean percentages of daily spawning were respectively 12% and 15% for IHHNV-infected and IHHNV-free females. The mean numbers of nauplii produced at each spawning were similar, respectively 99,000 and 102,000. These results suggest a possible impact of IHHNV on the reproductive physiology of female shrimp.

4. Discussion Because of their drastic impact on production, highly pathogenic viruses such as WSSV (Lo and Kou, 1998) or TSV (Lightner, 1996) are considered a serious problem by shrimp producers and are largely studied by epidemiologists and virologists (Lightner and Redman, 1998; Rajan et al., 2000). Endemic low virulence viruses, such as IHHNV, are comparatively poorly investigated and practically accepted by shrimp farmers. Such acceptance needs to be criticized since the lack of data related to IHHNV epidemiology may lead to a surprising increase of virus prevalence with subsequent productivity losses. As a matter of fact, nonpathogenic viruses can spread through wild and domesticated populations, this spread depending essentially on the routes of virus transmission and on the culture practices. The first objective of the present work was to determine the prevalence of IHHNV in wild and recently domesticated broodstock populations of L. vannamei in Ecuador and Panama. During the last 10 years, biotechnology based tools have been developed for very sensitive and specific diagnosis of pathogens in economically important marine invertebrates (Mialhe et al., 1995). Monoclonal antibody based immunoassays were developed (Mialhe et al., 1988) and later PCR-based diagnosis with a first assay for a scallop rickettsia (Le Gall and Mialhe, 1992; Kellner-Cousin et al., 1994). In order to get maximal accuracy in IHHNV prevalence determinations, a nested-PCR assay was presently applied

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for diagnosis using hemolymph samples that can be easily and non-traumatically obtained. Previous to this study, several clinical evaluations had shown that hemolymph is the best tissue sample for reliable diagnosis of IHHNV infection. In Ecuador, wild and recently domesticated broodstock showed similar IHHNV prevalences ranging between 47% and 63%. These similar values, relatively high, suggest that there is a strong connection between the natural and cultivated populations in terms of IHHNV spreading. As a matter of fact, in Ecuador, on one hand, a large proportion of cultivated larvae comes from nauplii produced from wild broodstock and, on the other hand, a large proportion of nauplii produced in the hatcheries from domesticated broodstock is discharged into the ocean. Such a massive discharge occurs because the hatcheries must be seeded in only 2 or 3 days in order to avoid problems of bacterial diseases (Otta et al., 2001) that are systematically observed when the seeding process is prolonged. One population located at Puerto Cayo in the Manabi Province was much less infected with 20% IHHNV prevalence. This low prevalence could be explained by the lack of hatcheries in this area. In Panama, a third generation of domesticated broodstock was found to be highly infected with a prevalence around 95%. This high prevalence could result from the broodstock feeding on tissues of IHHNV-infected shrimps. As a matter of fact, this procedure was used to try to select animals resistant to WSSV infection and seemed to be responsible for the horizontal transmission of IHHNV. The second objective of this work concerned the demonstration of vertical transmission of IHHNV. The nested-PCR analysis of ovaries and spermatophore-collected sperm from infected animals led to the suspicion that vertical transmission occurs through the females and eventually through the sperm. This characteristic relates IHHNV with a mosquito virus (Barreau, 1993), both classified inside the group of Brevidensovirus (Shike et al., 2000) of the Parvoviridae. The PCR-detection of viral DNA inside the ovaries has to be related to the previous histological detection of IHHNV virions in ovaries and inside the oocytes (Lotz, 1997). The vertical transmission of IHHNV through female L. vannamei was subsequently confirmed by analyzing embryos and larvae produced, respectively, by IHHNV-infected and IHHNV-free females fertilized by IHHNV-free males. The embryos and larvae produced from IHHNV-free females were free of virus whereas embryos and larvae produced from IHHNV-infected females were also found to be infected through the nested-PCR analysis. The level of IHHNV infection in the females was also considered by classifying them on the basis of a dot-blot analysis. In the case of females with the highest IHHNV infection, the embryos generally failed to develop and hatch. In order to determine possible correlations between the levels of IHHNV infection between the females and their nauplii, nested-PCR analysis was performed on serial dilutions of DNA extracted from the female hemolymph and from nauplii. By this procedure, it was possible to determine that infected females contain between 2.5 and 25 millions viral DNA molecules per milliliter of hemolymph. The number of viral DNA molecules was estimated between 100 and 10,000/ nauplius II with an apparent increase in the nauplii V. The increase of viral DNA, by 10 100 times, suggests that replication of IHHNV occurs during the nauplius stages. It must be noted that deformities were observed in IHHNV-infected nauplii.

