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SOJ Microbiology & Infectious Diseases
The amphizoic protozoa Acanthamoeba species are able to be free-living amoebae in environment or as parasites in humans and animals. These species comprise nearly 30 species and they have an increased role as human pathogens causing encephalitis in the nervous system or keratitis in the eyes. In diagnosis of a clinical sample of eye contact lens, light microscopy showed a heavy growth of amoeboid and Acanthamoeba trophozoites that occurred in agar plates and in cultivation flasks of patient sample and A. castellanii control as well. Fluorescence microscopy uncovered that visualised amoebae from patient and control samples emit fluorescence before- and after glutaraldehyde fixation. Polymerase Chain Reaction (PCR) and Gel electrophoresis identified that the isolated amoebic microorganisms were Acanthamoeba species. In conclusion, we have diagnosed a case of keratitis that caused by Acanthamoeba species by cultivating the eye contact lens sample using glutaraldehyde as fluorescent dye for fluorescence microscopy and PCR. Acanthamoeba trophozoites and cysts emit autofluorescence and glutaraldehyde can be used as fluorescent dye to enhance the autofluorescence of Acanthamoeba trophozoites and cysts for diagnosis purposes.
Keywords: Eye contact lens Acanthamoeba keratitis Fluorescence microscopy Glutaraldehyde as fluorescent dye Polymerase Chain Reaction Autofluorescence
Free-Living Amoebae (FLA) are environmental eukaryotes found worldwide in soil, air, and fresh or salt water. FLA comprise several genera such as Acanthamoeba, Balamuthia, Naegleria and Sappinia. The life cycle of Acanthamoeba has a reproductive trophozoite and a dormant cyst. The trophozoite has an oval to elongated shape with a diameter varies between 28 and 40 μm. The cyst has polygonal or star shape and measures between 15 to 28 μm in diameter. Naegleria species have an additional flagellate stage .
Acanthamoeba, Balamuthia, Naegleria and Sappinia are human pathogens causing infections in the central nervous system (CNS), eyes, lungs and skin [3,7,13,39]. Naegleria fowleri causes Primary Amoebic Meningoencephalitis (PAM), which is a fulminant, necrotizing and haemorrhagic meningoencephalitis and later leading to death [26,39].PAM is an acute infection and is generally fatal. Swimming in contaminated water exposes nasal mucosa to the organism, which can enter the CNS via the olfactory neuroepithelium and the cribriform plate. Most patients are healthy children or young adults .
Acanthamoeba and Balamuthia cause infections of the lungs and skin and Granulomatous Amoebic Encephalitis (GAE) . In contrast to PAM, GAE is a more subacute or chronic infection in immunocompromised or debilitated individuals of all ages [3,7,13,39,42]. The entry portal is thoughtfully to be the skin or lower respiratory tract, with subsequent haematogenous dissemination to the CNS .
Acanthamoeba Keratitis (AK) is the vision-threatening corneal disease that it was recognized at first time in 1973 in the United States . It is reported with an increased prevalence in different regions and countries year after year [9,16,23,24,34,35,38,44]. AK is usually an acute and progressive infection that becomes increasingly significant for human health worldwide . It is documented that keratitis has been led to blindness in 15% of untreated cases . The main risk factors that associated with AK are minor eye trauma and Contact Lens Wearing (CLW). Initially, patients suffer from severe ocular pain, photophobia, and a unilateral red eye .
Keratitis due to Acanthamoeba species is most commonly associated with poor contact lens hygiene . However, there have been reports showed that keratitis can be caused by Acanthamoeba species in the tropics in non-contact lens wearers [14,36]. Blindness of the affected eye is frequently related to significant diagnostic delay because patients are treated initially as viral, bacterial, or fungal keratitis .
Glutaraldehyde is a three carbon molecule terminated at both ends by aldehyde groups (HCO) covalent bonds with amine groups of any adjacent protein to build the cross-linking. The glutaraldehyde is an extremely efficient fixative to preserve cellular structure and the fixation by cross-linking is a method commonly used for light and electron microscopy .
However, the aldehyde groups of glutaraldehyde contain double bonds between carbon and oxygen atoms (O = C). Electrons of the double bonds will be excited when exposed to light during the fluorescence microscopy and returned very quickly to lower energy. This process results in emitting fluorescence, which is always occurred in the same frequency, and therefore, this is why it emits autofluorescence when it used as a fixative for fluorescent labeled microorganisms under fluorescence microscopy .
Doctoral thesis of the first author of this article uncovers that trophozoites as well as cysts of A. castellanii emit a detectable autofluorescence . This autofluorescence can be enhanced by glutaraldehyde fixation for Acanthamoeba detection purposes by fluorescence microscopy.
For diagnosis of Acanthamoeba in clinical sample of eye contact lens we used cultivation, glutaraldehyde as both fixative and fluorescent dye to visualize free living amoebae and Polymerase Chain Reaction (PCR) for identification of the isolated protozoa.
One milliliter cell suspensions from sample flask and A. castellanii positive control were centrifuged for 10 min at 300 x g. The pellets were suspended by 0.5 ml PBS and prepared to light and fluorescence microscopy for the demonstration of FLA cells.
One milliliter cell suspensions from sample flask and A. castellanii positive control were centrifuged for 10 min at 300 x g. The pellets were fixed with one milliliter 2.5% glutaraldehyde and examined by the fluorescence microscopy for the demonstration of fluorescent protozoa cells.
Two milliliters cell suspensions from sample flask and A. castellanii positive control were diluted in 8 ml of PBS, and centrifuged for 10 min at 300 g. The pellet were suspended in 2ml of PBS solution, DNA extracted using the Qiagen DNA mini kit (Qiagen, Hilden,Germany).
DNA was amplified utilizing two primers sets that were forward primer 5’- GGC CCA GAT CGT TTA CCG TGA A-3’ and reverse primer 5’-TCT CAC AAG CTG CTA GGG GAGTCA-3’as previously used by Pasricha, et al. . The amplification reaction was carried out in a final volume of 20 μl containing each primer at a concentration of 0.3 μM, 1.0× PCR golden buffer, 200 μM deoxyribonucleoside triphospate, 1.2 mM Mgcl2, 1.25 U/50 μl of Ampli Taq Gold (Sigma). The reaction was completed after 32 cycles of 95°C denaturation for 4 min, 55°C annealing for 20 sec, and 72°C for 10 sec extension. Final reaction products were analyzed by electrophoresis on agarose gel in 1× TBE buffer (Tri base, boric acid and EDTA (pH 8.0). The gel was stained in 0.1% SYBR Green bath, visualized by UV transillumination, and photographed using Polaroid films. DNA fragment 487 bp for Acanthamoeba was obtained as a final step.
Acanthamoeba keratitis (AK) was reported in 1973 in the United States . The keratitis is a disease in which amoebae invade the cornea of the eye and its causative agent Acanthamoeba species became well recognized as human pathogen that can lead to blindness if it is not treated . The reported first case of blindness due to Acanthamoeba was from Sudan and increasing of AK is observed in different regions and countries year after year [9,17,16,23,24,34,35,38,44].
Eye contact lens wearers typically seek medical help late, because they are used to minor irritations in the eye . In cases of severe infection, amoeba density is sometimes very high and the amoebae can already be detected by direct microscopy of the clinical samples without enrichment but by utilizing several staining methods such as methylene blue and Giemsa that enhance cellular intensity and differentiate between trophozoites and cysts of protozoa [22,38].
However, molecular biology methods such as mitochondrial DNA Restriction Fragment Length Polymorphism and zymodeme pattern analysis (isoenzyme electrophoresis) used in laboratory diagnosis of Acanthamoeba infection but these were not always reproducible methods [10,11,45].
The direct detection of the causative agent in a corneal scrape specimen is only the reliable diagnostic method for AK and the cultivation method remains the gold standard of Acanthamoeba laboratory diagnosis . However, if patients have already been pre-treated with antibiotics, the amoeba density is usually very low. Moreover, amoebae exhibit altered morphologies in these cases, even culture often remains negative and molecular techniques are indispensable. Reliable identification below the genus level requires genotyping . It is claimed that the most accepted technique for the diagnosis of AK, a part from the analysis of biopsies, is the use of in vivo confocal microscopy. It is a non-invasive tool with high sensitivity in cases of severe infection. But diagnosis confirmation requires other laboratory tests, mainly the cultivation of Acanthamoeba from corneal biopsy or the lens cases or contact lenses from the patient, as well as PCR or immunofluorescence assays .
In diagnosis of AK, Wilhelmus, et al. 1986 used Calco Flour White (CFW) staining and demonstrated amoebic cysts in corneal scrapings and keratectomy specimens from four patients with culture-proved Acanthamoeba keratitis and from one in which CFW was the only positive laboratory test. However, the CFW is a chemofluorescent dye with an affinity for the polysaccharide polymers of amoebic cysts .
