Before we get to the "rules", I want to say a few things. There are no rules when it come to making your work mean something, only suggestions so that you can make your own rules. Also, there are a few anime titles that would help you with adding symbolism, Serial Experiments Lain and Neon Genesis Evangelion. So, let the suggestions begin.

I. Expression is everything. If everything from the color to the text says that the person (or whatever) in your art is sad, but there is a big smile on the characters face and his/her/it's eyes art little crecents, the artwork is not getting the point across (unless, of course, the point is that the character is insane). Here are some examples of effective emotional expression.

a. Happy: Cresent-shaped or glassy eyes, anywhere from a small line of a smile ot a wide-open mouth smile (depending on the character), cheeks and nose sometimes blushed (blushing rules coming soon!)
b. Sad: Glassy eyes (either big or half-closed), knit eyebrowns, little frown (if the frown is too big, the character will look like they are faking it), lip more pronounced/pouty.
c. Surprised: Big eyes, open mouth in a frown. (this is for the serious anime, humerous anime is a little more...intense)
d. Terrified: Big eyes, small iris and pupil, sweat, twisted frown(showing teeth is always a plus).
e. Embarressed: Big eyes, small iris and pupil, blush size is directly proportional to how embarrassed the character is, small frown, raised knit eyebrows (^ shaped)
f. Confindent: Eyes closed a little less than half-way, sideways smirk, cocked head
g. Angry: Slit eyes, clenched jaw, knit eyebrows, tensed muscles (sharp canines add a frightening effect)
h. Determined: Knit eyebrows, big fierce eyes, small frown, a bit of a blush to the nose and cheeks helps add a bit of emotion to the character
i. Questioning: Big eyes, slightly raised eyebrows, small mouth.
j. Scared(not quite terror stricken): Big glassy eyes, raised knit eyebrows (making a ^ shape sort of)

II. Mixed emotions are sometimes more effective than one emotion because no one feel a single emotion at a time.
a. Anger/Sadness: Big glassy eyes, frown and/or gritting teeth, knit eyebrows
b. Anger/Surprise: Big eyes, small iris and pupil, knit eyebrowns, open mouth in a frown

III. Symbolism can come in many forms. It is the strongest force in art and can get a point across the way that nothing else can.
a. Text: Text is one of the more blatent and easy to spot kinds of symbolism, but it is no less a powerfull force. As in the example the most common way to use text is to have contridicting statements. The words "Motherless Children" seem to contridict the comforting statement: "God is in His Heaven and All's Right with the World". The character also contributes. She looks frightened, as though this contradiction is scaring her.
b. Images: Images are generally the most used type of symbolism. As in the example there are several things in the girls surroundings that lend to her personality and the meaning of the image. In the foreground, there is a spider eating a butterfly caught in it's web. Not only does the web contribute to the age of the room, but it shows the spider, a creature that often evokes fear, eating a butterly, a creature that is considered full of beauty and grace, as if the girls fear or the ugliness of her surrounding is eating away her beauty, inner or outer. The room and the broken glass are examples of how setting can be used as symbolism, which is explained below.
c. Texture, Color and Setting: Texture, color and setting are the most subtle and powerful types of symbolism. The example shows a young woman sitting on a broken wall over looking a pool and some buildings. But, on closer inspection, you may notice that the texture of her dress and the texture of the wall are the same. This symbolises that she is like a wall, she seems impenetrable, and yet she can be broken. You may also notice that the color of her eyes is a darker shade of the color of the wall, and that her hair is the color of the sky and her dress the color of the water in the pool. This is used to lead you to the point that the setting is symbolic of the character. Now, according to several books, water is symbolic of the womb and perhaps of the mother that the young woman never had. There are buildings in the background, but they are grey and seemingly devoid of life, showing her lonliness, but because all of this is half hidden by the wall, she does not show this on the outside. Also, the wall is damaged and she is injured showing that her wall is breaking down.

This lovely set was made by: