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Size and Appearance

Neon gobies are small and typical gobiioid in shape. Most Elacatinus spp. are less than 5cm (2 inches) total length. Neon gobies are black overall with a neon blue stripe extending from front of eye to the base of caudal fin.

Broodstock Care

Omnivorous and hardy they will do well in nearly any species-only or reef aquarium situation, but due to their size should not be kept with larger predatory fishes. Neon gobies do best in water temperatures below 26.5 decrees C (80 degrees F). Foods should include a variety of grated frozen shrimp, squid and fish, as well as commercial gelatin or pellet diets. Multiple feedings daily will condition neon gobies for spawning.

Pair Formation

Hermaphroditic sexual patterns are common in the family Gobiidae. I am not aware of a definitive classification of Elacatinus oceanops, but experience in our lab suggests that they are sequential hermaphrodites rather than simultaneous hermaphrodites.
Males are often larger and more slender. Females will possess a swollen abdomen, particularly when ripe with eggs.

Neon Gobies may be kept as groups of 6 or more individuals when provided with sufficient hiding spots, as these gobies can be quite quarrelsome. If they are to be kept as a pair, they should be observed closely during the first week after introduction. If fighting is excessive, one member of the pair should be swapped until marital harmony ensues. Groups of fewer than 6 individuals are not suggested, as pairs will begin to try to “evict” other gobies in their territory. Larger groups dampen and disperse these aggressive tendencies.

Spawning and Hatching

Spawning can occur as often as every fourteen days with plenty of feeding and warm water conditions. In their natural environment, demersal eggs are laid in small holes and crevices in the reef and under discarded bivalve shells. In captivity, small Tridacna sp shells serve well, as do halved clay flowerpots and short sections of half inch PVC pipe. Both parents tend eggs. Depending on temperature, hatching will commence in 6-8 days. Hatching occurs after dark.

Larval Rearing

Neon goby larvae are slightly shorter and substantially slimmer than clownfish larvae. The larval period ranges from 18-25 days depending on temperature and food type. The first diet is rotifers, followed by Artemia nauplii. The transition period is variable between these foods. Elacatinus larvae can be transitioned to Artemia as early as day 6, and while growth is more rapid, mortality is often high. Waiting until day 12-15 to begin Artemia feedings will delay metamorphosis by a few days, but will also significantly increase survivorship. We have successfully reared batches of neon goby hatchlings through metamorphosis only on rotifers, but metamorphosis took 30-35 days.

Below are descriptions of various methods used in aquaculture hatcheries, as well as an explanation of their typical applications, limitations, and in some cases, links to protocols for implementation.

STERILIZATION BY MEMBRANE FILTRATION:

Primarily utilized for small culture volumes, membrane filtration provides a high level of sterility while being extremely gentle to water chemistry. Sterilization is accomplished by forcing liquid through a filter that has a defined pore size, typically either 0.45 or 0.22 microns. This allows for the elimination of bacteria and fungi, which are too large to fit through the pores, without modification of the chemical constituents of the culture media. Effective for bacterial, fungal, and protozoan species, these filters are not generally effective against viruses as these are small enough to pass easily through the pores.

Membrane filters typically come in two flavors:
Syringe filters are handy for sterilizing very small volumes of liquid, less than 100ml. These filters come in convenient disposable cassettes that attach to the tip of syringes. Liquid is forced from the syringe through the filter and the exiting solution is sterilized.
Vacuum filtration setups are effective for larger volumes, up to a liter. These allow the culture media to be pulled through a larger filter membrane via a vacuum pump. The sterilized culture media is collected in a receiver vessel, often a side-arm Erlenmeyer flask. It is important to note that for either of these methods, all downstream vessels and apparatus that contact the sterilized media must be sterile themselves. Thus, it is common to use these methods only for making small stocks of solutions such as f/2 that are sensitive to other forms of sterilization, and may be stored in pre-sterilized, disposable containers.

HEAT STERILIZATION:

Heat sterilization, when properly performed, can be among the best methods of sterilization. However, many desirable constituents of culture media may be temperature sensitive and can be destroyed by heat. Most notable are vitamins, fertilizers, and antibiotic solutions, which are typically filter sterilized and added to heat sterilized media after it cools. In addition to being damaging to additives, high temperatures can cause undesirable precipitation of a variety of constituents of seawater, especially as temperatures approach boiling. Such precipitation may or may not have adverse effects on the culture, depending on the species and conditions that are utilized.