It is known that some unicellular organisms such as amoebae, free living amoebae and giardia species are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. The chitin is the carbohydrate polymer conveying the required structural toughness to the cyst wall. But microorganisms of Acanthamoeba genus are exceptions, as their endocysts are made of cellulose compared to the ecto cyst that composed of proteins, lipids, and putative carbohydrate components as lectin binding sites [5,6,29,41].The cellulose is a linear polymer of anhydrous glucose units. The hydroxyl groups of glucose units could be the coupling sites of the fluorescence dyes .
However, both polymers of chitin and cellulose form very similar crystalline macroscopic structures . Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1, 4-linked hexose backbones [21,12].
Chitin is an essential component of the cell walls and septa of all pathogenic fungi, and occurs in the cyst walls of pathogenic amoebae, the egg-shells and gut lining of parasitic nematodes and the exoskeletons of invertebrate vectors of human disease including mosquitoes, sand flies, ticks and snails . While CFW is a special fluorescent stain that binds strongly to structures containing cellulose and chitin and fungiflora Y stain binds both fungi and acanthamoebae [15,25,27,43,18] thus, the CFW and fungiflora Y stain are not specific dye for either Acanthamoebae or fungi.
Our current study utilized different methods such as amoeba cultivation, fluorescence microscopy and molecular methods to identify the causative agent of keratitis. These methods found that the causative agent was an amoeba belonged to Acanthamoeba genus and the identified Acanthamoeba found to emit autofluorescence. This finding was consistent with previous study by Abd, 2006 who found that fluorescence microscopy and flow cytometry analysis of A. castellanii cells population by FACSort, Becton Dickinson Immuno Systems, San Jose, CA, uncoverd that unlabeled A. castellanii cells adjusted for forward scatter (relative size) and side scatter FL1 (green colour) emitted detectable fluorescence, as in (Figure 2C and Figure 5).
Also our finding of the autofluorescence emitted by Acanthamoeba cells was consistent with findings of other researcher who found that chitin, collagen and elastin in animal cells or cellulose in plants and fungi emit autofluorescence when excited by ultraviolet, violet, or blue light under 400-520 nm utilizing commercial fluorescent dyes [2,31].
However, the commercial diagnostic fluorescent dyes such as calcoflour white stain and fungiflora Y stain include fluorescence molecules added to their structures and these molecules generate the fluorescence to differ from glutaraldehyde [15,18,25,27,43]. The glutaraldehyde molecule structure has electrons of the double bonds that will be excited when exposed to light and emit autofluorescence when returned very quickly to lower energy. Therefore glutaraldehyde is a fluorescent dye and a fixative too according to its chemical structure [40,19].
Utility of Acanthamoeba autofluorescence and glutaraldehyde enhanced fluorescence as a new method of diagnosis of Acanthamoeba trophozoites and cysts together with cultivation and molecular biology methods.
Emitting of green, red and blue autofluorescence uncovers new biological characteristics of Acanthamoeba.
Glutaraldehyde can be used as fixative and fluorescent dye for better visualization of protozoa and fungi.
Between 10 and 15 percent of all cases of prion disease are caused by mutations in the PRNP gene. Because they can run in families, these forms of prion disease are classified as familial. Familial prion diseases, which have overlapping signs and symptoms, include familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI).
The PRNP gene provides instructions for making a protein called prion protein (PrP). Although the precise function of this protein is unknown, researchers have proposed roles in several important processes. These include the transport of copper into cells, protection of brain cells (neurons) from injury (neuroprotection), and communication between neurons. In familial forms of prion disease, PRNP gene mutations result in the production of an abnormally shaped protein , known as PrP Sc , from one copy of the gene. In a process that is not fully understood, PrP Sc can attach (bind) to the normal protein (PrP C ) and promote its transformation into PrP Sc . The abnormal protein builds up in the brain, forming clumps that damage or destroy neurons. The loss of these cells creates microscopic sponge-like holes (vacuoles) in the brain, which leads to the signs and symptoms of prion disease.
The other 85 to 90 percent of cases of prion disease are classified as either sporadic or acquired. People with sporadic prion disease have no family history of the disease and no identified mutation in the PRNP gene. Sporadic disease occurs when PrP C spontaneously, and for unknown reasons, is transformed into PrP Sc . Sporadic forms of prion disease include sporadic Creutzfeldt-Jakob disease (sCJD), sporadic fatal insomnia (sFI), and variably protease-sensitive prionopathy (VPSPr).
Acquired prion disease results from exposure to PrP Sc from an outside source. For example, variant Creutzfeldt-Jakob disease (vCJD) is a type of acquired prion disease in humans that results from eating beef products containing PrP Sc from cattle with prion disease. In cows, this form of the disease is known as bovine spongiform encephalopathy (BSE) or, more commonly, "mad cow disease." Another example of an acquired human prion disease is kuru, which was identified in the South Fore population in Papua New Guinea. The disorder was transmitted when individuals ate affected human tissue during cannibalistic funeral rituals.
Rarely, prion disease can be transmitted by accidental exposure to PrP Sc -contaminated tissues during a medical procedure. This type of prion disease, which accounts for 1 to 2 percent of all cases, is classified as iatrogenic.
Learn more about the gene associated with Prion disease
Unicellular Eukaryotes as Models in Cell and Molecular Biology
3.5 Free-living forms as models for pathogenic forms
The cell membrane of Alveolata is covered with densely packed variant surface antigens (vsAG). This holds for free-living and pathogenic ciliates (the fish pathogen Ichthyophthirius ), Apicomplexa as well as for pathogenic flagellates. Apicomplexa encompass severe pathogens, such as Toxoplasma (potentially teratogenic) and Plasmodium (the malaria causing agent). Leishmania and Trypanosoma are examples of pathogenic flagellate genera causing different types of leishmaniosis and of trypanosomiasis, respectively, such as African sleeping disease (T. brucei) and Chagas disease (T. cruzi). All these protozoa can spontaneously and rapidly shed their vsAGs so that, in the case of parasites, the host's immune system cannot produce antibodies rapidly enough to cope with this molecular camouflage. The vsAG systems of nearly all pathogens and also of free-living ciliates and of fungi have in common that only one vsAG is expressed at a time (called the serotype) whereas other members of the multigene family are silent ( Deitsch et al., 2009 ). Since Apicomplexa are close relatives of ciliates, their vsAG proteins and their expression mechanisms reveal several similarities which legitimate a comparison of the antigenic systems between free-living and pathogenic forms ( Simon and Schmidt, 2007 ).
In many cases, surface proteins are attached to the cell surface by a glycosylphosphatidyl-inositol (GPI) anchor, from protozoa and yeast up to flowering plants and man ( Eisenhaber et al., 2003 Ferguson, 1999 Orlean and Menon, 2007 ). In Paramecium, the switch to a newly expressed vsAG requires removal of the old one which is cleaved off by a GPI-specific phospholipase C (PLC) and subsequently released into the medium, as demonstrated in Fig. 3.6 ( Klöppel et al., 2009 Müller et al., 2012 ). Therefore, expression of a pure new serotype-specific vsAG requires not only a complex mechanism of exclusive gene expression, as discussed below in Section 4.2.3 , but also an active release mechanisms and, to start with, a complex mechanism for surface attachment. Such release mechanisms by cleavage of GPI-anchors are not understood in depth in any of the different organisms. This is surprising especially since similar mechanisms have to be postulated even for mammals ( Simon and Kusch, 2013 ).
Figure 3.6 . Immunolocalization of variable surface antigens on P. tetraurelia cells. (A) Shows a cell expressing pure serotype 51A. Specific antibodies indicate the presence of the antigen on cilia (ci) and on the cortex (cx), that is, the nonciliary cell membrane. (B–D) A switch from serotype 51A (green fluorescence) to 51D (red fluorescence) has been induced via RNAi by experimental suppression of the old antigen, 51A. The new surface antigen can first be detected on the cortex membrane whereas the “old” antigen remains on the cilia, as shown in (B and C). In a late stage of serotype switching (D), some residual 51A antigen is detectable only on the tips of some cilia (lower half of the cell), whereas the upper part displays the new serotype (red) on cilia and the nonciliary cell membrane (cortex). Magnification 800 ×.
Figures (B–D) are from Simon et al. (2006).
It now appears of paramount importance to scrutinize the molecular background of all regulation steps involved in vsAG expression in free-living unicellular model systems. Here, its analysis is much easier to achieve than in parasites or in multicellular organisms.
