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s. souvorowiana , anatolica pseudiduma frivaldszky , and adesmia anomala dejeani gebler adults were collected in 2011 from fukang ( 44 24 n , 087 51 e , 444  m ) , which is about 100  km northeast of the geological center of asia .
0.5c , 30    6% relative humidity ( rh ) , and 16:8 ( l : d ) h photoperiod conditions and supplied with cabbage daily .
adult rearing , egg collection , larval rearing , and pupal collection were conducted following the methods of wang et  al .
all adults used in these experiments were obtained from both field collections and colonies maintained under laboratory conditions . to determine sexual dimorphism in external morphology ,
body parameters of adults ( including head width , pronotum width and length , elytra width , and body length ) of each sex were measured under stereomicroscope equipped with elements 3.0 software ( nikon smz-800 , nikon corporation , tokyo , japan ) and calibrated using an objective micrometer . statistical significance for adult body parameters
was determined using a student s two - tailed t - test with a significance level of p  <  0.05 .
the analysis was conducted by graphpad prism 4 software . when sexing by the tergal pigmentation pattern probing method , it was most effective to use the thumb and forefinger to hold each beetle with its head downward and against the microscope stage ( with its ventral side up ) , while using the other hand to manipulate the probe .
the probe , such as a separated arm of stainless tweezers ( 134-mm length ) , with the point blunted and polished , was inserted between the elytra and the abdomen , about 12  mm from the tip of the abdomen . by sliding the probe slightly beyond the apex of the abdomen , the edges of the last outer sternite and last outer tergite separate ( sappington and spurgeon 2000 ) .
pressure from the probe on the dorsum of the abdomen exposes the eighth abdominal tergite ( fig .
2.female s. souvorowiana adult being sexed by observing the posterior margin of the eighth tergite via probing .
pressure from a probe on the dorsum of the abdomen exposes the posterior edge of the eighth tergite .
female s. souvorowiana adult being sexed by observing the posterior margin of the eighth tergite via probing .
pressure from a probe on the dorsum of the abdomen exposes the posterior edge of the eighth tergite .
the regions lacking pigmentation are indicated by arrows . in the field , this observation can be conducted with the assistance of an eye loupe ( 10 ) .
sexed beetles were segregated , and confirmation of sex was carried out by dissecting the genitalia .
differences between male and female adults of s. souvorowiana are difficult to distinguish as this species does not have obvious sex - related external markings ( fig .
3 ) . there were no significant differences ( student s t - test , two tailed ) between males ( n  =  14 ) and females ( n  =  12 ) in the head width ( df  =  24 , t  =  0.19 , p  =  0.85 ) , head length ( df  =  24 , t  =  0.87 , p  =  0.39 ) , pronotum width ( df  =  24 , t  =  1.11 , p  =  0.28 ) , pronotum length ( df  =  24 , t  =  0.96 , p  =  0.35 ) , elytra width ( df  =  24 , t  =  0.59 , p  =  0.56 ) , elytra length ( df  =  24 , t  =  0.44 , p  =  0.66 ) , or body length ( df  =  24 , t  =  0.94 , p  =  0.35 ) ( fig .
similar results were found in other insects ( sappington and spurgeon 2000 , fairn et  al .
3.dorsal view of male ( left ) and female ( right ) adults of s. souvorowiana . 

4.variation in morphological characters of male ( n  =  14 ) and female ( n  =  12 ) s. souvorowiana adults . box and whiskers show the extreme minimum , lower quartile , median , upper quartile , and extreme maximum .
differences in means for body length and other body sizes are not statistically significant between sexes ( student s t - test , two tailed ) .
dorsal view of male ( left ) and female ( right ) adults of s. souvorowiana .
n  =  14 ) and female ( n  =  12 ) s. souvorowiana adults . box and whiskers show the extreme minimum , lower quartile , median , upper quartile , and extreme maximum .
differences in means for body length and other body sizes are not statistically significant between sexes ( student s t - test , two tailed ) .
after dissection of hundreds of the insects , we recognized the morphological differences in s. souvorowiana between the male and the female in pigmentation and the hairs on the eighth tergite ( table 1 ) .
the pigmentation occurred everywhere on the eighth abdominal tergite in males , whereas the pigmentation in females was absent ( white regions ) at the laterocaudal angles ( fig . 5 ) .
we speculate that the white regions ( lacking pigmentation , unsclerotic ) on female tergite may help to accurately perceive and accept the male aedeagus .
2 ) , using a polished probe ( a separated arm of the stainless tweezers , 134-mm length ) . based on this sexual - specific characteristic ,
a new sexing method was established , which we termed the tergal pigmentation pattern probing method . 

