Hyaluronan-estradiol nanogels as potential drug carriers to target ER+ breast cancer cell line.

An innovative hyaluronan-based nano-delivery system is proposed for the active targeting towards ER+ breast cancer. Hyaluronic acid (HA), an endogenous and bioactive anionic polysaccharide, is functionalized with estradiol (ES), a sexual hormone involved in the development of some hormone-dependent tumors, to give an amphiphilic derivative (HA-ES) able to spontaneously self-assemble in water to form soft nanoparticles or nanogels (NHs). The synthetic strategy used to obtain the polymer derivatives and the physico-chemical properties of the obtained nanogels (ES-NHs) are reported. ES-NHs ability to entrap hydrophobic molecules has also been investigated, by loading curcumin (CUR) and docetaxel (DTX), both able to inhibit the growth of ER+ breast cancer. The formulations are studied for their capability to inhibit the growth of the MCF-7 cell line, thus evaluating their efficacy and potential as a selective drug delivery systems. Our results demonstrate that ES-NHs have not toxic effects on the cell line, and that both ES-NHs/CUR and ES-NHs/DTX treatments inhibit MCF-7 cell growth, with ES-NHs/DTX effect higher than that of free DTX. Our findings support the use of ES-NHs to deliver drugs to ER+ breast cancer cells, assuming a receptor-dependent targeting.

Strategies to load therapeutics into polysaccharide-based nanogels with a focus on microfluidics: A review.

Nowadays nanoparticles are increasingly investigated for the targeted and controlled delivery of therapeutics, as suggested by the high number of research articles (2400 in 2000 vs 8500 in 2020). Among them, almost 2% investigated nanogels in 2020. Nanogels or nanohydrogels (NGs) are nanoparticles formed by a swollen three-dimensional network of synthetic polymers or natural macromolecules such as polysaccharides. NGs represent a highly versatile nanocarrier, able to deliver a number of therapeutics. Currently, NGs are undergoing clinical trials for the delivery of anti-cancer vaccines. Herein, the strategies to load low molecular weight drugs, (poly)peptides and genetic material into polysaccharide NGs as well as to formulate NGs-based vaccines are summarized, with a focus on the microfluidics approach.

Biodistribution and intracellular localization of hyaluronan and its nanogels. A strategy to target intracellular S. aureus in persistent skin infections.

Intracellular pathogens are a critical challenge for antimicrobial therapies. Staphylococcus aureus (S. aureus) causes approximately 85% of all skin and soft tissue infections in humans worldwide and more than 30% of patients develop chronic or recurrent infections within three months, even after appropriate antibacterial therapies. S. aureus is also one of the most common bacteria found in chronic wounds. Recent evidences suggest that S. aureus is able to persist within phagolysosomes of skin cells (i.e. keratinocytes, phagocytic cells), being protected from both the immune system and a number of antimicrobials. To overcome these limits, nano-formulations that enable targeted therapies against intracellular S. aureus might be developed. Herein, the biodistribution and intracellular localisation of hyaluronan (HA) and HA-based nanoparticles (nanogels, NHs) are investigated, both after intravenous (i.v.) injections (in mice) and topical administrations (in ex vivo human skin). Results indicate HA and NHs accumulate especially in skin and liver of mice after i.v. injection. After topical application on human skin explants, no penetration of both HA and NHs was detected in skin with intact stratum corneum. By contrast, in barrier-disrupted human skin (with partial removal and loosening of stratum corneum), HA and NHs penetrate to the viable epidermis and are taken up by keratinocytes. In mechanically produced wounds (skin without epidermis) they accumulate in wound tissue and are taken up by dermis cells, e.g. fibroblasts and phagocytic cells. Interestingly, in all cases, the cellular uptake is CD44-mediated. In vitro studies confirmed that after CD44-mediated uptake, both HA and NHs accumulate in lysosomes of dermal fibroblasts and macrophages, as previously reported for keratinocytes. Finally, the colocalisation between intracellular S. aureus and HA or NHs is demonstrated, in macrophages. Altogether, for the first time, these results strongly suggest that HA and HA-based NHs can provide a targeted therapy to intracellular S. aureus, in persistent skin or wound infections.

Halting hyaluronidase activity with hyaluronan-based nanohydrogels: development of versatile injectable formulations.