Michael D. Breed , Janice Moore , in Animal Behavior (Second Edition) , 2016
11.2 Evolution of Sex: Why Some Animals Are Called Male and Others Female
Biologists define maleness and femaleness by the relative size of the gametes that individuals produce. Species in which all gametes are the same size are termed isogamous and lack identifiable males and females. Isogamy is common in algae and protists, but virtually all animal species are anisogamous, producing small motile gametes, or sperm, and large gametes, or eggs. By convention, organisms producing large, nutrient-rich gametes are termed female, and organisms producing small, motile gametes are termed male. The isogamous nature of many unicellular organisms leads to the inference that isogamy may be the ancestral state for gametes. One look at an ostrich egg or a brooding octopus reveals that much has changed since that time. What caused this change? What caused the evolution of male and female?
By definition, a male produces small, motile gametes called sperm. Males produce large numbers of these gametes, with small investment in each.
By definition, a female produces large, relatively non-motile gametes called eggs. Females produce small numbers of these gametes, with large investment in each.
Isogamous species produce gametes that are all the same size. There is no differentiation of male and female. Anisogamous species produce gametes of two distinct sizes these species have males and females.
As is the case in all evolution, variation played a significant role in the evolution of gametes. Gametes that were slightly smaller than average were more nimble and faster than other gametes they could travel quickly to other gametes and fuse to produce diploid zygotes. In the world of motile gametes, if everything else were equal, small would often win the race to fertilization. True, the small gametes had reduced nutrient content for the zygote, but that could be balanced by fertilizing larger, nutrient-rich gametes. Meanwhile, large, nutrient-rich gametes also could influence the development of the zygote and could exert control over the fate of the offspring. Intermediate-sized gametes were neither nutrient-rich, nor were they particularly speedy. Thus, natural selection on gametes favored the extremes—small and large. This sort of selection, called disruptive selection, is thought to be the selective force that resulted in the sexes ( Figure 11.3 ).
Figure 11.3 . This diagram shows the effects of disruptive, stabilizing, and directional selection. The x-axis represents a measure of the trait—in this case, gamete size. The blue curve shows the distribution of the trait before selection the red curve shows the distribution of the trait after selection has had an effect. Under disruptive selection, animals producing either large or small gametes are favored by selection, while animals producing middle-sized gametes are selected against. Stabilizing selection reduces the variation in a trait by favoring a narrow range of phenotypes. Directional selection shifts the phenotype of the population in one direction.
Some animals are capable of producing both eggs and sperm these are hermaphrodites. Hermaphroditism can be further subdivided into animals such as some sea slugs that are simultaneously male and female (simultaneous hermaphrodites) and others that are first one sex and then the other (sequential hermaphrodites). For example, clownfish are males early in adult development and become females as they grow larger this is called protandrous, or male-first, hermaphroditism. In contrast, wrasses are females when smaller and become males as they grow larger this is called protogynous, or female-first, hermaphroditism.
A hermaphrodite is an animal that has both male and female reproductive organs and produces both eggs and sperm.
Protandrous hermaphrodites are male early in their lives and females later. Protogynous hermaphrodites are female early in their lives and males later.
Why does sex exist? Superficially, it may seem obvious that organisms should reproduce sexually, but sex carries a high evolutionary cost. Recall freshman biology lessons about diploid reproductive cells becoming haploid through a special cell division process called meiosis in other words, each time meiosis occurs, half the genome is “lost.” This is called the cost of meiosis. As a result, an asexually reproducing animal is able to pass along all of its genes to each of its offspring, but animals participating in sexual reproduction pass only half of their genes to each offspring. In addition, there is a cost associated with producing male and female offspring (both are necessary in sexually reproducing species), instead of putting all reproductive effort into asexually reproducing female offspring. Given this reasoning, why does evolution so often fail to favor organisms that transmit 100% of their genes to each offspring—that is, organisms that asexually reproduce? What forms of counterbalancing selection could favor sexual reproduction?
The cost of meiosis is the loss of half an animal’s genetic material when it produces haploid gametes.
Asexual reproduction is the production of offspring that are genetically identical to a single parent.
Sexual reproduction is the production of offspring by the combination of gametes from two parents.
In other words, why be sexual? The major argument for evolution and maintenance of sex is that the selective advantages from genetic recombination and genetic diversity among the offspring outweigh the costly nature of sex. In addition, deleterious genes do not accumulate in a sexual lineage. Indeed, this genetic diversification is so beneficial that it occurs in many organisms that do not have “male” and “female” forms, such as mating strains of protists. In a strict sense, then, sex refers to the mixing of genetic material, either within an organism (e.g., recombination) or between organisms. It is this mixing that proves to be highly advantageous. Gorelick and Heng 5 argue that the main functions of sexual reproduction are essentially genetic editing. Meiosis eliminates harmful changes and mutations and allows the repair of damage to DNA. Meiosis also fosters resetting accumulated epigenetic signals that limit gene expression. In Gorelick and Heng’s analysis, sexual reproduction through meiosis may actually reduce genetic variation by eliminating major chromosomal changes while maintaining small mutations. In either case, the recombinatorial phase of meiosis reshuffles genetic combinations, with the potential of producing offspring that are either more poorly or better suited for the environment.
Sexual reproduction is a fact of life for birds and mammals, which, with one or two rare exceptions, reproduce sexually. Hypothetically sexual reproduction is the ancestral state in vertebrates. There are nonetheless numerous examples of asexual reproduction in fish and a few examples in amphibia and reptiles.
Asexual reproduction is likely to evolve in environments that change little from generation to generation, because genetic recombination is not an advantage in predictably constant environments. Sometimes asexual reproduction crops up in invasive or pioneering species, in which single individuals may migrate to a suitable habitat if no potential mates are present, then asexual reproduction is obviously the only way to produce offspring, and selection will favor it. (Indeed, unless it arrived with fertilized gametes, a solitary invader that was incapable of asexual reproduction would leave few traces and no descendants.) It is possible that due to physiological and/or neurobiological constraints that tightly link reproductive physiology to survival, birds and mammals may have lost the ability to evolve to asexual reproduction.
Genetic diversity results from different offspring having different genetic combinations. Each egg from a female is genetically unique, and each sperm from a male is genetically unique. Thus, when a male and female mate, if they have multiple offspring, their offspring will differ genetically in other words, their offspring are genetically diverse.
Basal taxa are types of organisms that arose earlier in evolution. We use this term to avoid using the misleading words primitive or lower when describing animals.
Among invertebrates asexual reproduction is more common. In some invertebrate taxa there appears to be considerably more flexibility in terms of evolutionary switching between sexual and asexual patterns of reproduction. Given the ubiquity of sexual reproduction in animals and the fact that the more basal animal taxa reproduce sexually, the hypothesis that sexual reproduction is basal for animals as a whole is reasonable. Sea anemones produce dispersing offspring sexually, but when one of those offspring arrives on a rock and develops into an anemone, it can also reproduce by budding, producing clones of itself. This alternation of sexual and asexual reproduction allows a species to take advantage of maintaining genetic combinations in stable environments through asexual reproduction, while using sexual reproduction for recombination and offspring diversity when colonizing new habitats ( Figure 11.4 ). Similarly, in parts of their life cycles, many aphid species reproduce asexually ( Figure 11.5 ). In these organisms, sex seems to be facultative that is, it may or may not occur, depending on environmental stability. Stable environments favor asexuality.
Figure 11.4 . The effects of habitat stability on the evolution of reproduction. Stable habitats may favor asexual reproduction, while unstable (or unpredictable) habitats may favor sexual reproduction.
Figure 11.5 . Aphids such as these may reproduce asexually, resulting in clones of the insect.
Having established that sexual reproduction is common, and probably basal, in animals, it should be clear that the evolution of mating systems probably began before the evolutionary origin of animals, among their protistan predecessors in which proto-sperm swam in the ocean searching for proto-eggs to fertilize. This means that the use of chemical cues to find mates and competition among sperm are ancient behavioral features.
An egg is a gamete that is large and relatively immobile.
A sperm is a small and relatively mobile gamete.
Much of the theory that underlies the understanding of mating systems derives from the fact an egg is more costly to produce than a sperm. This means that females produce a few individually expensive gametes, while males can produce a huge number of relatively cheap gametes. Theoretically, then, eggs are valuable and worth protecting, while sperm are much less valuable. Another way of looking at this is to contemplate the numerical disparity between eggs and sperm: because fewer eggs are made than sperm, many, perhaps most, eggs should be fertilized, while many sperm will never fertilize an egg. The result of this observation is the evolutionary argument that, in general, females should protect their investment in eggs by caring for them, while males should be less likely to care for either their sperm or for their offspring. This is the evolutionary argument for the root of differences between the sexes. Laying aside sociocultural arguments about sex and gender roles in humans (recall the discussion of nature and nurture in Chapter 3 ), this characterization is a good starting point in exploring the evolution of mating systems across animal taxa.
