Virulence of an emerging pathogenic lineage of Vibrio nigripulchritudo is dependent on two plasmids

Vibrioses are the predominant bacterial infections in marine shrimp farms. Vibrio nigripulchritudo is an emerging pathogen of the cultured shrimp Litopenaeus stylirostris in New Caledonia and other regions in the Indo‐Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenic V. nigripulchritudo isolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulent V. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence‐linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence of V. nigripulchritudo in several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non‐pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage of V. nigripulchritudo.     

First occurrence of the yellow roughneck shrimp, <Emphasis Type="Italic">Rimapenaeus similis</Emphasis> (Smith, 1885) (Crustacea: Decapoda: Penaeidae) in the Mediterranean Sea (Tunisian waters)

During an experimental trawl survey carried out by the R/V “Hannibal” in June 2006 several specimens of the Western-Atlantic penaeid shrimp Rimapenaeus similis (Smith, 1885) were caught in the Gulf of Gabes (southern Tunisia, Central Mediterranean). This represents the first recording of Rimapenaeus similis in Tunisian waters and for the Mediterranean Sea. Biological information on the sampled population is also included.     

Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits

Vibrio nigripulchritudo is an emerging pathogen of farmed shrimp in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have suggested that pathogenicity is linked to particular lineages. Here, we performed high-throughput sequencing-based comparative genome analysis of 16 V. nigripulchritudo strains to explore the genomic diversity and evolutionary history of pathogen-containing lineages and to identify pathogen-specific genetic elements. Our phylogenetic analysis revealed three pathogen-containing V. nigripulchritudo clades, including two clades previously identified from New Caledonia and one novel clade comprising putatively pathogenic isolates from septicemic shrimp in Madagascar. The similar genetic distance between the three clades indicates that they have diverged from an ancestral population roughly at the same time and recombination analysis indicates that these genomes have, in the past, shared a common gene pool and exchanged genes. As each contemporary lineage is comprised of nearly identical strains, comparative genomics allowed differentiation of genetic elements specific to shrimp pathogenesis of varying severity. Notably, only a large plasmid present in all highly pathogenic (HP) strains encodes a toxin. Although less/non-pathogenic strains contain related plasmids, these are differentiated by a putative toxin locus. Expression of this gene by a non-pathogenic V. nigripulchritudo strain resulted in production of toxic culture supernatant, normally an exclusive feature of HP strains. Thus, this protein, here termed ‘nigritoxin'', is implicated to an extent that remains to be precisely determined in the toxicity of V. nigripulchritudo.     

Effect of Organic Acids on Shrimp Pathogen, <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

Shrimp farming accounts for more than 40% of the world shrimp production. Luminous vibriosis is a shrimp disease that causes major economic losses in the shrimp industry as a result of massive shrimp kills due to infection. Some farms in the South Asia use antibiotics to control Vibrio harveyi, a responsible pathogen for luminous vibriosis. However, the antibiotic-resistant strain was found recently in many shrimp farms, which makes it necessary to develop alternative pathogen control methods. Short-chain fatty acids are metabolic products of organisms, and they have been used as food preservatives for a long time. Organic acids are also commonly added in feeds in animal husbandry, but not in aquaculture. In this study, growth inhibitory effects of short-chain fatty acids, namely formic acid, acetic acid, propionic acid, and butyric acid, on V. harveyi were investigated. Among four acids, formic acid showed the strongest inhibitory effect followed by acetic acid, propionic acid, and butyric acid. The minimum inhibitory concentration (MIC) of 0.035% formic acid suppressed growth of V. harveyi. The major inhibitory mechanism seems to be the pH effect of organic acids. The effective concentration 50 (EC₅₀) values at 96 h inoculation for all organic acids were determined to be 0.023, 0.041, 0.03, and 0.066% for formic, acetic, propionic, and butyric acid, respectively. The laboratory study results are encouraging to formulate shrimp feeds with organic acids to control vibrio infection in shrimp aquaculture farms.     