Hyaluronan (HA) is among the most used biopolymers for viscosupplementation and dermocosmetics. However, the current injectable HA-based formulations present relevant limitations: I) unmodified HA is quickly degraded by endogenous hyaluronidases (HAase), resulting in short lasting properties; II) cross-linked HA, although shows enhanced stability against HAase, often contains toxic chemical cross-linkers. As such, herein, we present biocompatible self-assembled hyaluronan-cholesterol nanohydrogels (HA-CH NHs) able to bind to HAase and inhibit the enzyme activity in vitro, more efficiently than currently marketed HA-based cross-linked formulations (e.g. Jonexa™). HA-CH NHs inhibit HAase through a mixed mechanism, by which NHs bind to HAase with an affinity constant 7-fold higher than that of native HA. Similar NHs, based on gellan-CH, evidenced no binding to HAase, neither inhibition of the enzyme activity, suggesting this effect might be due to the specific binding of HA-CH to the active site of the enzyme. Therefore, HA-CH NHs were engineered into injectable hybrid HA mixtures or physical hydrogels, able to halt the enzymatic degradation of HA.

PVA/Dextran hydrogel patches as delivery system of antioxidant astaxanthin: a cardiovascular approach.

After myocardial infarction, the heart's mechanical properties and its intrinsic capability to recover are compromised. To improve this recovery, several groups have developed cardiac patches based on different biomaterials strategies. Here, we developed polyvinylalcohol/dextran (PVA/Dex) elastic hydrogel patches, obtained through the freeze thawing (FT) process, with the aim to deliver locally a potent natural antioxidant molecule, astaxanthin, and to assist the heart's response against the generated myofibril stress. Extensive rheological and dynamo-mechanical characterization of the effect of the PVA molecular weight, number of freeze-thawing cycles and Dex addition on the mechanical properties of the resulting hydrogels, were carried out. Hydrogel systems based on PVA 145 kDa and PVA 47 kDa blended with Dex 40 kDa, were chosen as the most promising candidates for this application. In order to improve astaxanthin solubility, an inclusion system using hydroxypropyl-ß-cyclodextrin was prepared. This system was posteriorly loaded within the PVA/Dex hydrogels. PVA145/Dex 1FT and PVA47/Dex 3FT showed the best rheological and mechanical properties when compared to the other studied systems; environmental scanning electron microscope and confocal imaging evidenced a porous structure of the hydrogels allowing astaxanthin release. In vitro cellular behavior was analyzed after 24 h of contact with astaxanthin-loaded hydrogels. In vivo subcutaneous biocompatibility was performed in rats using PVA145/Dex 1FT, as the best compromise between mechanical support and astaxanthin delivery. Finally, ex vivo and in vivo experiments showed good mechanical and compatibility properties of this hydrogel. The obtained results showed that the studied materials have a potential to be used as myocardial patches to assist infarcted heart mechanical function and to reduce oxidative stress by the in situ release of astaxanthin.

Productive performance and blood profiles of laying hens fed Hermetia illucens larvae meal as total replacement of soybean meal from 24 to 45 weeks of age.

The aim of the research was to study the effects of an insect meal from Hermetia illucens larvae (HILM) as complete replacement of soybean meal (SBM) on productive performance and blood profiles of laying hens, from 24 to 45 wk of age. A total of 108 24-week-old Lohmann Brown Classic laying hens was equally divided into 2 groups (54 hens/group, 9 replicates of 6 hens/group). From 24 to 45 wk of age, the groups were fed 2 different isoproteic and isoenergetic diets: the control group (SBM) was fed a corn-soybean meal based diet, while in the HILM group the soybean meal was completely replaced by Hermetia illucens larvae meal. Feed intake, number of eggs produced, and egg weight were recorded weekly along the trial. At 45 wk of age, blood samples were collected from 2 hens per replicate. The use of HIML led to a more favorable (P < 0.01) feed conversion ratio in hens but lay percentage, feed intake, average egg weight, and egg mass were higher (P < 0.01) in hens fed the SBM diet. Hens fed insect meal produced a higher percentage of eggs from small (S), medium (M), and extra-large (XL) classes (P < 0.01) than SBM, while the SBM group had a higher percentage of eggs from the large (L) class (P < 0.01). The levels of globulin and albumin to globulin ratio were, respectively, higher and lower (P < 0.05) in HILM than the SBM group. Cholesterol and triglycerides were higher (P < 0.05 and P < 0.01, respectively) in hens from SBM than in the HILM group. Blood levels of Ca were higher (P < 0.01) in hens fed insect meal, while creatinine was higher (P < 0.01) in blood of hens fed SBM. Hermetia illucens larvae meal can be a suitable alternative protein source for laying hens even if the complete replacement of soybean meal needs further investigation to avoid the negative effects on feed intake.