These concepts lead to the often repeated prediction that females should be very choosy about who fertilizes their gametes, whereas males should be relatively indiscriminate in their mating. But is this really always true? Males that invest substantially in offspring care may also exert careful choice of mates. Either sex may have secondary sexual characteristics such as ornamentation or weaponry that exists for combat or territoriality. These characteristics may distract from parental care because of the hormonal state that underlies combat and territoriality does not prime the animal for parental care.
This is an important point that will form the core for a large part of the discussion in this chapter. Indeed, when these predictions are not met, there are frequently other forces at work, such as circumstances that limit male options for mating with limited mating options, the number of potential matings for the males is more or less equal to the number of potential matings for females, and male choosiness about mates should equal that of females. Males that invest more than females do in offspring are often more choosy than females ( Figure 11.6 ).
Figure 11.6 . Female mormon crickets eat spermatophores such as this one. Spermatophores are sperm containers that males of many arthropod species produce in the case of the mormon cricket, the spermatophore is nutrient-rich and left with the female after mating. Because the mormon cricket spermatophore is costly to make, a male carefully chooses his mate.
Photos: Darryl Gwynne (left) and Michael Breed (right).
Of Special Interest: The Red Queen and the Evolution of Sex
How can sex as a reproductive mechanism be explained in the face of the cost of meiosis? The key may lie in the evolutionary arms race between species and their diseases and parasites. Sex provides new genetic combinations to fight disease, but does a species ever truly get ahead of its diseases and parasites?
In Lewis Carroll’s Through the Looking Glass, Alice finds herself in the company of the Red Queen ( Figure 11.7 ). The charming story of Alice climbing a hill with the Red Queen is accompanied by the Red Queen’s confusing explanation of how hills can become valleys can become hills. In the same conversation Alice learns that you have to run faster and faster just to keep up with the competition:
Alice didn’t dare to argue the point, but went on: “And I thought I’d try and find my way to the top of that hill — ”
“When you say ‘hill,’” the Queen interrupted, “I could show you hills, in comparison with which you’d call that a valley.”
“Well, in our country,” said Alice, still panting a little, “you’d generally get to somewhere else—if you ran very fast for a long time, as we’ve been doing.”
“A slow sort of country!” said the Queen. “Now here, you see, it takes all the running you can do, to keep in the same place. If you want to get somewhere else, you must run at least twice as fast as that!”
Figure 11.7 . The Red Queen lectures Alice about the difficulties of running as fast as you can, only to realize that you are not really moving forward relative to your surroundings.
Leigh van Valen introduced the Red Queen to evolutionary biology, pointing out that a race which becomes faster and faster without a sign of a winner is a perfect analogy for coevolutionary arms races ( Figure 11.8 ). 6 In such coevolutionary races, for example, a prey species evolves a defense, the predator evolves to defeat the defense, the predator evolves another defense, and on and on…. Moving fast seems to never do more than get you back to the starting point, but stopping during an evolutionary arms race leads to extinction. The same principle can apply to the evolution of sex.
Figure 11.8 . The steps involved in an evolutionary arms race. Each time a species evolves an innovation (step 1) the other species evolves a counter-innovation. The first species evolves a counter-counter-innovation. This continues over evolutionary time repeating itself so that neither species ever truly wins the race. In this chapter the red queen hypothesis applies to the evolution of sex, but it can also apply to predator–prey coevolution.
Many evolutionary biologists see the Red Queen’s tale as relevant to the evolution of sex, explaining that the value of sexual reproduction fluctuates over time genetic recombination gained from sexual reproduction produces new defenses against diseases and parasites. When disease and parasite pressure are low, the cost of meiosis and the benefit of proven genetic combinations favor asexual reproduction, but when diseases and parasites surge, sexual reproduction is favored. Thus, the race goes up and down hill, and the fast-moving species are again condemned to run in the same place.
Why are sex ratios often 1:1 in populations? Evolutionary theory developed by R. A. Fisher 7 and expanded by R. L. Trivers 8 states that within a population the investment in male and female gametes should be equal. This theory concerns sex ratios in populations and applies to both parental investment in caring for offspring in species that do so and to parental investment in sperm and eggs alone for species that provide no additional care. At the population level, equal time and energy should be invested in male and female offspring, but the number of male and female offspring may differ. The amount of investment in males and females at the population level is what matters. Because this equality is at the population level, rather than the individual level, individuals may produce male- or female-biased broods, as long as across the population, the ratio is equal. As Fisher explained, if the ratio of males to females deviates very much from 1:1, the rarer sex becomes more valuable, and selection favors individual parents increasing production of the rarer sex. This causes an evolutionary balancing effect which maintains sex ratio to 1:1.
Equal investment in males and females is based on the assumption of equal genetic relationship of the parent to its male and female offspring. Nature does not always honor this assumption. There are at least three major exceptions to this condition of equal genetic relationship:
In ants, bees, and wasps, females are more related to their sisters than to their daughters ( Chapters 13 and 14 Chapter 13 Chapter 14 ). This disparity in genetic relationship leads to possible asymmetries in investment in males and females.
Local mate competition happens when brothers end up competing with each other to mate. In this case theory predicts that the population sex ratio should be strongly female biased, so that only enough males are produced to ensure that every female mates.
Local resource competition occurs when females remain near their birth site and males disperse. The females end up competing with sisters for resources, while males probably do not compete with their brothers. This results in male-biased sex ratios.
Prevalence and Risk Factors Associated with Sexually Transmitted Diseases (STDs) in Sikkim
The population of Sikkim is a unique blend of multi-tribal and metropolitan culture. However, till date, no data regarding prevalence of sexually transmitted diseases (henceforth abbreviated as STDs) among this population is available and hence requires attention. Hence the objective is to determine the prevalence of STDs in Sikkim and to describe associated risk factors. A cross-sectional study involving ‘Questionnaire-based anonymous feedback system’ was followed to collect data from 2,000 individuals across the society. The four most common STDs, gonorrhea, syphilis, chlamydia and HIV, were considered for the study. Total 69 (3.6 %) cases of STDs were found in 1,918 individuals was affected by at least one of the STDs, out of which 43 were males and 26 were females. Cases of gonorrhea, syphilis, chlamydiasis and HIV were 25, 22, 4 and 18 respectively. Out of total 69 cases of STDs, 20 individuals were also suffering from some kind of hepatitis. Addictions like alcoholism, smoking and drugs were also found in significant number, with 1,019 (>50 %) individuals with at least one of these addictions. Relative risk analysis indicates that gender-wise females are more vulnerable to STDs than males. The number of partners, addictions, especially alcohol and drug abuse, also contribute to STD cases. STDs act as a significant risk factor in transmitting some of the types of hepatitis. In such cases, females are more vulnerable than males. The results suggest that new community health programs are essential for both, HIV and non-HIV STDs in Sikkim.
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Impact of selected Infectious diseases on reproductive - PowerPoint PPT Presentation
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Although genes and hormones are the most well characterized mediators of sex differences in immune responses, environmental factors can also modulate the functioning of the immune system differentially between males and females.
Nutrition. The nutritional environment of the fetus can have differential effects depending on its sex. Maternal micronutrient supplementation during pregnancy in a Gambian placebo-controlled study reported sex differences in CpG methylation of genes involved in immunity and defence against infection (for example, genes encoding CD4, defensins and genes associated with IFN signalling), and female fetuses were most affected in the supplemented group whereas males were most affected in the non-supplemented group 97 . The study demonstrates that sex-differential developmental trajectories commence in utero and persist to 9 months of age, indicating long-term epigenetic reprogramming in relation to nutrition during pregnancy. A high-fat diet also enhances, whereas prenatal exposure to famine reduces, placental gene expression and DNA hypomethylation to a greater extent in female than in male fetuses 98 . Several studies also suggest that the immunomodulatory effects of breast milk may benefit infant females more than males, with breastfeeding reducing the risk of neonatal respiratory tract infection in female but not male infants 99 .
There is accumulating evidence that micronutrients act differently in males and females. Perinatal and postnatal vitamin B, vitamin C and vitamin E supplements are associated with a 32% reduction in mortality among females but not males in a randomized placebo-controlled trial of Tanzanian mothers infected with HIV 100 . Studies conducted in African and Asian infants suggest that females may benefit more than males from maternal micronutrient supplements 101,102 . Vitamin A supplementation (VAS), given with measles vaccination to children between 6 and 23 months of age, have sex differential immunomodulatory effects compared to a placebo, including decreased leukocyte subsets in males, and increased numbers of leukocytes and IFNγ production by ex vivo stimulated cells from females 103 .