Characterization of some fish and shrimp spoiling bacteria

The classification of some important groups of bacteria involved in fish and shrimp spoilage was studied. Trimethylamine is produced byPseudomonas putrefaciens, a “non-defined” group resemblingPs. putrefaciens, Photobacterium spp. and someMoraxella-like bacteria. Hypoxanthine is produced by the same groups of bacteria except the last named and also by the “typical shrimp spoilers” (presumptiveAlteromonas). Strong off-odours are produced on fresh fish byPs. putrefaciens, dextroseoxidativePseudomonas spp. (Groups I and 11 according to Shewan, Hobbs and Hodgkiss, 1960), the above mentioned “non-defined” group and by only some of the “typical shrimp spoilers”, whereasMoraxella-like bacteria andPhotobacterium spp. failed to produce strong odours. Strong off-odours are produced on boiled shrimp by the “typical shrimp spoilers” (presumptiveAltermonas),Ps. putrefaciens, the dextrose-oxidativePseudomonas spp. and the “non-defined” group;Moraxella-like bacteria produced less offensive odours or none, nor didPhotobacterium.     

Development of a haemolysin gene‐based multiplex PCR for simultaneous detection of Vibrio campbellii,Vibrio harveyi and Vibrio parahaemolyticus

Aim: To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species‐specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost‐effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.     

Seaweed meal as a protein source for the white shrimp Litopenaeus vannamei

The rhodophytes Hypnea cervicornis and Cryptonemia crenulata are abundant along the Brazilian coastline and are rich in nutrients. They may therefore be used as a source of protein in shrimp diets. The aim of the present study was to test this hypothesis. The experiment was conducted in a laboratory, where 10-day-old post-larvae aged underwent 7 days of acclimation in a 1,000 L tank. They were then kept in plastic aquariums, each containing 10 L, and 20 larvae were fed daily (10% of biomass) in four equal portions with one of four diets (five repetitions of each) for a period of 45 days. All diets contained 30% crude protein (isoprotein) and 300 kcal 100 g⁻¹ (isocaloric), with different percentages of seaweed powder: Diet “A” 39%; Diet “B” 26%, Diet “C” 13%, and Diet “D” without seaweed (control diet). Algae were collected, rinsed, dried and ground up for the feed formulations. Weight of the animals was measured at the beginning of the experiment and at 15-day intervals to assess their growth. The physico-chemical variables of the water were measured every 2 days. Final biomass, biomass gain and specific growth rate (SGR) exhibited no significant differences between treatments (P > 0.05). Survival rate was equal under the four experimental conditions, being consistent within four decimal places 95.2% to 97.00% (P > 0.05). Diets “A” and “B”, with a greater content of algae, exhibited better feed conversion (1.79:1 and 1.82:1) than Diets “C” and “D” (2.04:1 and 2.08:1) (P < 0.05). The physical-chemical variables of the water showed no significant variation and remained within the standards necessary for the wellbeing of the animals. If sufficient biomass of beached algae can be practically and economically collected, it may be used as a component in the making of shrimp feed.     

Effect of probiotic Pediococcus acidilactici on antioxidant defences and oxidative stress of Litopenaeus stylirostris under Vibrio nigripulchritudo challenge

Antioxidant defences and induced oxidative stress tissue damage of the blue shrimp Litopenaeus stylirostris, under challenge with Vibrio nigripulchritudo, were investigated for a 72-h period. For this purpose, L. stylirostris were first infected by immersion with pathogenic V. nigripulchritudo strain SFn1 and then antioxidant defences: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx), Total antioxidant status (TAS), glutathiones and induced tissue damage (MDA and carbonyl proteins) were determined in the digestive gland at 0, 12, 24, 48 and 72 h post-infection (h.p.i.). In the meantime, TAS was also measured in the blood. Infection level of the shrimps during the challenge was followed by determining V. nigripulchritudo prevalence and load in the haemolymph of the shrimps. Changes in all these parameters during the 72-h.p.i. period were recorded for control shrimps and shrimps previously fed for one month with probiotic Pediococcus acidilactici MA18/5M at 10⁷ CFU g^?1 of feed.Our results showed that immersion with V. nigripulchritudo led to maximal infection level in the haemolymph at 24 h.p.i. preceding the mortality peak recorded at 48 h.p.i. Significant decreases in the antioxidant defences were detected from 24 h.p.i. and beyond that time infection leaded to increases in oxidative stress level and tissue damage. Compared to control group, shrimps fed the probiotic diet showed lower infection (20% instead of 45% at 24 h.p.i. in the control group) and mortality (25% instead of 41.7% in the control group) levels. Moreover, infected shrimp fed the probiotic compared to uninfected control shrimps exhibited very similar antioxidant status and oxidative stress level. Compared to the infected control group, shrimps fed the probiotic sustained higher antioxidant defences and lower oxidative stress level.This study shows that bacterial infection leads to oxidative stress in L. stylirostris and highlighted a beneficial effect of P. acidilactici, suggesting both a competitive exclusion effect leading to a reduction of the infection level and/or an enhancement of the antioxidant status of the shrimps.     