Gel-embedded niosomes: preparation, characterization and release studies of a new system for topical drug delivery.

In the present paper physical gels, prepared with two polysaccharides, Xanthan and Locust Bean Gum, and loaded with non-ionic surfactant vesicles, are described. The vesicles, composed by Tween20 and cholesterol or by Tween85 and Span20, were loaded with Monoammonium glycyrrhizinate for release experiments. Size and zeta (ζ)-potential of the vesicles were evaluated and the new systems were characterized by rheological and dynamo-mechanical measurements. For an appropriate comparison, a Carbopol gel and a commercial gel for topical applications were also tested. The new formulations showed mechanical properties comparable with those of the commercial product indicating their suitability for topical applications. In vitro release experiments showed that the polysaccharide network protects the integrity of the vesicles and leads to their slow release without disruption of the aggregated structures. Furthermore, being the vesicles composed of molecules possessing enhancing properties, the permeation of the loaded drugs topically delivered can be improved. Thus, the new systems combine the advantages of matrices for a modified release (polymeric component) and those of an easier permeability across the skin (vesicle components). Finally, shelf live experiments indicated that the tested gel/vesicle formulations were stable over 1 year with no need of preservatives.

Prediction of digestible energy and gross energy digestibility of feeds and diets in ostriches.

1. The aim of this study was to assess regression equations able to predict the digestible energy (DE) and gross energy digestibility (GEd) of feed ingredients and diets for ostriches. 2. Results of chemical-nutritional characteristics from 17 ingredients (two varieties of maize, two barleys, oat, triticale, wheat bran, soybean meal, sunflower meal, beet pulp, maize silage, alfalfa hay, 4 alfalfa meals and lupin) and 12 experimental diets were used in a stepwise procedure. 3. Acid detergent lignin (ADL) was the first independent variable included in the model to predict the DE of all the samples (R(2) = 0.65 and Residual Standard Deviation (RSD) 1.02). When the concentration of ash, acid detergent fibre (ADF) and crude fibre were included in the model, the R(2) value of the regression equation increased (from 0.65 to 0.85) and RSD decreased (from 1.02 to 0.48). 4. The ADL concentration was also the first independent variable chosen by the stepwise regression analysis for the estimation of GEd from chemical-nutritional characteristics of feeds, explaining 57% of the total GEd variation. The concentrations of crude protein and ADF included at the second and third steps in the model increased the R(2) (up to 0.70 and 0.73, respectively) and decreased the RSD values (from 0.29 to 0.21 and 0.20, respectively). When other variables as crude fibre, ash and gross energy were included in the model, the coefficient of determination and the RSD strongly improved (0.85 and 0.12, respectively).

Chasing bacteria within the cells using levofloxacin-loaded hyaluronic acid nanohydrogels.

In the present work, an innovative approach based on the delivery of levofloxacin (LVF) from polysaccharide nanohydrogels for the treatment of bacterial intracellular infections is described. The nanohydrogels (NHs) were obtained by self-assembling of the hyaluronic acid-cholesterol amphiphilic chains in aqueous environment. LVF, a fluoroquinolone antibiotic scarcely efficient in intracellular infections, was entrapped within such NHs by nanoprecipitation, thus forming a drug delivery system (LVF-NHs) that was tested for its activity on different bacteria strains. The MIC values of levofloxacin-loaded nanohydrogels were determined for Staphylococcus aureus and Pseudomonas aeruginosa strains and compared to those obtained using free LVF. The intracellular antimicrobial activity of LVF-NHs and free LVF was compared on HeLa epithelial cell line infected by the above mentioned bacteria, and the increase in antibacterial efficacy of LVF-NHs with respect to that of free LVF was evidenced. The obtained results allow to conclude that this new approach can be considered as really promising method for intracellular infection treatments.

Effect of mannanoligosaccharides supplementation on caecal microbial activity of rabbits.