Microbiota. A perturbed microbiome — referred to as dysbiosis — contributes to various disease processes including inflammation and diabetes. Sex influences the host microbiome outside of the reproductive tract, which probably involves sex steroid hormones 104,105 . During early life, sex does not influence the microbiome composition. Deep sequencing of colonic contents in pre-pubescent mice report no sex difference in bacterial community composition, suggesting that sex does not influence the microbiome in this age group 106 . A number of mouse studies, however, show sex differences in host gene expression in the gastrointestinal tract before puberty, demonstrating that sex-specific gene regulation occurs even in the absence of high levels of circulating sex hormones 106 . After puberty, female rodents have lower frequencies of Bacteroidetes than males 104,105 .
In a mouse model of spontaneous type 1 diabetes, adoptive transfer of gut commensals from male mice into females resulted in systemic hormonal changes and protected against disease 104,105 . Similar to what is seen in mice, the human female microbiome is less abundant in Bacteroidetes spp. than males 107 . A study specifically analysing for a sex–diet interaction in diverse vertebrate species including fish, mice, and humans confirmed that diet has sex-specific effects on the gut microbiome in two species of fish, affects Fusobacteria spp. levels in humans, but does not seem to affect the microbiome in laboratory mice 108 . The lack of effect of diet on sex difference in the gut microbiome in laboratory mice may reflect the highly simplified diets they are fed and the artificial environment in which they are maintained 109 .Whether sex differences in the effects of diet on the gut microbiome in humans contributes to sex differences in diseases associated with dysbiosis, such as inflammatory bowel disease requires consideration. These data also imply that therapeutic approaches to treat diseases associated with dysbiosis may need to be different for males and females.
15.25: Fungal and Protozoan Diseases of the Reproductive System - Biology
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Morone genus Morone, hybrids [Moronidae] FAO Names: En - Striped bass, hybrid, Fr - Bar d'Amérique, hybride, Es - Lubina estriada, híbrida Biological features
The fishes that make up the hybrids of the genus Morone are all within the Moronidae family, which is a small group of freshwater (white bass and yellow bass) and anadromous estuarine (striped bass and white perch) and marine percoids (striped bass) found naturally from the Mississippi River drainage system to the East Coast of the United States and Canada. Also included in this family are the European-North African species of Dicentrarchus represented by two species (D. labrax and D. punctatus). There is only one reported case of intentional congeneric cross hybridization between the two genera and the offspring were not viable or were triploids.
The general features of Morone include having a medium to large size the body is either moderately deep or elongate and terete or compressed dependent on the species (in striped bass the body depth is less than ⅓ of body length, and it is more than ⅓ in the other species and hybrids the mouth is moderate to large with a terminal lower jaw jutting forward of the snout opercules have at least one well-developed spine maxillary teeth are small and there are one to two hyoid tooth patches on the tongue scales are ctenoid and the lateral line is complete extending into the caudal fin there may or may not be several lateral stripes found on the fish that may be complete or broken in appearance dorsal fins are separate or slightly joined with fin spines stout that vary among species but typically are 7-8 in first dorsal and one anterior in second dorsal that may be joined or separate, there are 3 spines in the anal fin of varying length that is species dependent the caudal fin is emarginated or forked dependent on the species and, the pelvic fin is thoracic with the pectoral fins located high on the side. The hybrids of Morone are intermediaries of the parental species and typically deeper in body than striped bass with 7-8 broken stripes laterally found on the sides.
Size differences are significant in that the white bass, white perch, and yellow bass are small maturing in the 0.5 kg range with record fish being approximately 3 kg, 1.38 kg, and 1.36 kg, respectively. Netted striped bass, however, have been reported in the range of 54.5 kg with the record fish caught on fishing tackle being 31.8 kg for a freshwater system and 37.2 kg for saltwater: over 10 times the size of white bass. The record hybrid fish caught by angling was 12.5 kg.
Hybrid eggs 2.5 hours post fertilization.
Photo by R. M. Harrell
Larval hybrid striped bass at hatch.
Photo by R. M. Harrell
First feeding larvae. Photo by R. M. Harrell Live harvested hybrid striped bass.
Photo by D. W. Webster
Fingerlings. Photo by R. M. Harrell (modified)
Although one of the pure parents of hybrid Morone, striped bass, have been artificially cultured since the 1880s, Morone aquaculture really did not become established as a science until the 1960s. Prior to that time, production was essentially dependent on one hatchery system in the coastal area of the Roanoke River, North Carolina in the United States This hatchery was dependent on collecting naturally-ovulating female striped bass and spermiating males on the spawning grounds just below the hatchery location.
By 1962 hormone-induced spawning of striped bass was successful in South Carolina, and by 1965 the first Morone hybrid was artificially made by combining the eggs of striped bass with the sperm of white bass creating a palmetto bass. The original objective to produce the hybrid was to provide a fish that could occupy the open-water areas of these new reservoirs the same way white bass typically do, but had the potential to obtain the size of a striped bass and create an open-water fishery. Coupled to this logic was the failure of striped bass to do well in the relatively shallow, warm-water, man-made reservoirs which is prime habitat for the white bass. Management biologists were hoping to capitalize on the anticipated increased vigor often found in hybrid crosses, and they were looking for a fish that would be more tolerant of warmer temperatures, lower oxygen concentrations, and smaller sized reservoirs (<500 ha) that had been proven to be barriers in establishing inland striped bass populations.
Ultimately, it was confirmed that hybrid Morone did indeed exhibit hybrid vigor, had excellent potential for management, and they produced an exceptional recreational fishery. Today, with the exception of Alaska and Idaho, 48 of the 50 States have natural or introduced populations of striped bass or its hybrids. These fish are a popular recreational fish and over 200 million fingerlings (40-125 mm) are produced annually in the United States for stocking.
Aquaculture for hybrid Morone food-fish production began in the United States in the 1970s when in 1973 the first commercial hybrid striped bass production facility was started. They produced about 9 000 kg but failed in 1974. In 1977 a second farm was established and produced around 13 200 kg, but it also failed within three years. By 1980 several farms were in operation and in 2000 annual production was approaching 5 000 tonnes. Production in Asia began in 1996, while production in Europe began in 2004.
Main producer countries
According to FAO statistics (2013) the producers countries are United States, Israel and Italy, as shown in the map below.
Main producer countries of striped bass, hybrid (FAO Fishery Statistics, 2013) Habitat and biology
The various hybrids of Morone have different requirements for larval-rearing, and are strongly linked to the female parent of the cross. Once the fish are readily accepting artificial diets the production methods are similar regardless of the parental species. Production is divided into different phases hatchery (seed supply), fingerling production (nursery), and grow-out (ongrowing).
Morone are eurythermic (4&ndash30 o C). Fingerling production and grow-out to market-size fish allows for much more flexibility in biological requirements, while larval rearing is more exacting. All Morone are dioecious, group-synchronous, iteroparous, spring spawners mostly in freshwater tributaries and in coastal areas above the tidal zones. Spawning is usually initiated with increasing spring water temperature and ranges from 12&ndash24 o C with peak spawning being around 18&ndash20 o C. All Morone spawn in freshwater.
Traditionally, wild-caught fish from natural spawning grounds were used as broodstock for all Morone culture including making hybrids. Because the females were naturally close to spawning only administration of ovulatory hormones, such as human chorionic gonadotropin, was needed to stimulate ovulation. However, in recent years, the life cycle has been closed and using a combination of maturation hormonal implants and photoperiod manipulation, most Morone can be induced to spawn at least twice per year.
In nature, striped bass and white perch are pelagic spawners while the other Morone spawn near shore, usually around vegetation and/or rocky substrates. Striped bass, white perch, and hybrid eggs are non-adhesive and demersal with a specific gravity greater than freshwater. White bass and yellow bass eggs are adhesive. Mature striped bass eggs are about 1.5 mm in diameter while white bass eggs are about 0.75 mm. A water flow of approximately 30 cm/sec is required for keeping the striped bass and palmetto bass eggs in suspension. Egg development is fast and usually requires about 36 hours to hatch at 20 o C.
Newly hatched larval striped bass (prolarvae) are typically 4&ndash7 mm in total length (TL) while white bass larvae are considerably smaller (ca. 3&ndash5 mm TL). The larvae of hybrids are closer to that of striped bass than white bass but are highly dependent on the female of the cross. Larval Morone have a yolk sac that contains a large oil globule that helps maintain buoyancy in the water column. Mouth parts are typically developed by 3&ndash5 days post-hatch and they begin feeding on small zooplankton.
Rarely do first-feeding larval Morone accept artificial food. Therefore most the nursery production of fingerlings is usually a separate segment of the aquaculture of the species and their hybrids and is conducted in outdoor pond systems where natural zooplankton populations are manipulated and managed. Due to size differences of the larvae and exacting live food size requirements rarely are there completely closed life-cycle operations. Larval white bass, white perch, yellow bass and hybrids of these crosses with the female being the parent require very small zooplankton (i.e., rotifers) for first feeding. Larval striped bass and palmetto bass hybrids can be started on first instar nauplii of Artemia. All Morone larvae do better with their first food sources containing high levels of highly unsaturated fatty acid enrichments especially EPA and DHA.