the eighth tergite of female has two white regions lacking pigmentation at its laterocaudal angles ( arrows ) ; however , the eighth tergite of male is covered with dark pigment all over .
meanwhile , hairs on the eighth tergite of the male are denser than those of the female . 

table 1.keys for sexing adults of s. souvorowianacharactermalefemalepigmentation on the eighth tergitepresent all overabsent at the laterocaudal angleshairs on the eighth tergitedensesparse dorsal view of the eight tergites in s. souvorowiana adults .
the eighth tergite of female has two white regions lacking pigmentation at its laterocaudal angles ( arrows ) ; however , the eighth tergite of male is covered with dark pigment all over .
meanwhile , hairs on the eighth tergite of the male are denser than those of the female .
keys for sexing adults of s. souvorowiana at the beginning of this study , we attempted to expose the eighth tergite as fully as possible to gain a complete view . with our experience
obtained , it became unnecessary to completely separate the seventh sternite and tergite to see the whole eighth tergite .
moreover , hairs on the eighth tergite of male were denser than that of female ( fig . 5 ) . without exception ,
live male and female adults of s. souvorowiana ( n  =  200 total ) were nondestructively distinguished by the tergal pigmentation pattern probing method , as was true with a sample of 100 dead individuals as well .
the experienced personnel in our laboratory can routinely discriminate live beetles about 810 per minute .
in addition to the pigmentation patterns , the density of hairs on the eighth tergite was helpful for sex determination . also , the sexing experiment could be conducted with 100% accuracy using an eye loupe ( 10 ) in the field ( n  =  30 ) . for students with good eyesight
, beetles could be sexed with naked eyes . with the tergal pigmentation pattern probing method
, there was no need to squeeze the abdomen of insect or withdraw the sternite from the abdomen with forceps .
in addition , the eighth sternite usually bent into the last external visible sternite and was difficult to be observed .
the nondestructive method we developed for sex determination of the adult darkling beetle s. souvorowiana is as accurate as dissection methods .
this method is also applicable to other species of tenebrionid beetles , such as a. 
 pseudiduma and a. 
 anomala dejeani ( fig .
it is possible that this method would be useful for identification of more species in tenebrionids .
further work is required to determine the effectiveness of this method for predicting sex in other tenebrionid beetles . 

arrows indicate the white regions . moreover , the colors on the eighth tergites of the females are lighter than those of the males . 
 
arrows indicate the white regions . moreover , the colors on the eighth tergites of the females are lighter than those of the males .
the darkling beetle , sternoplax souvorowiana ( reitter ) ( coleoptera : tenebrionidae ) , is flightless and lives in the guerbantonggut desert in northwestern china . its special eggshell structure , 
 day - active habit , large body size , short life cycle , and ease of rearing under laboratory conditions make it an excellent model for advanced studies on desert adaptation . determining the sex of this beetle is usually complicated by the lack of a discreet , externally visible gender - specific character . to date 
 , dissection has been used for sex identification in this species , whereas a nondestructive means is needed for further studies of sexual dimorphism . here , a new method based on the difference of the pigmentation pattern on the eighth tergite of each sex is described and illustrated . 
 this method can be quickly learned , is nondestructive , is 100% accurate , and is fast enough for most applications in both the field and the laboratory . 
 experienced users in our laboratory routinely sex 810 beetles per minute .