A total of 200 weaned (35 days) hybrid Hyla rabbits were randomly divided among five groups housed in bicellular cages (20 cages per group). Between 35 and 60 days of age, the groups were submitted to the following treatments: group ANT (positive control) fed a basal diet supplemented with antibiotics (colistin sulphate, 144 mg/kg; tylosin, 100 mg/kg; and oxytetracyclin, 1000 mg/kg); groups MOS_0.5, MOS_1.0 and MOS_1.5 fed the basal diet supplemented with 0.5, 1.0 and 1.5 g/kg mannanoligosaccharides (MOS), respectively; another group fed the basal diet without antibiotics or mannanoligosaccarides supplementation (negative control). Along the trial, an episode of epizootyc rabbit enteropathy occurs so that in the control group mortality rate was very high (78%) and survivor rabbits showed severe symptoms of disease (diarrhoea). Thus, the control group was discarded from the trial. At 60 days of age, samples of caecal content were collected from 10 rabbits per group and used as inocula for an in vitro gas production trial. At the end of fermentation (120 h of incubation), organic matter digestibility (OMd), cumulative gas production, fermentation kinetics, pH, volatile fatty acid (VFA) and NH3 productions were measured. Inoculum from MOS_1.0 rabbits showed the significant higher values of OMd (64.21%, P < 0.05), gas production (262.32 ml/g, P < 0.05), acetate (96.99 mmol/g OM, P < 0.05) and butyrate (26.21 mmol/g OM, P < 0.05) than the other groups. Slight differences were recorded among the groups ANT, MOS_0.5 and MOS_1.5. In addition, branched chain acids, in proportion to total VFAs, were significantly higher in MOS_1.0 inoculum (0.04, P < 0.05). MOS are able to affect fermentation activity of caecal micro-organism, but their activities seem not proportional to their level in the diet.

Injectable and in situ gelling hydrogels for modified protein release.

A novel injectable polysaccharide system based on calcium Alginate (Ca-Alg) hydrogel and two Dextran methacrylate derivatives (DexMA) was recently developed. The resulting Interpenetrating Polymer Network showed a synergistic mechanical behavior that can be exploited to target the hydrogel properties towards specific biomedical needs. In the present paper, hydrogels composed of 3% (w/v) Ca-Alg and Dextran (Mw 40 x 10(4) and 500 x 10(4)), derivatized with methacrylic groups (derivatization degrees 5 and 30%) at concentrations 5% (w/v), were characterized. The data reported here evidenced that Mw and derivatization degree of Dex chains can deeply affect the mechanical as well a model protein (Horseradish peroxidase) delivery rate. The enzymatic activity of such model protein was never significantly altered by the adopted experimental conditions.

Effect of diet on the metabolic profile of ostriches (Struthio camelus var. domesticus).

In order to study the metabolic profile of ostriches in relation to diet, 40 animals of both sexes were divided equally into two groups and fed two diets ad libitum consisting, on a dry matter basis, of the same commercial concentrate (60%) for the two groups and of corn silage (group A) or alfalfa hay (group B). In the morning, after about 12 h of fasting, blood was collected from the wing vein. The following haematological parameters were determined with an automatic system (Ektachem 250 analyser, Kodak): glucose, cholesterol, triglycerides, lactate (LAC), total protein (TP), uric acid, total bilirubin (Tbil), creatinine (CREA), calcium (Ca), magnesium (Mg), phosphorus (P), sodium (Na), potassium (K), chloride (Cl-), iron (Fe), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), cholinesterase (ChE), alpha-amylase (Amyl), lipase (LIP) and gamma-glutamyltrasferase (GGT). Diet significantly affected some parameters of the metabolic profile. Indeed, owing to the presence of alfalfa hay in the diet, group B showed, in comparison to group A, significantly higher values of uric acid (222.5 vs 387.5 mmol/L, p < 0.01), GGT (8.50 vs 11.3 U/L, p < 0.05), Tbil (8.50 vs 10.7 mmol/L, p < 0.05), Ca (2.41 vs 2.83 micromol/L, p < 0.01), Mg (1.01 vs 1.18 micromol/L, p < 0.05) and K (2.71 vs 3.16 micromol/L, p < 0.01). The levels of creatinine (27.3 vs 32.6 mmol/L, p < 0.05) and AST (344.9 vs 461.4 U/l, p < 0.01) were also higher for group B.