Molecular epidemiology of Vibrio nigripulchritudo, a pathogen of cultured penaeid shrimp (Litopenaeus stylirostris) in New Caledonia

A collection of 57 isolates of Vibrio nigripulchritudo from either diseased or healthy shrimp and from shrimp farms environment was studied in order to gain a better understanding of the epidemiology of this pathogen, notably isolated from two distinct shrimp disease complexes. Molecular typing using two different techniques, arbitrarily primed PCR (AP-PCR) and multi-locus sequence typing (MLST), studied together with experimental pathology data allowed a relevant epidemiological insight into this possibly emerging pathogen. Additionally, results obtained with the two molecular typing techniques were congruent and allowed discriminating the strains associated with the "Summer Syndrome" from strains isolated from other contexts, especially the other shrimp vibriosis "Syndrome 93". These results highlight that the "Summer Syndrome" is most probably caused by an emergent clonal pathogen that therefore deserves surveillance and that AP-PCR can satisfactorily be used for that purpose.     

An in vitro and in vivo assessment of the potential of Vibrio spp. as probiotics for the Pacific White shrimp,Litopenaeus vannamei

Aims: The objective of the work was to determine whether known strains of nonpathogenic vibrios can act as probiotics for the control of Vibrio infections in the Pacific white shrimp, Litopenaeus vannamei. Methods and Results: Of the ten species tested, only Vibrio alginolyticus (NCIMB 1339) and Vibrio gazogenes (NCIMB 2250) showed antagonistic activity towards a panel of shrimp pathogenic vibrios. In the case of V. alginolyticus, this activity depended on the presence of live bacteria while in V. gazogenes both live and dead bacteria showed anti‐Vibrio activity. Injection of shrimp with either V. alginolyticus or V. gazogenes at 3 × 10⁷ or 3 × 10⁵ total bacteria per shrimp resulted in mortality with higher levels in the case of V. alginolyticus (100% mortality 18 h postinjection of 3 × 10⁷ bacteria). Juvenile shrimp were fed commercial diets top‐coated with either chitin (an immune stimulant) or chitin + V. gazogenes. Both chitin and V. gazogenes caused a significant decline in the number of Vibrio‐like bacteria in the fore and hind gut, and changes were also seen in the hepatosomatic index (a measure of digestive health) and the total number of blood cells in circulation. Analysis of mid/hindgut and faecal samples obtained using terminal restriction fragment length polymorphism showed that the gut microbiota of shrimp has limited bacterial diversity and that after 8 weeks exposure to the experimental diets there were significant changes in the microbial flora of the GI tract of shrimp as a result of the presence of V. gazogenes. Conclusions: Of the vibrios tested, V. gazogenes has potential as a probiotic for the control of bacterial diseases in shrimp. Significance and Impact of the Study: Overall, this study shows the promise of V. gazogenes together with chitin to improve the health and welfare of shrimp under aquaculture conditions.     

Detection of Microbial Pathogens in Shellfish with Multiplex PCR

Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl₂ and primer annealing temperature of 55°C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelex™ 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was ≤10¹–10² cells following a “double” multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneComb™ (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The colorimetric GeneComb™ assay avoids use of hazardous materials inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed by colorimetric GeneComb™ DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish. Received: 17 November 1997 / Accepted: 17 February 1998     

Development of a single base extension-tag microarray for the detection of pathogenic Vibrio species in seafood