Preflexion larval (hatch&ndash12 mm) Morone and hybrids quickly develop into postflexion fry (12&ndash25 mm), then fingerlings (>25 mm), with the latter having complete fin complements, usually within 30 days post-hatch dependent on food availability and temperature. As they grow they typically have a down-stream movement, especially striped bass and white perch. In coastal areas striped bass and white perch enter the estuaries and spend most of their life there, moving upstream to spawn annually once mature. In some coastal river systems larger striped bass, especially females, move off-shore and migrate up and down the East Coast of the United States overwintering before moving back into estuaries and freshwater tributaries to spawn in Spring. There is an introduced striped bass population on the West Coast of the United States that follow a similar pattern. There are no reports of hybrid Morone being captured in near-shore coastal waters.
In broodstock hatchery operations mature fish (both striped bass and white bass for hybrid production and parental stock domestication and selection) are maintained at low densities in 3-10 ppt salinity and fed high-quality protein and fat diets. Water quality is maintained by filtration, ozonation, oxygenation, and degasification. Photoperiod and temperature is controlled and appropriately manipulated along with hormonal implants to cycle spawning.
Production cycle of striped bass, hybridThe production systems of hybrid Morone culture are very specialized and typically broken down into three stages: seed (hatchery production), nursery (fingerling production), and ongrowing (grow-out). The seed stage lasts days to weeks unless controlled, out-of-season production is occurring then it can be a year-round process. The nursery stage lasts 1&ndash10 months dependent on whether a phase I (30&ndash75 mm) fish or an advanced phase II fingerling (100&ndash200 mm) is required. The ongrowing stage is market driven (typically 0.75&ndash1.5 kg) and can take 10&ndash24 months or more depending on size required and production system (indoor vs. outdoor) used.
Because the target fish of this information sheet is a hybrid between two species of Morone the only source of seed is by artificial production from a hatchery system. The two major crosses of hybrid Morone are the palmetto bass (striped bass ♀ X white bass ♂) and its reciprocal cross,sunshine bass (white bass ♀ X striped bass ♂). These two crosses will be the example of production discussions of hybrid from this point forward.
Regardless of the cross produced, hybrids must be made by manually stripping the eggs from the female of choice and then manually fertilizing them with sperm from the male of choice as volitional tank spawning rarely occurs to make F1 hybrids. The dry method of fertilization is the preferred method because once water is added sperm motility is very short-lived (usually less than 2 minutes). Gynogenetic and triploid hybrid Morone have been created, but the process is technical and beyond the scope of this report.
Historically, these crosses were made with broodstock collected from the natural spawning grounds in the spring of the year (late February to early June dependent on location). In the past decade or so, however, many hatcheries have been developing their own lines of broodstock and are becoming less dependent on gravid, running-ripe, wild broodstock. This shift is especially true where the parental Morone are not a native species and not readily available. Some hatcheries are also using domesticated fish in concert with photoperiod and hormonal manipulation to conduct out-of-season spawning.
Once fertilized, the egg incubation of hybrid Morone typically occurs in MacDonald hatching jars, which minimizes space requirements. Domesticated seed suppliers producing palmetto bass have an advantage due to the size of the eggs and the fact that striped bass eggs are non-adhesive, which lend themselves to circular tank incubation options. Also palmetto bass larvae are larger at first feeding thansunshine bass (see below).
Given that white bass eggs used to makesunshine bass are adhesive there are limitations to incubation systems, which require some means to break-down the adhesive matrix surrounding the eggs (i.e., tannic acid treatment 150&ndash300 mg/L for 7&ndash12 minutes). This step in incubation is important because of the probability of fungi attacking dead eggs and spreading to viable eggs during incubation, which can cause catastrophic losses. There is no approved treatment for these fungi for fish in the United States. Thus, use of tanks for incubation ofsunshine bass, while possible, is more problematic. Whensunshine bass eggs are incubated in MacDonald jars and the resultant embryos hatch they swim up and out of the jar into an aquaria set-up with a standpipe and a mesh screen of appropriate size to prevent loss of larvae down the drain. Diligence must be provided to prevent the fine-mesh screen from clogging and the water overflowing the standpipe.
Regarding choice ofsunshine bass versus palmetto bass as the hybrid of choice there are several considerations. Palmetto bass have larger eggs and resultant larvae, and therefore at first feeding are able to consume first instar Artemia nauplii. Conversely,sunshine bass larvae must be started on a smaller live food supply, usually rotifers at a density >300 rotifers/L. This prey-size difference is important because rotifers are more expensive and complicated to raise than Artemia, and thussunshine bass are harder to grow in indoor tank culture. Secondly, female striped bass yield hundreds of thousands of eggs, whereas female white bass produce only tens of thousands, and therefore fewer striped bass females are needed to produce the same number of larvae of palmetto bass. Regardless of these two positive aspects of palmetto bass, thesunshine bass is the hybrid of choice for seed suppliers because female white bass mature faster (average of 2.5 years versus 4-6 for striped bass females), they are easier to handle in spawning, and their ovulation is less synchronous (once a female striped bass ovulates all the eggs are spawned completely within a very short window so timing of ovulation is crucial). Also, a majority of male striped bass are mature at 2 years of age.
At hatch, bothsunshine bass and palmetto bass larvae have an endogenous source of energy in the yolk-sac and oil globule. This stage is known as prolarvae. In reality, due to size differences in overall total length, the time between endogenous and exogenous feeding is similar among the two crosses: 4&ndash5 days post-hatch at 20 o C.
Typically, prolarval hybrid Morone are kept in hatchery systems until the mouthparts are developed and the larvae begin to feed on live food sources (4&ndash7 days post hatch dependent on temperature). It is not unusual for prolarvae to be shipped to nursery producers such that when they arrive they are close to or ready to initiate exogenous feeding.
Essentially all of the phase I stage of hybrid Morone nursery production is conducted outdoors in specialized earthen ponds. These ponds are specifically designed to drain easily into a catch-basin either located within the pond proper or at the exit of the drainage system. Supply water is almost always from natural freshwater or brackish (< 10 ppt) sources because it has a readily available source of zooplankton that is critical for larval survival success. It is essential, however, when using natural water systems (rivers, ponds, lakes, or estuaries) that the incoming water be filtered with a fine-mesh screen (200-400 μm to prevent the introduction of other fish eggs or larvae that may prey upon the hybrid Morone larvae), and that timing of stocking the nursery ponds coincides with the early successional patterns of zooplankton dynamics progressing from rotifers to cladocerans and copepods. Thus, not only does the water source need to have the right zooplankton populations as an inoculate seed source, the pond must be appropriately fertilized to manage the phytoplankton-zooplankton dynamics.
Because Morone larvae are sensitive to light and variations in water quality, stocking is usually done at twilight when pond oxygen levels are still high. Tempering of shipping water with pond water is critical especially with regard to temperature, salinity, and pH. Stocking densities are dependent on the size needed at harvest. For phase I production, larval stocking densities range from 125 000&ndash1 000 000 larvae per hectare with 30&ndash60 day harvest sizes of 25&ndash40 mm and 450&ndash3 500 fish/kg. The smaller densities yield larger fish but there is usually a larger size diversity of the fingerlings. Most ongrowing producers want larger fish for stocking so the lower densities yielding the larger fish at harvest are the typical approach. Each nursery producer understands what their pond systems will yield in time, size, and quantity of fish.
The major limitation on phase I production in length of time is in relating prey size and availability to growing fingerlings. Once the fish reach the 40&ndash50 mm size (
1 g) they switch from planktivorous feeding to a piscivorous nature and begin cannibalizing each other. Here it is crucial to either train the fish to feed on artificial diets while still in the pond or harvest and grade the fish and train them to feed in indoor tanks.
Traditionally, once phase I fish are harvested, graded, and trained to feed they can be restocked for phase II production to produce larger-sized fish (75&ndash250 mm), stocked directly into tanks for intensive production, or graded and stocked directly into grow-out ponds (avoiding phase II production) to produce market-sized fish. This latter method is known as direct stock.
Direct stock can be accomplished by selecting out the larger (
3 g) fish from phase I production that invariably results from differential growth or the smaller phase I fish are held until they reach
3 g and are graded for uniform size and then stocked into final grow-out ponds. Once the 3 g fish are stocked into grow-out ponds they must be trained or retrained to accept artificial diets in the pond and fed until they reach market-size (18&ndash24 months). Direct stock avoids harvesting phase II fish, which is a costly step in labour and potential loss of fish due to additional excessive handling and stress.