Use of faeces as an alternative inoculum to caecal content to study in vitro feed digestibility in domesticated ostriches (Struthio camelus var. domesticus).

1. In order to find an alternative source of inoculum to caecal content for studying the in vitro feed digestibility in domesticated ostriches (Struthio camelus var. domesticus), caecal content and faeces of 4 male birds were used as inocula for an in vitro gas production trial. 2. About 1 g of each of 5 substrates (maize silage, CS; alfalfa hay, AH; barley, BG; soybean meal, SM; beet pulp, BP) was weighed, in quadruplicate per inoculum, in 120 ml flasks; 75 ml of anaerobic medium and 4 ml of reducing solution were added and flasks were kept at 39 degrees C. Caecal content and faeces were diluted respectively 1 : 2 (CI) and 1 : 4 (FI) with an anaerobic medium and were injected into the respective flasks (10 ml). 3. Gas production was recorded 22 times up to 120 h of incubation and fermentation characteristics (for instance, degraded organic matter, OMd; potential gas production, A; maximum fermentation rate, Rmax; time at which it is reached, Tmax; pH; volatile fatty acid, VFA; ammonia) were studied for each inoculum and substrate. 4. CI and FI showed significant differences in Tmax (16.37 vs 18.47 h, respectively), propionic (16.47 vs 12.07 mmoles/l) and butyric acid (6.50 vs 7.98 mmoles/l) and ammonia concentration (17.18 vs 19.95 mmoles/l). The substrates, according to their chemical composition, showed different fermentation characteristics. However, the regression equations able to estimate some fermentation characteristics of the caecum from those of faeces were statistically significant and showed R2-values ranging from 0.87 to 0.99. 5. The differences in fermentation pathways of the two inocula did not appear to influence the rate and extent of OM digestion. Faecal fermentation predicted rates and extent of OM digestion by caecal fermentation in ostriches; consequently, the faeces could be considered as an alternative to caecal content to study feed digestibility in the species, although there is a need to undertake further research.

Reversible immobilization of engineered molecules by Ni-NTA chelators.

Electrochemical synthesis of nickel-nitrilotriacetic acid (Ni-NTA) chelators, for subsequent immobilization of (His)(6)-tagged proteins (Photosystem II (PSII) as model molecule), on Au or Au-graphite electrodes is compared to chemical synthesis. Results show: (i) higher Ni-NTA surface density, (ii) shorter treatment time (1-12 min vs. 16 h normally needed for self-assembled monolayer (SAM)), (iii) possibility of addressing the chelator to only one Au electrode, in a sensor micro-array.

Effect of collection rhythms and season on rabbit semen production.

The effects of collection regimen and time of year on rabbit semen production were determined in this study. A total of 14 crossbreed Hyla bucks were used in winter and summer. In each season, rabbits were assigned to two groups. In group 1, (n = 7) rabbits were subjected to an extensive collection regimen (two ejaculates per male, once daily/week) and in group 2, (n = 7) a semi-intensive semen collection regimen was performed (two ejaculates per male, twice weekly). The traits recorded for each sample were libido, volume, pH, motility, sperm concentration, percentage of alive spermatozoa and sperm abnormalities. The results obtained in this study indicate that when increasing collection frequency, the rate of useful collections decreased (from 0.81 +/- 0.017 to 0.69 +/- 0.016; p < 0.01). The rate of useful collection also decreased in the transition from winter to summer (from 0.79 +/- 0.018 to 0.70 +/- 0.017; p < 0.01). Among the ejaculate characteristics studied, only volume/ejaculate (from 0.64 +/- 0.015 to 0.53 +/- 0.017; p < 0.01) and spermatozoa/ml (from 406 +/- 15 to 359 +/- 13 million; p < 0.01) appeared negatively affected by collection. In winter fewer volume/ejaculates were produced (0.55 +/- 0.015 vs 0.60 +/- 0.016 ml; p < 0.01) and fewer spermatozoa/ml (360 +/- 14 vs 394 +/- 16 million; p < 0.01) than in summer. The doses produced per ejaculate decreased as collection frequency increased, but the number of doses produced per week was higher in the semi-intensive than the extensive rhythm (26.5 +/- 2.1 vs 20.9 +/- 1.5; p < 0.01). The results suggest that a semi-intensive rhythm may be viewed favourably.

Physical and chemical quality of ostrich eggs during the laying season.