In this study, a single base extension-tag array on glass slides (SBE-TAGS) microarray was established to detect the seven leading seafood-borne pathogens, including Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio mimicus, Vibrio alginolyticus, Vibrio anguillarum, and Vibrio harveyi. Three multiplex PCR assays were developed to specifically target the following species with individual gene markers, which are aadS, tdh, and trh for V. parahaemolyticus; col, toxR, and vvh for V. alginolyticus, V. mimicus, and V. vulnificus; and empA, vhh1, and tcpA for V. anguillarum, V. harveyi, and V. cholerae, respectively. The purified PCR products were used as template DNA for single base extension-tag reactions, labeled with Cy3 fluorescent dye and hybridized to DNA microarrays. The detection specificity of this microarray method was 100%, with the sensitivity for pure genomic DNA at 200 fg to 2 pg per reaction. Application of the DNA microarray methodology to 55 naturally contaminated seafood samples (shrimp, fish, and oysters) revealed the presence of V. parahaemolyticus at 50.9% and V. alginolyticus at 32.7%. This corresponds with traditional assays (microbiological and biochemical tests) except one sample which was identified as negative in V. parahaemolyticus by the microarray assay but as positive by the conventional method. Therefore, a combination of multiplex PCR with DNA microarray hybridization based on SBE-TAGS ensures rapid and accurate detection of pathogenic Vibrio species in seafood, thereby providing safer seafood products for consumers at a low financial burden to the aquaculture industry.     

Language and Emotional Knowledge: A Case Study on Ability and Disability in Williams Syndrome

Williams Syndrome provides a striking test case for discourses on disability, because the characteristics associated with Williams Syndrome involve a combination of “abilities” and “disabilities”. For example, Williams Syndrome is associated with disabilities in mathematics and spatial cognition. However, Williams Syndrome individuals also tend to “have a unique strength in their expressive language skills”, and are socially outgoing and unselfconscious when meeting new people. Children with Williams are said to be “typically unafraid of strangers and show a greater interest in contact with adults than with their peers.” This apparently keen social knowledge is a counterexample to the discussion of disability among academic philosophers, especially philosophers in the early modern period. Locke infamously used the example of disability to claim that Descartes’ arguments in favor of innate ideas were incorrect. On the contrary, Williams Syndrome may stand as an example of innate social knowledge; something that could benefit current discourse in philosophy, disability theory, and medical ethics.

<Emphasis Type="Italic">MEFV</Emphasis> heterogeneity in Turkish Familial Mediterranean Fever patients

Turkey is one of the few countries in the world where Familial Mediterranean Fever (FMF), an autoinflammatory disease caused by mutations in MEFV, the gene encoding pyrin, is not rare. Many interesting studies regarding the genetics of Familial Mediterranean Fever in Turkey have been already published. Despite that different MEFV genetic profiles have been revealed for Turkish FMF patients, deriving from different regions of Turkey, a systematic population genetics analysis has not been carried out yet. The present study aims to investigate the population genetics of MEFV in Turkish FMF patients so as to additionally facilitate the clinical interpretation of individualized genetic data. All relevant studies have been recruited by searching PubMed with the terms “MEFV”, “FMF”, and “Turkey”. Seven of them, including 3,061 FMF patients, contained all necessary data concerning allelic and genotypic frequencies of the 4 commonest MEFV mutations in Turkey (M694V, V726A, M680I, E148Q). From all 6,122 MEFV alleles analyzed, the M694V mutation was recognized in 15.6–52.2% (mean 29.3%), the V726A in 1.5–9.7% (mean 4.8%), the M680I in 1.5–15.5% (mean 7.6%), and the E148Q in 3.2–13.9% (mean 5.5%). Unidentified mutations ranged from 0–42.9% (mean 16.8%). No mutations were found in 0–54.5% (mean 36.0%) of the patients. The allelic and genotypic frequencies of the most frequent mutation (M694V) showed aberration of the Hardy–Weinberg law for all 7 populations studied. By application of the Arlequin 2.0 population genetics software, the Fixation index (F _ST) was found to be 0.09994, thus demonstrating that the observed variability is mainly within (90.006%) and not among (9.994%) populations (P < 0.00001). Moreover, the global test of differentiation demonstrated that every population differs from each other (P < 0.00325). Finally, the Ewens–Watterson test of selective neutrality yielded to statistical significance in only 3 populations. In conclusion, Turkish FMF patients are characterized by an increased genetic heterogeneity, explained by the intrapopulation differentiation. Thus, the regional origin should be regarded as a determining factor in the diagnosis of FMF in Turkish patients.     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.     