The traditional method requires grading the fish and training them to accept artificial diets &ldquoin house&rdquo then restocking phase I fish (
1.5&ndash2 g) in the same phase I ponds from which they were harvested then fed and held for up to 10 months when they are then harvested before the next phase I nursery season. Stocking densities for the traditional phase II production are in the 10 000&ndash250 000 fish/ha range (most producers stock in the 25 000&ndash60 000 fish/ha range) and density is related to desired size at harvest (i.e., lower densities yield larger size fish). Stocking densities in the direct stock method are approximately 9 250&ndash10 000 larvae per ha because these fish will not be harvested until they reach market-size (ca 0.75&ndash1.5 kg).
The traditional phase II production has an economic advantage in that it affords the use of the specialized nursery ponds year-round where the second phase of production yields smaller numbers but higher value fish owing to size. During phase II production and direct stock strategies, as long as fish are feeding (usually temperatures above 16 o C) they are fed at least once daily with a high protein (30&ndash50 percent), high fat (10&ndash16 percent). The goal is to get as much growth as possible within this growing the fish can &ldquooverwinter&rdquo with minimal mortalities and/or weight loss. Outdoor feeding usually ceases when water temperatures get below 6 o C.
Typical phase II harvest size initially stocked at densities in the 20 000&ndash30 000 larvae per hectare range (stocked at a size of
650 fish/kg) yield fish in the 125&ndash225 g range. Obviously a trade-off exists between phase II production and the direct stock method with respect to biological maximum growth and economic maximum growth. These variables are site and operator dependent.
Over the past 10 years 70&ndash90 percent of all ongrowing methods of hybrid Morone has occurred in earthen ponds. Regardless of whether the producer is using the direct stock method or stocking phase II fish, the goal is to reach market size before the end of the second growing season. Based on a mean survival of 80 percent, yield per production pond typically ranges between 1.6 and 1.7 tonnes.
Because of the carrying capacity of the pond supplemental aeration is essential. Supplemental aeration can be in the form of paddlewheels, airlift pumps, bubbler systems or even adding freshwater from an external source. All these supplemental efforts impact economic yield as they either run off electricity or diesel fuel, which must be factored in production costs.
When the pond carrying capacity is being approached water quality is crucial. Shifts in unionized ammonia, pH, nitrite and even total gas saturation can lead to stress and mortality. Stressed fish tend to cease feeding and become susceptible to secondary disease infections. Constant attention must be provided at all stages of production.
Stocking densities for ongrowing techniques are similar to that for direct stock approaches (9 250&ndash10 000 fish per ha). Second-year growth of hybrids should yield 1 kg fish by 18 months of production from larvae to market.
In the United States in 2014 about 17 percent of the fish were produced in closed or semi-closed recirculating aquaculture systems where water quality, temperature, oxygen, and feeding was carefully controlled. Under these conditions fish typically reach market-sized fish in 10&ndash12 months because the fish are still growing during winter months where fish in ponds essentially cease growth. Stocking densities are system and redundancy back-up systems dependent and are too specific to be covered here. Likewise in 2014 about 2 percent of the market-size hybrid Morone production occurred in cage culture. Because fish in cages are exposed to the same weather conditions as those in ponds their typical grow-out times are similar. Stocking densities are also site, cage or net-pen size, and back-up aeration capacity dependent and is not covered here.
Harvesting phase II fish is accomplished in the same manner as phase I fish. The pond is dewatered and the fish are collected in a catch basin with nets, graded, and placed on transport trucks to be taken to the grow-out producers or to a holding facility for later shipping. If the pond is too large and expensive to dewater other options to harvest include using large haul seines. Haul seines can be size selective and, dependent on the mesh size used, a population can be &ldquothinned&rdquo periodically to maintain uniform size. This technique is useful with hybrid Morone owing to their hybrid vigor and faster growth rates. In using haul seines, boom-loaded baskets dip the concentrated fish out of the nets and then loaded on a transport trucks, or the fish are crowded into a fish pump or fish.
Ongrowing fish harvest is often carried out over several months. In tank and cage-culture systems harvest is simple wherein fish are crowded by screens and netted out for thinning or complete harvest, grading, and, ultimately, processing. In pond systems fish are harvested by use of a 4 cm or larger, soft-mesh, knotless seine. Using the smaller mesh size all market-sized fish will be captured and smaller than market-size fish will escape through the mesh. Market-sizes range from small (0.5&ndash0.7 kg), medium (0.7&ndash1.0 kg), and large (>1 kg) fish. Using larger mesh seines affords the opportunity to be size selective and respond to market demand. Partial harvest affords the smaller fish the opportunity to continue to grow without undue competition from the larger fish. Partial harvest also provides fish availability over a longer period.
On-grown fish can also be harvested a fish elevator system. This system is simply a mechanically operated "Archimedes Screw" contained within a fiberglass or PVC pipe wherein the fish are lifted from the pond and off-loaded on a truck or tank system. It can have a built-in grader distribution system that sorts fish by size and distributes them to separate holding tanks or hauling trucks. This system minimizes tissue damage, prevents unsightly wounds, abrasions, and/or scale losses that may impact the quality of the fish at market.
Hybrid striped bass are marketed and processed in two primary fashions: live market and fresh, whole-fish on ice. There is a growing trend in the Individual Quick Freezing (IQF) flash-frozen, fillet market, but it is secondary to the two primary methods. Regardless, because of the hybrid Morone&rsquos high metabolic rate and susceptibility to handling stress care must be taken to avoid product quality and shelf-life problems. Thus, harvested fish should be handled as gently as possible with minimal physical contact as they will &ldquobruise&rdquo easily and develop stress-related reddened areas on the flesh due to petechial hemorrhaging.
If fish are live hauled, particularly in warm weather, they should be placed in a cooled, salt-water system (isosmotic
7&ndash10 ppt) with pure oxygen supplied to the tank to minimize stress. Live haul trucks typically have 2 500&ndash4 500 kg capacities but density should be considered in proportion to distance to market or processing facility and the amount of ice needed to keep temperatures down and oxygen needed to keep dissolved oxygen at saturation or higher level.
Alternatively, fish can be packed on ice in boxes at the harvest site and loaded onto a refrigerated truck for transport. In this case immediately &ldquochill-killing&rdquo the fish at harvest by placing them in an ice brine may be advantageous. First, it quickly calms the fish down and prevents further injury to itself or other fish. Second, it quickly lowers body temperature, which helps maintain ice levels in packing boxes and prolongs the freshness of the fish tissue.
The amount of ice needed for transport is dependent on the initial temperature of the fish, the adequacy of the insulation in the transport unit, and the amount of time the fish need to be on ice. In general, with good fish-ice contact, 0.33 kg of ice will reduce the temperature of a 1 kg fish from 27 o C to 2 o C over a 4-6 hour period. If longer periods of time are needed to keep the fish iced (e.g., 12 h) then a 1:1 ratio of ice to fish weight is recommended. Fresh fish that have been adequately chilled and iced immediately can be held on ice for 8&ndash9 days and still maintain high quality of the flesh. Ideally flesh should be maintained as close to 0 C as possible.
In processing, the fish should be bled as soon as possible. After exsanguination, fish are washed and eviscerated. The head may or may not be retained dependent on market choice (e.g., fish is marketed &ldquoin the round&rdquo). For fish that are not to be skinned, scaling should be done even before filleting. The filleting can be done manually or mechanically, but mechanical filleting usually produces a lower yield. With hybrid Morone fillets yields are about 29&ndash50 percent of the whole fish. Other processing yields can be seen in the table below.
Processing yields fromsunshine bass (from Coale et al., 1993)
Product Form Percent Yield Whole fish 100 Dressed, with gills 90.5 Dressed, without gills 85.4 Fillet, with rib bones 45.4 Fillet, with skin 41.9 Fillet, without rib bone, skinless 32 Fillet, without rib bone, skinless, trimmed 29.5 Solid waste: Frame 48.8 Skin 19.8 Viscera 9.5
Fixed costs for production systems are dependent on the production segment(s) with which the producer is involved (e.g., land purchase or water column leases for pond, cage or net-pen, intensive tank culture) water supply (e.g., wells or pumps for surface water systems) loan costs and permitting requirements listed by the country in which the producer is located and who owns the land and water supply systems. Variable costs include electricity, feed, seed supply (contingent upon production segment), production labour, other labour needs, packaging, processing, distribution, maintenance, supplies, medicines (where available), chemicals, and insurance. In recent years feed has seen the biggest increase in annual costs with shifts as high as 17 percent. The second highest increase in variable costs, at least within the United States, has been crop and liability insurance with annual increases approaching 12 percent. Transportation costs from the farm to the processing facility or market are also a considerable variable cost.