1. Fifteen ostrich females were monitored for a full breeding season. At 30, 70, 110, 150 and 210 d from the beginning of laying, one egg per female was taken to the laboratory to assess its physical and nutritive characteristics. 2. The average length was 15.4 cm, while the width was 12.9 cm. Average shell thickness ranged from 2.20 mm at the equator to 2.24 mm at the small pole. The three principal components of the egg (albumen, yolk and shell) amounted to 57.1, 23.3 and 19.6%, respectively. None of the above variables, despite showing some significant differences between sampling periods, showed a clear trend linked to the laying season. 3. In contrast, the dry matter of the albumen showed a correlation with the sampling period, increasing from 11-1% in the first period to 12.0% in the last. The crude protein content ranged from 47.7 to 48.2%, the fat from 43.8 to 44.2% and the ash between 5.2 and 5.5% of dry matter. 4. The fatty acid composition of the yolk showed the presence of C18:1 (from 122.0 to 136.4 mg/g), followed by C16:0 (from 40.8 to 46.0 mg/g), C18:0 (from 19.2 to 21.2 mg/g) and C18:2 (from 17.1 to 19.6 mg/g). The content of cholesterol/g of yolk was between 10.6 and 10.9 mg. 5. In conclusion, compared with the hen's egg, the ostrich egg has similar chemical and nutritive characteristics, but a higher unsaturated/saturated fatty acid ratio and lower cholesterol content.

Chemical speciation drives hydrothermal vent ecology.

The physiology and biochemistry of many taxa inhabiting deep-sea hydrothermal vents have been elucidated; however, the physicochemical factors controlling the distribution of these organisms at a given vent site remain an enigma after 20 years of research. The chemical speciation of particular elements has been suggested as key to controlling biological community structure in these extreme aquatic environments. Implementation of electrochemical technology has allowed us to make in situ measurements of chemical speciation at vents located at the East Pacific Rise (9 degrees 50' N) and on a scale relevant to the biology. Here we report that significant differences in oxygen, iron and sulphur speciation strongly correlate with the distribution of specific taxa in different microhabitats. In higher temperature (> 30 degrees C) microhabitats, the appreciable formation of soluble iron-sulphide molecular clusters markedly reduces the availability of free H2S/HS- to vent (micro)organisms, thus controlling the available habitat.

Determination of apparent digestibility coefficients in 6-, 12- and 18-week-old ostriches.

1. The apparent digestibility coefficients (ADC) of a diet consisting of a concentrate and of alfalfa grass in a 4:1 ratio were measured in 6, 12-and 18-week-old ostriches. 2. These coefficients were measured in 4 animals eating ad libitum using both a total collection method, and internal marker methods with acid detergent lignin (ADL) and with acid insoluble ash (AIA). 3. The ADC of dry matter, organic matter, crude protein, ether extract and energy were not influenced by the age of the ostriches. The ADC of structural carbohydrates, however, were significantly higher (P<0.01) in the 12-and 18-week-old animals. 4. The results obtained by the total collection and ADL methods were similar. The AIA method, however, overestimated the values due to the very high marker recovery rate (128.0%).

Genetic variation among endosymbionts of widely distributed vestimentiferan tubeworms.

Vestimentiferan tubeworms thriving in sulfidic deep-sea hydrothermal vents and cold seeps are constrained by their nutritional reliance on chemoautotrophic endosymbionts. In a recent phylogenetic study using 16S ribosomal DNA, we found that endosymbionts from vent and seep habitats form two distinct clades with little variation within each clade. In the present study, we used two different approaches to assess the genetic variation among biogeographically distinct vestimentiferan symbionts. DNA sequences were obtained for the noncoding, internal transcribed spacer (ITS) regions of the rRNA operons of symbionts associated with six different genera of vestimentiferan tubeworms. ITS sequences from endosymbionts of host genera collected from different habitats and widely distributed vent sites were surprisingly conserved. Because the ITS region was not sufficient for distinguishing endosymbionts from different habitats or locations, we used a DNA fingerprinting technique, repetitive-extragenic-palindrome PCR (REP-PCR), to reveal differences in the distribution of repetitive sequences in the genomes of the bacterial endosymbionts. Most of the endosymbionts displayed unique REP-PCR patterns. A cladogram generated from these fingerprints reflected relationships that may be influenced by a variety of factors, including host genera, geographic location, and bottom type.