Use of microsatellite DNA and otolith Sr:Ca ratios to infer genetic relationships and migration history of four morphotypes of <Emphasis Type="Italic">Rhinogobius</Emphasis> sp. OR

The genetic diversity and relationship among four morphotypes of Rhinogobius sp. OR, Gobiidae (“Tōshoku,” “Shinjiko,” “Gi-tōshoku,” and “Shimahire”) were investigated with seven microsatellite DNA loci, and amphidromy of these morphotypes was verified by strontium (Sr) and calcium (Ca) deposition in the otolith. Samples of “Tōshoku,” “Shinjiko,” “Gi-tōshoku,” and “Shimahire” were collected from, respectively, three, three, two, and four locations in Japan. Microsatellite analysis detected high genetic diversity (based on the number of alleles, allelic richness, and average observed heterozygosity) in the “Tōshoku” and “Shinjiko” morphotypes relative to the “Shimahire” morphotype; the “Gi-tōshoku” morphotype had an intermediate level of variation. Almost all pairwise F _ST values were significantly different from zero (P < 0.001), except between two populations of “Tōshoku.” Clear genetic independence was observed between the “Shinjiko” and “Shimahire” morphotypes in the Maruyama River. A principle component analysis based on microsatellite data indicated that the “Tōshoku,” “Shinjiko,” and “Gi-tōshoku” morphotypes were genetically similar. Furthermore, the three populations of “Tōshoku” were closely related each other, and two of those collected from the Lake Biwa system were a single population. There was, however, a high degree of genetic differentiation between “Shimahire” and the other morphotypes; moreover, there was high genetic divergence among four populations of the “Shimahire” morphotype. Amphidromous migratory histories were indicated by Sr:Ca ratios in two of three populations of the “Shinjiko” morphotype and in one of two “Gi-tōshoku” morphotypes, whereas all populations of the “Shimahire” morphotype were freshwater residents. The large genetic divergence and low genetic diversity in “Shimahire” are likely related to migration history.     

In vitro separation of a rose chimera

“Fairmount 1 thorny” (“FM1 thorny”) (a Rosa multiflora Thunb ex. J. Murr.) and a thornless sport of “FM1 thorny” (“Fairmount 1” (“FM1”)) were established in vitro to investigate chimeral segregation under various levels of BA and to obtain a pure thornless rose. While the chimeral thornless sport was expected to segregate in vitro and yield both thorny and thornless plantlets, “FM1 thorny” was to yield only thorny plants. “FM1” segregated in vitro into its constituent genotypes and yielded thorny and thornless plantlets, suggesting that “FM1” is chimeral. “FM1 thorny” produced only thorny plants in vitro. These results indicate that the “FM1 thorny” clone was not chimeral (pure thorny) and that the thornless regenerates of “FM1” did not develop via somaclonal variation. There was a significant linear relationship between increasing BA concentration and the percentage of thorny plants. Among a population of 690 tissue culture derived plants from all the BA experiments, 6 plants were classified as pure thornless plants 1 year later.     

Polymorphism of canonical and noncanonical <Emphasis Type="Italic">gypsy</Emphasis> sequences in different species of <Emphasis Type="Italic">Drosophila </Emphasis><Emphasis Type="Italic">melanogaster</Emphasis> subgroup: possible evolutionary relations

Mobile genetic elements constitute a substantial part of eukaryotic genome and play an important role in its organization and functioning. Co-evolution of retrotransposons and their hosts resulted in the establishment of control systems employing mechanisms of RNA interference that seem to be impossible to evade. However, “active” copies of endogenous retrovirus gypsy escape cellular control in some cases, while its evolutionary elder “inactive” variants do not. To clarify the evolutionary relationship between “active” and “inactive” gypsy we combined two approaches: the analysis of gypsy sequences, isolated from G32 Drosophila melanogaster strain and from different Drosophila species of the melanogaster subgroup, as well as the study of databases, available on the Internet. No signs of “intermediate” (between “active” and “inactive”) gypsy form were found in GenBank, and four full-size G32 gypsy copies demonstrated a convergence that presumably involves gene conversion. No “active” gypsy were revealed among PCR generated gypsy ORF3 sequences from the various Drosophila species indicating that “active” gypsy appeared in some population of D. melanogaster and then started to spread out. Analysis of sequences flanking gypsy variants in G32 revealed their predominantly heterochromatic location. Discrepancy between the structure of actual gypsy sites in G32 and corresponding sequences in database might indicate significant inter-strain heterochromatin diversity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.