Other limiting factors that need consideration are diseases and approved disease treatment proper food storage to prevent the establishment of mould or infestation of insects the presence of fish eating birds and a means to control them snakes, alligators, and turtles and back-up generator systems in case of power failures when supplemental oxygenation and/or water flow systems are needed.
Good husbandry techniques can help avoid most hybrid Morone diseases. Many of these diseases are as a response to some external stressor that has made the fish more susceptible to infection with a disease agent. Thus, the key to disease management is stress avoidance.
In some cases antibiotics and other pharmaceuticals have been used in treatment but their inclusion in this table does not imply an FAO recommendation.
- For all measures no chemicals can be used in the United States for control of infectious diseased fish for human consumption. Thus, health maintenance, disease prevention, quarantine, and maintaining husbandry practices with good water quality is essential to good fish health.
- In all cases with bacterial infections avoid stressing animals in captivity and maintain good water quality at proper stocking densities. Disinfecting water in recirculating systems with UV or Ozone helps. In freshwater systems prophylactic treatments with Na Cl (0.5-2 percent) or Potassium Permanganate (2-5 ppm) for differing periods of time may be helpful. Check to see which medicated feeds and/or vaccinations are allowed for systemic infections.
- Because protozoans are ubiquitous and are most problematic during stressful conditions such as handling or poor water quality, utilization of best management practices in husbandry and water quality management is important. Insure good water quality, moderate stocking densities, good nutrition, and proper handling will mitigate the probability of protozoan infections.
Most federal, provincial, and state agencies responsible for managing the natural resource of the country of interest will have some disease-diagnostic services. In academia, most veterinary colleges and universities will be a source of assistance. There are limited numbers of private practices that deal with fish diseases so it is important to work with the state or provincial licensing authorities to locate a certified disease specialist.
Within the United States primary contacts would be through the U.S. Department of Agriculture Animal Health and Plant Inspection Service and their regional Fisheries Research Centers the U.S. Department of Interior Fish and Wildlife Service and the U.S. Geological Survey fishery research units and the Department of Commerce National Oceanic and Atmospheric Administration Aquaculture Division.
In the United States, 2014 pricing of hybrid Morone for live fish was USD 4.37/kg, which was down from 2013 FOB (Free On Board) prices of USD 4.43/kg. In contrast whole fish on ice in 2013 brought an average retail price of USD 3.60/kg while it increased in 2014 to USD 5.25/kg. Overall in the United States FOB pricing for whole fish (on ice) had increased 2.4 percent/year since 1996.
In 2014 the United States, industry total farm-gate market segment exceeded USD 33.2 million. Individual farm-gate market segment values were as follows: On-ice (whole) &ndash USD 21.9 million Live &ndash USD 8.7 million Seed stock &ndash USD 323.5 thousand Nursery fingerlings &ndash USD 6.8 thousand. Thus, the largest market share value-wise is with whole fish in the round on ice. On a global basis there was a significant jump in market value that exceeded USD 50 million while previously the highest global value was around USD 37 million.
With the exception of 2013, hybrid Morone culture production has seen a decline to steady rate production in the past decade, but production in Europe and Asia is expected to continue into the near future as more domesticated broodstock are developed, and they become less dependent on United States seed suppliers. Within the United States the per capita consumption of cultured seafood is expected to reach 55 percent in 2015 and projected to grow. Hybrid Morone is able to compete well with other marine species and within the United States its largest competitor is the striped bass from wild-caught fisheries, which is seasonal and imports of marine species such as European seabass (Dicentrarchus labrax) and spotted seabass (D. punctatus), barramundi (Lates calcarifer) and other Lates spp., Patagonian toothfish (Dissostichus eleginoides), and fish such as common dolphinfish (Coryphaena hippurus). There appears to be strong pricing growth expectations especially as hybrid Morone begin to be exported more to Europe and Southeast Asia. Overall on a global basis there is an expectation in production growth and competitive pricing. European and Asian production appears to be all consumed locally with little to no export. There is limited export of hybrid Morone of market-sized fish.
With regard to needed areas of research the six identified priority areas by the hybrid Morone industry are as follows: 1) understand and improve the nutritional needs of the different hybrid and parents being cultured 2) improved survival and growth of larval fish especially in indoor tank systems 3) improvements in controlled reproduction 4) better understanding of genetic variability across natural geographical ranges of broodstock 5) detailed information on parental strains and, 6) development of DNA markers and genetic maps that affords better selection efforts (to date over 500 microsatellite markers have been identified).
Strain evaluations of parental crosses are still on-going at several United States academic institutions. There are on-going efforts in the United States to domesticate a marine strain of striped bass that will be more suited to off-shore net-pen operations that, when successful, will be a year-round competitor for hybrid Morone production capacity.
Within the United States there is little effort to domesticate white bass males or females because they are so readily able to be captured as gravid and running-ripe individuals from the wild. Some effort is ongoing to domesticate both striped bass and white bass in Europe and Asia. Hormonal maturation and ovulation control research is still on-going but current technology, along with photoperiod-thermal manipulation to extend spawning seasons and provide out-of-season spawning, appear to be adequate.
Diet and nutrition research are critical to continued success and improvement in the industry. It appears proper balance of the dietary amino acid profiles, supplements with high omega-3 fatty acid, and substitutes for fish meal and oils are high priorities. Research with striped bass diets has demonstrated squid substitutes for fish meal and oils up to 25 percent is successful. Some efforts have been undertaken to genetically select for white bass that produce a larger mouth gape to remove the need for the rotifer batch culture approach and increase opportunities to fully close the life cycle of broodstock to indoor systems.
Male gamete storage has become more successful with both cold-banking and long-term cryopreservation. This fact will aid in the growth of the hybrid industry because often it is difficult to synchronize spawning of the two parents required to produce hybrids due to slight temporal spawning differences between species. Having cryopreserved semen alleviates that concern.
From an economic perspective sustained growth will be dependent on improved success of closed, high-density production systems, improved access and affordability to and of crop insurance to protect against catastrophic losses, and reasonable access to both public and private funding as incentives for construction and reasonable consideration to loan repayments.
The main concerns associated with hybrid Morone culture are similar to those of any predatory fish species. These include ecological, environmental, and genetic. From an ecological perspective because hybrid Morone can disrupt the natural ecology if they escape into the environment, especially outside of their natural range.
Environmental concerns relate to use of antibiotics, waste discharges, and processing by-products. The use of antibiotics and some prophylactic chemicals to treat the water source can release non-metabolized antibiotics into the environment raising the concern of antibiotic resistant strains of bacteria. Likewise, there are concerns about the potential of bioaccumulation of prophylactic chemicals in sediments and other areas associated with aquaculture site locations. Similarly, because the diets are high in protein and fish digestibility levels generally are inefficient, there is a constant concern of organic waste and nitrogen and phosphorous build-up in effluents or underneath open-water net pens or cages. Processing waste disposal is another important environmental consideration. Most operations are constantly looking for waste utilization options and some innovative approaches are now being tested.
Last are genetic concerns. Hybrid Morone are not sterile and several generations and backcrosses have been found under both artificial production settings and in natural environments where hybrids have been stocked with sympatric parental species. Thus, while not common, backcrosses and putative F2s have been collected from the spawning grounds of other Morone. So the potential of outcrossing with pure parents is real and should be considered before large-scale operations are put into place &ndash precautions in design and redundancy to prevent escape are therefore important.
Recognition of the risks associated with any form of aquaculture should be integral to any planning and development considerations. Precautionary steps, if appropriately taken, should mitigate any major concern responsible agencies may have in permitting and approving aquaculture operations regardless of the target species being considered.
Aquaculture is a growing global food production sector and has strong links to natural resource management and concerns for environmental protection. Because hybrid Morone are a vertebrate and their culture is usually in concentrated intensive husbandry conditions there are growing concerns for animal welfare. Production operations should proactively take steps to minimize any undue stress to the animals and minimize what might be perceived as animal cruelty. Such considerations as hunger, discomfort, pain, injury, or disease are all listed in the arena of animal cruelty. This concern is especially prevalent in Europe and is growing annually within the United States Therefore, ethical production of all phases of the aquaculture operation is important. Proper steps taken to ensure animal welfare can also be an excellent marketing tool and aquaculture associations that police themselves and certify that operations take critical steps to ensure animal welfare may well be a branding opportunity that adds value to the final product.
Certain market segments such as European countries will not accept genetically modified organism. Thus, as research continues to move forward we must find other means to improve performance and examine ways to produce fish that grow faster, are more disease resistant, and have better food conversion ratios. At the moment interspecific hybrids are not shunned by consumers, but there are regulatory issues that must be considered and permission to produce procured before operations are initiated.
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