Autoclaving is the most common and effective method for sterilizing moderate amounts of material, especially in a laboratory setting. Requiring specialized equipment, material is heated under pressure in the presence of steam. Given an adequately long exposure time, this is an effective method of destroying bacteria, fungi, spores, and viruses. The most common exposure conditions are 121 degrees C at 15psi. Similar levels of sterility can be attained in a pressure cooker without the expense of an autoclave.
Boiling is a moderately effective method of sterilization. It does a good job of killing most bacteria, viruses, and fungi. However, it often is not successful at destroying the environmentally resistant spores produced by some species. To ensure complete killing of spores, it may be necessary to boil the medium on 2 or 3 consecutive days, allowing the medium to cool between treatments.
Pasteurization is not quite as assured a method for complete destruction of microorganisms as autoclaving, but properly executed can reach nearly the same kill rates without the problems of precipitation that may occur with boiling or autoclaving. Pasteurization can be accomplished by heating the solution to 80 degrees Celsius, allowing the solution to cool naturally, then heating again, generally 24 hours later. This may be repeated a third time for extra safety.

CHEMICAL STERILIZATION

The most economical and convenient method for sterilizing moderate to large volumes of water is chemical sterilization. This is most often accomplished through the addition of strong oxidizing agents such as chlorine, or by dropping the pH below 4 through the use of hydrochloric or muriatic acid. Highly effective, these methods are affected by factors such as temperature, contact time, dissolved organics etc., therefore, sterilization parameters will need to be adjusted in response to these conditions.
It is important to return the pH to normal before using the media if acid-sterilization was employed, and chlorine solutions must be neutralized, generally through additions of sodium thiosulfate. Chemical sterilization may destroy additives such as vitamins, antibiotics, and fertilizers, so sterile stocks of these should be added only after neutralization of the chlorine or acid.

**Be sure to familiarize yourself with proper chemical handling techniques before attempting these procedures. Always wear protective goggles and gloves when handling any chemicals**

1.  Collect a known quantity of water to be sterilized.  If water is turbid, it may be necessary to prefilter for clarity.  This can be accomplished by dripping the water through a few coffee filters.

2.  Add 0.5ml unscented laundry bleach per liter of water.

3.  Seal the vessel and swirl the media so that all internal surfaces are wet.

4.  Allow the vessel to sit at room temperature for at least 6 hours, preferably 12-24.  The vessel may be stored for long period of time with the bleach inside, provided the vessel remains uncontaminated.

5) Prepare a 1M stock solution of Sodium Thiosulfate.  The most commonly available crystalline form is the pentahydrate, and should be added at 248 grams per liter.  This solution may be filter sterilized or autoclaved to ensure sterility.

6.  To de-chlorinate the water, add 0.1ml of 1M sodium thiosulfate per liter of water.

7.  Aerate at least 2 hours.

8.  Confirm that no residual chlorine exists before using the medium.  Dip tests for pools work well, as do the DPD reagent available in convenient liquid or powder pillow-packets.

Acid Sterilization

**If you are not familiar with the safety hazards associated with handling concentrated acids, DO NOT attempt this method**

1. Collect a known quantity of water to be sterilized.  If water is turbid, it may be necessary to prefilter for clarity.  This can be accomplished by dripping the water through a few coffee filters.

2.  Add sufficient hydrochloric acid or muriatic acid to reduce the pH of the solution to 3.6.  The amount needed will vary depending on the concentration of the acid stock, the initial pH of the water, and its buffering capacity, which may also be affected by residual calcium deposits in the vessel.  A good starting point for muriatic acid (about 32% concentration) is 2ml/L.

3.  Confirm pH with a pH meter or narrow range test strips.

4.  Swirl the sterilized solution so that all internal surfaces are moistened.

5.  Allowing a sufficient sterilization time (2-4 hours).

6.  Return the pH to normal with sodium bicarbonate (baking soda).  A good starting point is 1/8 tsp/L.

6.  Confirm pH is appropriate for culture with a pH meter or narrow range test strips.

Probably their most distinctive feature, the feature from which they derive their name, is the appearance of “pom poms” held on a pair of their legs. The pom poms are actually a pair of anemones from the Genus Bunodeopsis. Pom Pom crabs use these stinging anemones as a form of defense, assuming a boxer’s posture in response to threats. Even more remarkable, as the crab molts, these anemones must be moved from the old exoskeleton and mounted to the new chelipeds! No wonder these are a favorite organism for reef aquarium owners.