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The lack of IHHNV in females identified as virus-negative on the basis of a nested-PCR analysis led to consider the application of this procedure to prevent the vertical transmission of the virus. The analyses were performed after eyestalk ablation and the first spawning since this double stress induces the replication of the virus. Similar observations have been made in P. monodon for WSSV replication (Peng et al., 2001). The reliability of this procedure has been evaluated by repeating the nested-PCR analyses after some supplementary spawnings. Since almost 90% of females found to be IHHNV-free after the first nested-PCR diagnosis was confirmed free of virus after the second analysis, it may be considered that the present screening procedure is reliable and effective in preventing IHHNV vertical transmission. The identification of IHHNV-free females would not only improve nauplius quality but also production of nauplii since it was established that IHHNV-free females produced about 25% more nauplii. Moreover, the size disparity in the growth of juveniles was greatly reduced in cultivated shrimps that corresponded to nauplii produced from IHHNVfree females by comparison to those corresponding to IHHNV-infected females, with all the females coming from the same broodstock (E. Castillo, personal communication). This type of screening procedure is presently being applied not only for IHHNV but also simultaneously for WSSV in order to select shrimps free of virus in spite of high prevalence in the farms. Shrimps that are also free of septicemia are also being selected to constitute broodstock. As a matter of fact, such multi-pathogen-free surviving shrimps may present some resistance characteristics that make them candidates to be used in breeding programs. Acknowledgements The scientific team at Concepto Azul is very grateful to Wil Van der Knaap, Wageningen University, The Netherlands, for his helpful reviewing of the manuscript. References
` Barreau, C., 1993. Etude des interactions entre un nouveau virus entomopathogene et des moustiques vecteurs dagents transmissibles: Potentialites dutilitisation en lutte biologique. PhD Universite de Montpellier II, pp. 185. Bell, T.A., Lightner, D.V., 1984. IHHN virus: infectivity and pathogenicity studies in Penaeus styliostris and Penaeus vannamei. Aquaculture 38, 185 194. Bonami, J.R., Trumper, B., Mari, J., Brehelin, M., Lightner, D.V., 1990. Purification and characterization of the infectious hypodermal and haematopoietic necrosis virus of penaeid shrimps. J. Gen. Virol. 71, 2637 2664. ` Destoumieux, D., Munos, M., Cosseau, C., Rodriguez, J., Bulet, P., Comps, M., Bachere, E., 2000. Penaeidins, antimicrobial peptides with chitin-binding activity are produced and stored in shrimp granulocytes and released after microbial challenge. J. Cell Sci. 113, 461 469. Kalagayan, H., Godin, D., Kanna, R., Hagino, G., Sweeney, J., Wyban, J., 1991. IHHN virus as an etiological factors in Runt-Deformity Syndrome (RDS) of juvenile Penaeus vannamei cultured in Hawaii. J. World Aquac. Soc. 22, 235 243. Kellner-Cousin, K., Boulo, V., Lacroix, I., Mialhe, E., Mathieu, M., 1994. Use of monoclonal antibodies for identification of growthcontrolling neuropeptides in the mussel Mytilus edulis (Mollusca: Bivalvia). Comp. Biochem. Physiol. 109A, 689 698.

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