As you can see clearly in the picture below, this specimen of Lybia tesselata is carrying a fine clutch of eggs along the pleopods. This picture was taken approximately 24 hours after the eggs were deposited. With such odd looking parents, we are all quite excited to start working with the larvae. Check back often, we plan to update with lots of pictures as the project progresses.

Let Nature Keep your Marine Aquarium Clean and Healthy.
If you have been looking to add some serious oomph to your clean-up crew, or give your sand bed or refugium some needed diversity, look no further. This is your opportunity to get some of the most beneficial organisms available for a marine tank. Best of all, these organisms are aquacultured in fish-free systems, so they will arrive in excellent health, accustomed to spending their whole life in aquariums, and most importantly, will be free of the parasites that wild-caught specimens may carry. And because they are aquacutured, no organisms will have been taken from the reefs. These organisms have been selected to fulfill overlapping and complementary cleaning duties that will leave your sandbed healthy and clean. In most healthy aquariums, they will continue to breed and will adjust their populations to the level of pollution in your tank. ALL Species Offered Here Are Reef Safe.

Potassium permanganate is a staple chemical in the aquaculture industry. It has been widely used as a method of removing oxygen-consuming dissolved organic chemicals from water, increasing oxidation-reduction potential, and controlling nuisance hair algae. It is also used as a dip for removing parasites from freshwater fish. We use it regularly for treating rapid tissue necrosis (RTN) in corals and preventing bacterial infections. It is also highly effective at removing other pests, and now it seems that it has potential for removing both the adults and egg masses of coral-eating aeolid nudibranchs.

Care should be taken whenever one is using potassium permanganate. An overdose in a reef tank will quickly kill every inhabitant. Treatment for ectoparasites, rapid tissue necrosis or aeolid nudibranchs should always be performed as a separate dip, never in a display aquarium.

Arizona Aquaculture Solutions produces our own line of aqueous potassium permanganate for home aquarists to use. Unlike the dry form of the chemical which must be shipped as a hazardous material, aqueous potassium permanganate can be shipped without difficulty.

Several genes and gene clusters have been identified in Vibrio cholerae that have aided in the understanding of how biofilms are formed and what environmental factors are determinants in their formation. The first gene involved is in the vps gene cluster. This gene cluster is involved in the creation of the VPS exopolysaccharide, a primary component in the biofilm matrix. The vps genes are clustered in two regions within the Vibrio cholerae genome, with vpsA-K in one region, and vpsL-q in another. Transposon inactivation mutants were screened, leading to the description of several of the genes in this cluster.

Although the function of vpsA and vpsL have not been fully described, mutations in these genes lead to a reversion from R-type colonies to L-type with low expression of VPS exopolysaccharide. . Several regulatory genes have also been described. VpsR is a positive regulator, and HapR and CytR are negative regulators. Recently, a second positive regulator has also been found, termed vpsT, it is a transcriptional activator that works in concert with vpsR to regulate each others expression, and both can also autoregulate their own expression. It is only with both of these genes activated that maximal biofilm is deposited. These regulatory genes show homology to typical two-component regulatory systems, although the sensor kinases have yet to be identified.

Utilizing a vpsL deletion mutant, it was also discovered that there are at least two pathways associated with biofilm production in Vibrio cholerae, and that they each respond to different environmental cues. These pathways were termed vps-dependent or vps-independent. The vps dependent pathway requires a nutrient rich media with the presence of pre-formed monosaccharides in the media. There is no calcium requirement for this pathway. The O-antigen is also required for vps-dependent formation. The vps independent pathway does not require the presence of monosaccharides in the media and can be expressed on minimal media, so long as milimolar amounts of calcium is present. It has been hypothesized that because Vibrio cholerae is associated with both marine and estuarine environments, different mechanisms are needed for survival in these environments.

THE VPS-DEPENDENT PATHWAY

The vps-dependent pathway is associated with nigh nutrient concentrations and is able to grow with low salinity and low calcium concentrations, as one would expect during rainfall and runoff entering a positive estuary. Nutrient concentrations are highly variable in this environment and the biofilm may even play a role in the accumulation and storage of nutrients for starvation conditions. On the other hand, the vps-independent pathway only has a requirement for calcium. This less selective pathway would be expressed in the marine environment, where vibrios are often found colonizing nutrient sources such as the chitinous exoskeletons of planktonic organisms.

An additional gene has been found that plays a role in producing the proper structures for biofilm development. It has been termed mbaA, for maintenance of biofilm architecture. In vps-dependent biofilm formation, the vps gene clusters are activated by the presence of a mannose-sensitive hemagglutinin type IV pilus (MSHA). Once initial attachment has occurred, the EPS is synthesized as the cells move via their flagella, and additional planktonic cells are recruited for settlement. The gene mbaA is not associated with these early stages of biofilm development. Instead, they appear to be regulators that control the amount of matrix EPS that is being produced in mature biofilms.

To find the mbaA gene, a mini-Tn10 mutant was isolated by researchers from a screen that produced abnormally robust biofilms. The insertion was determined to be at the 315th codon in a 2,376-bp open reading frame with no determined function. Sequence analysis indicated that this is likely a three-gene operon. Based on the identification of this mutant, an mbaA deletion mutant was created. These mutants showed extremely high levels of EPS being secreted, and none of the biofilms had the peaks, valleys, and channels associated with mature biofilms. Although the biofilms being formed were vigorous, when mbaA mutants were constructed with defects in either the MSHA or flagella genes, no biofilms were formed. This indicates that early requirements for biofilm formation are not circumvented by the mbaA mutation, and its presence occurs later in formation. Similarly, deletions in the vps gene clusters that are associated with defects in biofilm formation were not overcome by the mbaA deletion. It was also determined that the increased EPS production was not associated with either increased cell density or cell division, again supporting the hypothesis that mbaA is a regulator of EPS production.

SUMMARY

Based on these findings, it is apparent that biofilm formation is a critical factor in the environmental survival of Vibrio cholerae, and is likely a strong determinant of pathogenesis in many primarily marine pathogens, especially those associated with coral beaching, wound infections of fish, and secondary infections following parasite attack. The presence of two distinct and independently regulated pathways for the formation of these structures demonstrates their importance. With different structural types created in response to varying environmental conditions, these biofilms are able to survive starvation conditions, create ideal conditions for exchange of genetic information via horizontal gene transfer, and maximize survival when attached to the exoskeletons of planktonic crustaceans for long-term oceanic survival and dispersal. Similarly, survival within a biofilm while attached to the chitinous exoskeletons might facilitate transport and survival through the GI tract when ingested by larger organisms, allowing the possible infection of a new host.

In the next sections, we will look at how biofilms may aid in coral pathogenesis, aquaculture mortalities, and filtration systems.

In the first sections we looked at a survey of the attributes of biofilms as survival mechanisms and as generalized virulence attributes. In this section, we will be examining the molecular mechanisms that control biofilm formation, and the role of these mechanisms in the pathogenesis of Vibrio species bacteria. Vibrios are found in nearly every marine aquarium, and are opportunistic pathogens of many ornamental fishes. It is also becoming increasingly evident that Vibrios are responsible for a variety of coral diseases, and are also potential pathogens of human reefkeepers. As such, their effects in aquarium settings are being studied in great detail. Some bits of this section do tend to get a bit thick, but I will attempt to sum all of this up at the end.

The genus Vibrio is a diverse group of bacteria found in myriad microcosms within the marine environment. Vibrios have been associated with disease in intensive aquaculture systems, coral bleaching, they are even found in the light organs of marine fishes and are a resident of the teeth of the great white shark. The best-known member of this group is Vibrio cholerae, the causative agent of human cholera. This is a classic disease of mankind and continues to be a significant cause of morbidity and mortality in developing nations.

Unlike other pathogens that utilize biofilms to evade the host immune response, biofilms do not appear to play a role in the disease cholera. This infection is defined by toxin production and does generally have a carrier or chronic state. Instead, toxin production causes fluid production causing copious diarrhea and associated release of the bacteria back into the environment. As a self-limiting disease, cholera would have little use for biofilm genes in the host. However, these bacteria are strongly associated with biofilm development in the marine environment.

Vibrio cholerae can assume two forms when plated on laboratory media. The most common form is the smooth or luminescent (L) form. Occasionally colonies assume a wrinkled or rugose (R) form. It appears that the rugose form is actually a biofilm form. The smooth form is typically isolated from patients infected with Vibrio cholerae. When grown on media that stresses the bacteria, L colonies become R colonies at a high rate. When these R colonies are placed back on rich media, they revert to an L form at a rate of 1.5X10-5. When these bacteria were compared using fingerprint analysis, they were found to be genetically identical. This indicates that the bacteria are not distinct genotypes, but rather undergo a phase variation triggered by stress through an unidentified mechanism.